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E2F6 is a Common Repressor of Meiosis-Specific Genes

Permanent Link: http://ufdc.ufl.edu/UFE0021593/00001

Material Information

Title: E2F6 is a Common Repressor of Meiosis-Specific Genes
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Kehoe, Sarah M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: ant4, e2f6, meiosis, repression, transcription
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Genes whose protein products function specifically during meiosis are strictly repressed in somatic cells. Their aberrant expression may lead to mitotic catastrophe and predispose cells to oncogenic transformation. Although previous studies have demonstrated various mechanisms delineating repression of individual meiosis-specific genes, the existence and identity of a master regulatory protein which coordinately represses multiple meiosis-specific genes in somatic tissues remains to be elucidated. E2F6, a member of the E2F family of transcription factors, was recently shown to play an essential role in the repression of a few meiosis-specific genes including Stag3 and Smc1?. Here we report that E2F6 is required for the repression of another meiosis-specific gene, Ant4, in somatic cells. This discovery prompted us to investigate whether meiosis-specific genes in general, require E2F6 for their repression. We compiled a list of 24 meiosis-specific genes and observed that 19 of them (79.2%) have the core E2F6-binding element (TCCCGC) within 200bp upstream of their transcription initiation sites. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%) and the mathematical probability of random occurrence (9.3%). Further, using embryonic stem cells, we demonstrate that E2F6 indeed binds to the promoters and represses the transcription of these meiosis-specific genes. These data suggest that E2F6 serves as a common repressor of meiosis-specific genes. Of interest, E2F6 deficiency alone did not derepress most of these meiosis-specific genes in somatic cells, suggesting that a functional redundancy exists whereby other safeguards are in place to ensure the repression of most meiosis-specific genes in somatic cells.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Sarah M Kehoe.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Terada, Naohiro.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021593:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021593/00001

Material Information

Title: E2F6 is a Common Repressor of Meiosis-Specific Genes
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Kehoe, Sarah M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: ant4, e2f6, meiosis, repression, transcription
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Genes whose protein products function specifically during meiosis are strictly repressed in somatic cells. Their aberrant expression may lead to mitotic catastrophe and predispose cells to oncogenic transformation. Although previous studies have demonstrated various mechanisms delineating repression of individual meiosis-specific genes, the existence and identity of a master regulatory protein which coordinately represses multiple meiosis-specific genes in somatic tissues remains to be elucidated. E2F6, a member of the E2F family of transcription factors, was recently shown to play an essential role in the repression of a few meiosis-specific genes including Stag3 and Smc1?. Here we report that E2F6 is required for the repression of another meiosis-specific gene, Ant4, in somatic cells. This discovery prompted us to investigate whether meiosis-specific genes in general, require E2F6 for their repression. We compiled a list of 24 meiosis-specific genes and observed that 19 of them (79.2%) have the core E2F6-binding element (TCCCGC) within 200bp upstream of their transcription initiation sites. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%) and the mathematical probability of random occurrence (9.3%). Further, using embryonic stem cells, we demonstrate that E2F6 indeed binds to the promoters and represses the transcription of these meiosis-specific genes. These data suggest that E2F6 serves as a common repressor of meiosis-specific genes. Of interest, E2F6 deficiency alone did not derepress most of these meiosis-specific genes in somatic cells, suggesting that a functional redundancy exists whereby other safeguards are in place to ensure the repression of most meiosis-specific genes in somatic cells.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Sarah M Kehoe.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Terada, Naohiro.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021593:00001


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a4f8bb0550c5dfa0f8e7264969a5df0842c06a84







E2F6 IS A COMMON REPRESSOR OF MEIOSIS-SPECIFIC GENES


By

SARAH M. KEHOE















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007





































O 2007 Sarah M. Kehoe



























To my dad, who instilled in me his lifelong passion for learning










ACKNOWLEDGMENTS

I would first like to thank my mentor, Dr. Naohiro Terada, for always being optimistic, no

matter how confusing science may seem at times. His positive attitude, encouragement, and

perseverance will not be forgotten. Dr. Terada' s office door being always open for scientific

discussions and his willingness to meet regularly with his graduate students despite his busy

schedule, exemplifies his dedication. I would also like to thank all of the members of the Terada

lab, both past and present, for their support. In particular, I'd like to thank lab manager, Amy

Meacham, for her frequent assistance, her reliable smile, and her friendship. I would especially

like to thank former graduate student, Amar Singh, for serving as my mentor at the lab bench; he

was a wonderful teacher who answered my many questions and helped me to get my feet off the

g~round.

I am also grateful for my committee members, Dr. Joirg Bungert, Dr. Hideko Kasahara, Dr.

Paul Oh, and Dr. Jim Resnick, for their helpful suggestions, feedback, and expertise. I would

like to thank our collaborator, Dr. Stefan Gaubatz at the University of Wurzburg for providing

invaluable materials and sound advice for my proj ect. Without him, I would not have had the

resources necessary to come to the conclusions that I did.

Further, I want to thank my parents, Robert and Patricia, and sister, Karen, for their

unconditional love and support. Likewise, I'd like to thank my husband, Joe, for always being

there for me at the end of every day. His humor and encouragement have been important to

surviving graduate school. Lastly, I'd like to thank my horse, Sundance, for keeping my life

balanced and my stress levels at bay. Without being able to escape to the barn and ride my horse

on days when the complexity of science seemed too great to ever make headway, I would never

have made it this far.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................. ...............7..___ .....


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 INTRODUCTION ................. ...............12.......... ......


Transcriptional Control of Gene Expression ................. ...............12........... ...
The General Transcription Machinery .............. ...............13....
Gene Regulatory Proteins ................. ...............15........... ....
The E2F Family of Transcription Factors .............. .....................16
The Activating E2Fs............... ............ ........1
Pocket Protein-Dependent Repressive E2Fs .............. ...............20....
Pocket Protein-Independent Repressive E2Fs............... ...............22..
Summation ................. ...............25.................


2 MATERIALS AND METHODS .............. ...............30....


Cell Culture and Creation of Stable Cell Lines .............. ...............30....
Gel Mobility Shift Assay ................. ...............30.......... ....
Chromatin Immunoprecipitation .............. ...............31....
Real-Time Quantitative PCR ............... .........__ ...............32...
Plasmid Construction and Site-Directed Mutagenesis .............. ...............33....
Transient Transfection and Reporter Assays ................. ...............34 ......... ...
Immunoblotting .................. ...............35..
Reverse Transcription-PCR ............ ......__ ...............35....
Com putational Analysis........ ... .. ... .. ................... ...............3
Bisulfite Sequencing and Combined Bisulfite Restriction Analysis (COBRA) .....................37
Immunostaining ............ _...... ._ ...............38....

3 REPRESSION OF ANT4 GENE EXPRESSION BY E2F6 ................. ................. ...._41


Motivation............... ...............4

Background ................. ...............41.................
Re sults ................ ...............46.................
Discussion ................. ...............51.................














4 REPRESSION OF ADDITIONAL MEIOSIS-SPECIFIC GENES BY E2F6.......................74


Motivation............... ...............7

Back ground ................. ...............74.......... ......
Re sults ................ ...............77.................
Discussion ................. ...............79.................


5 THE ROLE OF E2F6 DURING MALE MEIOSIS AND SPERMATOGENESIS ...............91


Motivation............... ...............9

Back ground ................. ...............9.. 1..............
Re sults ................ ...............96.................
Discussion ................. ...............97.................


6 CONCLUSIONS AND SIGNIFICANCE ................. ...............102...............


LIST OF REFERENCES ................. ...............104................


BIOGRAPHICAL SKETCH ................. ...............117......... ......










LIST OF TABLES


Table page

2-1 Primers used for analyzing ChlP experiments with semi-quantitative PCR .....................39

2-2 Primers used for semi-quantitative RT-PCR .............. ...............40....

4-1 Locations of E2F6 TFBS (TCCCGC) within upstream promoter regions of meiosis-
specific genes relative to their transcription initiation sites ................. ............ .........86










LIST OF FIGURES


Figure page

1-1 Five ways in which regulatory proteins can inhibit transcription. .........._.... ........_......27

1-2 The E2F family proteins and their core functional domains............... ...............28

1-3 Changes in the composition and location of E2F complexes as cells enter the cell
cycle. ............. ...............29.....

3-1 Ant4 encodes a novel isoform of adenine nucleotide translocase. ................ ........._......59

3-2 The conservation of Spl1 binding sites in the Ant4 promoter..........._.._.._ ....._.._.. ......60

3-3 The proximal promoter region of the Ant4 gene has a conserved E2F6 binding site........61

3-4 Gel mobility shift assay of E2F6 binding to the Ant4 promoter ................. ..........._...__.62

3-5 The proximal promoter region of Ant4 is bound by endogenous E2f6 in
undifferentiated WT R1 ES cells. ............. ...............63.....

3-6 E2F6-mediated repression of Ant4 requires an intact transcription factor binding site. ...64

3-7 E2F6-mediated repression of Ant4 requires an intact C-terminal and DNA binding
domain............... ...............65.

3-8 Ant4 transcription is repressed by stable E2F6 overexpression in ES cells. ................... ...66

3-9 Ant4 transcription is derepressed in E2f6- MEFs. Semi-quantitative RT-PCR
analysis is shown for E2f6, Ant4, and B-actin expression in WT MEFs and E2f6-/
M EFs. ........... ..... ._ ...............67...

3-10 The polycomb proteins Eed and Ezh2 bind to the Ant4 promoter in D6 R1 EBs.
Chromatin immunoprecipitation (ChIP) was performed using anti- E2f6, Eed, Ezh2,
Dnmt3, Suzl2, and acetylated histone H3 (AH3) antibodies. ............. .....................6

3-11 Ant4 is derepressed in NIH-3T3s after treatment with 5-AZA-DC but not TSA. 5CLM
or 10 CLM of AZA-DC were added either alone or in combination with 200nm TSA
for 65 hours in NIH-3T3s. ............. ...............69.....

3-12 CpG methylation at the Ant4 promoter is partially reduced in E2f6- MEFs. A)
COBRA analysis of the methylation status of Ant4 in various tissue types. .............._.......70

3-13 Tuba3 methylation decreases in E2f6- mouse tail DNA. ............. .....................7

3-14 E2F6 overexpression does not increase CpG methylation at the Ant4 promoter. .............72

3-15 The "All" OR "None" model of E2F4 compensation. ......_................. ................73










4-1 Frequency of appearance of E2F6 TFB S within upstream regions of genes relative to
their transcription initiation sites. ............. ...............87.....

4-2 E2f6 binds to the promoters of meiosis-specific genes. .......................... ...............88

4-3 Many meiosis-specific genes are repressed by E2F6. Semi-quantitative RT-PCR
analysis of meiosis-specific gene expression in R1, HA-E2F6, and HA-AC-E2F6 ES
cell s. ............. ...............89.....

4-4 Limited meiosis-specific genes are derepressed in E2f6- MEFs. Semi-quantitative
RT-PCR analysis of meiosis-specific gene expression in WT testis from 6-week old
mice, R1 ES cells, WT MEFs, and E2f6- MEFs. ............. ...............90.....

5-1 E2fl and E2f6 mRNA transcript l evels in the te sti s .......... ................ ................10

5-2 Immunohistochemical analysis of E2f6 expression in mouse testis. ............... ... ............101









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

E2F6 IS A COMMON REPRESSOR OF MEIOSIS-SPECIFIC GENES

By

Sarah M. Kehoe

December 2007

Chair: Naohiro Terada
Major: Medical Sciences--Molecular Cell Biology

Genes whose protein products function specifically during meiosis are strictly repressed

in somatic cells. Their aberrant expression may lead to mitotic catastrophe and predispose cells

to oncogenic transformation. Although previous studies have demonstrated various mechanisms

delineating repression of individual meiosis-specific genes, the existence and identity of a master

regulatory protein which coordinately represses multiple meiosis-specific genes in somatic

tissues remains to be elucidated. E2F6, a member of the E2F family of transcription factors, was

recently shown to play an essential role in the repression of a few meiosis-specific genes

including Stag3 and Smclf. Here we report that E2F6 is required for the repression of another

meiosis-specific gene, Ant4, in somatic cells.

This discovery prompted us to investigate whether meiosis-specific genes in general,

require E2F6 for their repression. We compiled a list of 24 meiosis-specific genes and observed

that 19 of them (79.2%) have the core E2F6-binding element (TCCCGC) within 200bp upstream

of their transcription initiation sites. This was significantly higher than the frequency found in

the promoters of all mouse genes (15.4%) and the mathematical probability of random

occurrence (9.3%). Further, using embryonic stem cells, we demonstrate that E2F6 indeed binds

to the promoters and represses the transcription of these meiosis-specific genes. These data










suggest that E2F6 serves as a common repressor of meiosis-specific genes. Of interest, E2F6

deficiency alone did not derepress most of these meiosis-specific genes in somatic cells,

suggesting that a functional redundancy exists whereby other safeguards are in place to ensure

the repression of most meiosis-specific genes in somatic cells.









CHAPTER 1
INTTRODUCTION

Transcriptional Control of Gene Expression

With the discovery of the structure of DNA by Watson and Crick in the early 1950s

(Watson & Crick 1953) came the understanding that every cell in the body, although diverse,

contains the same genetic information as all other cells. Prior to this breakthrough, it was

difficult to fathom that two cell types with extreme differences in shape and function, such as

cardiomyocytes and neurons, could have been coded from the same genome. In fact, it was

speculated that different cell types actually contained different sets of genes due to a selective

loss of unnecessary genes as the cells differentiated (Alberts et al. 2002). Today, we know that

the vast diversity of cell types found in the body is a result of differences in gene expression

rather than a consequence of changing the presence of nucleotide sequences in a cell's genome.

Experimental evidence leading to this conclusion involved taking the nucleus from a fully

differentiated frog cell and inj ecting it into a frog egg whose nucleus had been removed (Gurdon

1962). The donor nucleus was able to direct the recipient egg to produce a normal tadpole that

contained a full range of differentiated cell types, thereby proving that the genome must be

preserved in the differentiated donor cell. Similar experiments done in various species of

mammals including: sheep (Campbell et al. 1996, Wilmut et al. 1997), cattle (Cibelli et al. 1998,

Kato et al. 1998), mice (Wakayama et al. 1998), goat (Baguisi et al. 1999), pig (Polejaeva et al.

2000), cat (Shin et al. 2002), rabbit (Chesne et al 2002), horse (Galli et al 2003), and

endangered wolves (Kim et al. 2007), have further validated that the genome is upheld in every

somatic cell.

Given that the human genome encodes approximately 35,000 genes (Pennisi 2001),

specialized cell types have the difficult j ob of selectively expressing the genes that they need to










support the unique properties of their cell type. If this were not difficult enough, the ability to

express the appropriate genes is further complicated by the fact that the eukaryotic genome is

packaged into nucleosomes and higher order forms of chromatin which can restrict a gene's

accessibility. Fortunately, cells are equipped with the molecular machinery needed to overcome

such obstacles. This machinery controls gene expression by operating at various levels using

different mechanisms: 1) on a transcriptional level by controlling how often a gene is transcribed

to RNA, 2) on an RNA processing level by regulating RNA transcript splicing, 3) on a location

basis by selecting which messenger RNA (mRNA) in the nucleus gets transported to the

cytoplasm for protein synthesis, 4) on a degradation basis by selectively destabilizing certain

mRNA molecules in the cytoplasm, 5) on a translational level by selecting which mRNAs are

translated into proteins by ribosomes, and 6) on a post-translational level by affecting protein

activity via activation, inactivation, degradation, or compartmentalization (Alberts et al. 2002).

For the remainder of this dissertation, I will focus on the transcriptional regulation of gene

expression.

The General Transcription Machinery

At the heart of transcription is the enzyme, RNA polymerase. RNA polymerase moves

along the DNA, unwinding the DNA helix ahead of it to expose a template strand of DNA so

that incoming ribonucleotides can complementary base pair and form a growing RNA chain.

This RNA chain is then released and depending on the type of RNA, goes on to be processed

accordingly. In this study, we focus on mRNA which is transcribed by RNA polymerase II, and

after splicing goes on to be translated into protein. In order for the RNA polymerase to

transcribe a gene accurately, it must be able to recognize where in the genome to start and end

transcription. The starting point of transcription is indicated by a special sequence of nucleotides

known as the promoter and when the RNA polymerase comes across this sequence, it binds










tightly to it. The polymerase then begins transcription and continues to produce an RNA

transcript until it encounters a termination signal. This end signal then enables the release of the

newly made RNA chain and the polymerase from the DNA.

The observation that unlike bacterial RNA polymerase, purified eukaryotic RNA

polymerase II could not initiate transcription in vitro led to the discovery and purification of

additional required factors (Weil et al. 1979). These factors are referred to as general

transcription factors because they assemble on all promoters used by RNA Polymerase II. Their

job is to help the RNA polymerase position itself correctly at the promoter, to assist in pulling

apart the DNA strands, and to release the RNA Polymerase from the promoter into elongation

mode once transcription has begun. These general transcription factors assemble at the promoter

region in a specific order. First, the general transcription factor, TFIID, binds to the TATA box,

a short DNA sequence located 25 nucleotides upstream from the transcription start site that is

composed primarily of T and A nucleotides. The subunit of TFIID that recognizes the TATA

box is called TBP (for TATA-binding protein). TFIID binding to the TATA box causes a

distortion in the DNA which brings DNA sequences on both sides of the distortion together to

allow for subsequent protein assembly. However, many promoters do not contain TATA boxes.

In some large groups of genes, such as housekeeping genes, the TATA box is often absent and

the corresponding promoters are referred to as TATA-less promoters. In these promoters, the

exact position of the transcription start site and the binding of the general transcription factors is

often controlled by an initiator (Inr) nucleotide sequence in the transcription initiation region, or

by a downstream promoter element (DPE) which is typically observed 30bp downstream of the

transcription start site (Smale 1997). Sequences which have the capacity to function as Inrs or

DPEs are less characterized than the TATA-box sequence.









After TFIID binding, other general transcription factors including, TFIIA, TFIIB, TFIIE,

TFIIF, and TFIIH along with the RNA polymerase then assemble at the promoter. This cohort of

factors at the promoter is termed the transcription initiation complex. After the RNA polymerase

is correctly positioned at the promoter, it then gains access to the transcription initiation site with

the help of TFIIH' s DNA helicase activity. TFIIH then phosphorylates the tail of the RNA

polymerase causing a conformational change which releases RNA polymerase from the

promoter. RNA polymerase then transcribes along the template DNA strand causing the

formation of an elongating RNA transcript (Alberts et al. 2002).

Gene Regulatory Proteins

Although the general transcriptional machinery for protein-encoded genes is the same for

most eukaryotic promoters, gene regulatory proteins on the other hand are diverse. These are the

proteins which regulate gene expression by binding to short stretches of DNA of a defined

sequence and in turn, control the accessibility of the general transcriptional machinery to specific

promoters. It is estimated that 5-10% of the coding capacity of a mammalian genome is devoted

to the synthesis of these regulatory proteins (Alberts et al. 2002). This high percentage is not

surprising given the vast diversity of cell types found within the body, the constantly changing

environmental cues imparted on a cell, and the fact that each gene is regulated by a set of

regulatory proteins which are themselves products of genes that are regulated by a whole other

set of proteins. Further complexity results from the fact that these regulatory proteins have been

shown to regulate transcription at distant sites, often thousands of base pairs away from the

promoter. The explanation for this is that DNA can form loops to allow the distantly bound

regulatory proteins to come into contact with proteins bound at the promoter. An additional level

of complexity comes from the combinatorial nature of transcriptional regulation, where a









regulatory protein's function as either an activator or a repressor is often determined not by the

protein itself but by the proteins with which it interacts (Alberts et at. 2002).

Although the diversity and complexity of transcriptional regulatory protein networks

appears overwhelming, progress has been made to elucidate some of the mechanisms by which

regulatory proteins activate and repress transcription. As illustrated in Figure 1-1, regulatory

proteins can inhibit transcription via several mechanisms. These include: 1) competing with

activator proteins for binding to the same regulatory DNA sequence, 2) binding to the activation

domain of an activator protein thereby preventing it from carrying out its activator functions

even though it is still capable of binding to DNA, 3) blocking assembly of the transcription

initiation complex, 4) recruiting chromatin remodeling complexes or histone modifying enzymes

which form more compacted chromatin, and 5) recruiting deacetylases and other histone tail

modifying enzymes to add repressive chromatin marks to histones as well as recruiting DNA

methyltransferases to methylate DNA and lock a promoter in an inactive state (Alberts et at.

2002). Similar mechanisms are used by activators to produce the opposite effect of

transcriptional activation. The remainder of this chapter will focus on a specific family of

regulatory proteins known as E2Fs and will discuss their involvement in regulating transcription.

The E2F Family of Transcription Factors

E2F transcription factors regulate the expression of genes whose protein products are

essential for cell cycle progression, cellular proliferation, DNA repair, and differentiation

(Dimova et at. 2005, Trimarchi & Lees 2002). E2F was originally identified as a cellular DNA-

binding protein that could bind to the sequence, TTTCGCGC, within the adenovirus E2

promoter (Yee et at. 1987). Concurrently, La Thangue and coworkers identified DRTF l, a

transcription factor which had the same consensus DNA-binding site as E2F during studies of

embryonic stem cell differentiation (La Thangue & Rigby 1987). It is now clear that E2F and









DRTF 1 are the same factor. In order to bind to DNA, E2F requires a binding partner known as

dimerization partner (DP). The E2F and DP proteins heterodimerize at which point E2F

becomes functional. Additionally, E2F associates with and is regulated by the retinoblastoma

(RB) protein. RB was the first tumor suppressor protein ever identified and it is absent or

mutated in at least one-third of all human tumors (Weinberg 1992). In particular, it is known for

being mutated in hereditary and sporadic retinoblastoma. The importance of the RB-E2F

interaction is demonstrated by the finding that all naturally occurring RB mutants isolated from

human tumors lack the ability to bind and negatively regulate E2F.

Currently, there are eight known members of the E2F family (DeGregori & Johnson

2006). E2F 1 was the first member to be cloned. It was isolated by three independent groups

through E2Fli's ability to bind to RB. Subsequently, seven additional members of the E2F family

were discovered as a result of their homology to E2F 1 or because of their ability to bind to RB.

However, some of these E2Fs are unable to bind to RB. Figure 1-2 illustrates the protein binding

domains of the E2F family members and shows that only E2Fs 1-5 possess an RB binding

domain. This domain allows for E2Fs 1-5 to interact with RB and/or other related pocket

proteins. E2Fs 1-5 are also the only E2Fs that have a transcriptional activation domain.

However, only E2Fs 1-3a serve as transcriptional activators whereas E2Fs 3b-5 are

transcriptional repressors despite the presence of an activation domain. E2Fs 6-8 are also

repressors but they are considered to be pocket protein-independent transcriptional repressors.

E2Fs 6-8 lack both a transactivation domain and a RB binding domain, but they do contain a

highly conserved DNA binding domain. E2Fs 1-6 also contain a dimerization domain for their

interaction with the DP proteins, but this domain is absent in E2F7 and E2F8. Generally, the

various types of protein domains possessed by individual E2F family members dictate their









specific functions. Below we will describe the attributes of the three main classes of E2Fs: 1)

activating E2Fs, 2) pocket protein-dependent repressive E2Fs, and 3) pocket protein-independent

repressive E2Fs.

The Activating E2Fs

The activating E2Fs, E2Fl1-3 a, are potent transcriptional activators of E2F-responsive

genes. Their key purpose is to activate genes which are essential for cellular proliferation.

Briefly, cellular proliferation is regulated by the cell cycle which consists of four phases: G1 (the

transition phase to prepare for DNA replication), S (the replication phase), G2 (the transition

phase to prepare for cell division), and M (the division phase). Cells which are not cycling are

considered to be in a quiescent GO phase. E2Fs 1-3a activate target genes at the Gl/S boundary

of the cell cycle, thereby promoting S-phase entry (Schwartz et al. 1993). Interestingly,

overexpression of any one of these E2Fs is sufficient to induce quiescent cells to re-enter the cell

cycle (Johnson et al. 1993, Qin et al. 1994, Lukas et al. 1996). Conversely, reducing the levels

of individual activating E2Fs can result in cell-cycle arrest (DeGregori et al. 2006). When all

three activating E2Fs are mutated, cellular proliferation is completely blocked (Wu et al. 2001).

Given their strong ability to alter cellular proliferation, it is not surprising that these E2Fs

are tightly regulated and that their expression and activity is restricted to specific windows

during the cell cycle. The activating E2Fs exhibit maximum expression levels during late G1

and early S phase (DeGregori et al. 2006). During this time, the activity of the E2F protein is

also raised and it coincides with a wave of transcription of E2F-responsive genes. The amount

of E2F protein activity is largely determined by the interactions between the E2Fs and RB. RB

binding to E2Fs 1-3a can inhibit the activation of E2F-responsive genes in one of two ways.

First, due to the fact that the RB binding domain is located in the transactivation domain of E2F










proteins (see Figure 1-2); RB binding can physically mask the activation domain. This

association prevents the E2F-DP heterodimer from recruiting the general transcription factor,

TFIID, thereby inhibiting E2Fs' ability to activate. The other way by which RB blocks E2F

activity is through active repression as a consequence of recruiting HDAC, SWI/SNF,

SUV39H1, and other chromatin modifying enzymes to the promoters of E2F-responsive genes.

RB-mediated repression is relinquished as quiescent cells are stimulated to enter the cell cycle

due to phosphorylation of RB by cell cycle-dependent kinases. Upon RB phosphorylation, E2F

is released. This sudden abundance of "free E2Fs" results in the activation of E2F-responsive

genes during late G1 of the cell cycle (Trimarchi et al. 2001). During this time, E2F-responsive

promoters are bound by activating E2Fs and this binding is often associated with

transcriptionally permissive modifications in local chromatin.

In addition to their roles in promoting cell cycle progression, activating E2Fs are also

important during development. This is evidenced by the various E2F mutant mouse models. For

instance, E2fl-/- mice are viable and fertile but have tissue-specific abnormalities which include:

an excess of T cells, exocrine gland dysplasia, and the development of testicular atrophy (Field et

al. 1996, Yamasaki et al. 1996). Also, E2fl-/- mice develop various tumors between 8 and 18

months of age. Although E2F 1 overexpression in tissue culture cells stimulates cell proliferation

and can be oncogenic (Singh et al. 1994), the E2fl-/- mouse clearly demonstrates that E2F 1 can

also function as a tumor suppressor. E2f2 mutant mice display enhanced T lymphocyte

proliferation which leads to the development of autoimmunity (Murga et al. 2001). E2fl/E2f2

double-knockout mice exhibit severely impaired hematopoiesis due to defective S phase

progression in hematopoietic progenitor populations (Li et al. 2003). Combined E2fl and E2f2

loss also results in polyploidy in the liver, salivary gland, and exocrine pancreas. E2f3-/- mice









are shown to die in utero and the few that survive to adulthood die prematurely due to congestive

heart failure (Cloud et al. 2002). It should be noted here that E2f3- mice are deficient in both

E2f3a and E2f3b isoforms. From the phenotype of E2fl E2f3 double-mutant mice (Cloud et al

2002), it is clear that almost all of the developmental and age-related defects arising in the

individual E2fl or E2f3 mutant mice are exacerbated by the combined mutation. Interestingly

however, these mice did not have an increased incidence of tumor formation, revealing that

tumor suppression is a specific property of E2F 1 and not E2F3. Overall, in addition to their roles

in the induction of proliferation, the activating E2Fs have crucial functions during development.

Pocket Protein-Dependent Repressive E2Fs

Repressing E2Fs that possess an RB binding domain are termed pocket protein-

dependent repressive E2Fs. This class of E2Fs consists of E2Fs 3b-5. It has been suggested that

their primary roles are to repress growth promoting E2F-responsive genes during GO/early G1

and to induce cell cycle exit and terminal differentiation (DeGregori et al. 2006). They diverge

considerably in sequence from the activating E2Fs in that they lack most of the sequence that is

amino-terminal to the DNA binding domain. Further, they use their RB binding domain to

interact with additional RB pocket protein family members. E2F4 interacts with pl07, pl30, and

RB whereas E2F5 interacts mostly with pl30. These repressive E2Fs are further distinguished

from the activating E2Fs by their expression patterns. Unlike the activating E2Fs, repressive

E2Fs are constitutively expressed and can be found in nearly equivalent levels in both quiescent

and proliferating cells. In fact, E2F4 is expressed at higher levels than any other E2F and it

accounts for at least half of the E2F-pocket protein activity observed in vivo (Trimarchi et al.

2002). Interestingly, while the protein levels of these E2Fs do not change rapidly, substantial

changes in their subcellular location have been observed during the cell cycle.









Unlike the activating E2Fs which are constitutively nuclear, E2Fs 4-5 are predominately

cytoplasmic. This difference in location is attributed to the fact that activating E2F proteins have

a nuclear localization signal whereas repressive E2Fs have a nuclear export signal. However,

when the repressive E2Fs associate with pocket proteins, it is sufficient to induce their nuclear

localization. Once in the nucleus, repressive E2F-pocket protein complexes can be found

abundantly and this occurs during GO/G1 phases of the cell cycle (Figure 1-3). During this time,

these repressive E2F complexes are bound to the promoters of many E2F-responsive genes and

their binding is often correlated with repressive chromatin modifications. Then around mid to

late G1 in the cell cycle, the repressive E2F complexes are released from the promoters. This

release is due to the separation of the repressive E2F from its pocket protein. The timing of the

release is often correlated with the upregulation of activating E2Fs which may subsequently

replace repressing E2F protein occupancy at E2F-target promoters during S phase. At this point,

the repressive E2Fs have been exported back out to the cytoplasm, rendering them inactive

(Muller et al. 1997).

The role of E2Fs 3b-5 in the repression of E2F-responsive genes was elucidated when

these proteins were ablated from cell lines. Mouse embryonic fibroblasts (MEFs) deficient in

E2f4 and/or E2f5 have defects in their ability to exit the cell cycle in response to various growth-

arrest signals including pl6 overexpression and contact inhibition (DeGregori et al. 2006).

However, these mutant cells are able to respond to growth stimulatory signals and there is no

change in their proliferative capacity. Thus, the loss of these repressive E2Fs impairs the

repression of E2F-responsive genes and therefore the ability to exit the cell cycle.

In agreement with the phenotype of E2f4- MEFs, cellular proliferation in E2f4- mice is

also normal. However, E2f4- mice display defects in terminal differentiation. This is









evidenced by several phenotypic abnormalities in E2f4-/- mice: 1) fetal anemia and erythrocyte

maturation defects, 2) a lack of mature hematopoietic cell types but an abundance of immature

cells, and 3) a thinning of gut epithelium due to a reduction in the density of villi (Rempel et al.

2000, Humbert et al. 2000). These observations suggest a critical role for E2F4 in controlling

the maturation of cells in a number of different tissues. A knockout of the other pocket protein-

dependent repressive E2F, E2f5, results in mice which are viable but develop hydrocephalus due

to an overproduction of cerebral spinal fluid resulting from a choroid plexus defect (Lindeman et

al. 1998). Thus, E2F4 and E2F5 clearly have individual roles during development. However,

the neonatal lethal phenotype of E2f4/E2f5 double-knockout mice (Gaubatz et al. 2000)

demonstrates that E2F4 and E2F5 must also have some overlapping functions in biological

processes that are essential to survival. Moreover, the individual and combined knockout

phenotypes of both mice and cell lines show that these pocket protein-dependent repressive E2Fs

are dispensable for cellular proliferation but are required for cell cycle exit and terminal

differentiation.

Pocket Protein-Independent Repressive E2Fs

The previously described E2F proteins' roles as transcriptional activators or repressors

depended upon whether or not they were bound to a pocket protein. Conversely, pocket protein-

independent repressive E2Fs, E2Fs 6-8, lack an RB binding domain (Figure 1-2) and therefore

repress E2F-responsive genes through mechanisms which do not require pocket proteins. These

E2Fs are localized to the nucleus and their expression fluctuates during the cell cycle. In

contrast to pocket protein-dependent repressive E2Fs, E2F6 expression is low in serum-starved,

quiescent cells. Instead, E2F6 expression is increased during re-entry into the cell cycle with

peak levels in late Gl/early S phase. From that point, E2F6 remains expressed throughout the

cell cycle. Similarly, E2F7 and E2F8 are also expressed in a cell growth-dependent manner,









accumulating as cells enter the cell cycle and with peak levels found during S phase. This

expression pattern suggests that these E2Fs may serve as components of a negative feedback

system to counterbalance activating E2Fs during the cell cycle. The finding that E2Fs 6-8 bind

to E2F-responsive promoters during S phase agrees with this idea. Additionally, these E2Fs may

be involved in defining the distinction between Gl/S- and G2/M- regulated transcription. This

role was proposed after the observation that E2F6 selectively binds and represses only those

E2F-responsive genes that were activated at the Gl/S boundary and not the G2/M boundary of

the cell cycle (Giagrande et al. 2004).

Overexpression studies have further elucidated the roles of pocket-protein independent

E2Fs. Ectopic expression of either E2F6 or E2F7 is able to block endogenous E2F-responsive

gene transcription as well as block the transactivating activity of ectopic E2F 1 (Cartwright et al.

1998, Gaubatz et al. 1998, Bruin et al 2003). Further, E2F6 overexpression leads to the

accumulation of cells in S-phase (Cartwright et al. 1998). In agreement with this observation is

the prediction that following cellular damage, expression of E2F6 may be upregulated to inhibit

E2F-dependent transcription in S-phase and therefore, delay the progression of the cells through

S phase until either the damage has been repaired or the cell has been targeted for apoptosis.

Additionally, E2F6 overexpression in quiescent cells delays re-entry into the cell cycle (Gaubatz

et al. 1998). This is consistent with the detection of an E2F6 chromatin-modifying complex at

cell cycle-regulated promoters during GO. However, E2f6-/- MEFs display no defects in cellular

proliferation (Storre et al 2002). It is speculated that proliferation remains normal because many

of these cell cycle promoters are also occupied by repressive E2F4 complexes, suggesting that

E2F6 is redundant. E2F7 or E2F8 overexpression significantly slows down cellular proliferation

in MEFs (Bruin et al. 2003, Maiti et al. 2005). Ectopic expression of E2F8 has also been shown









to ablate DNA replication of cells in S phase which may reflect the downregulation of E2F-target

genes required for DNA synthesis. Taken together, these observations propose that pocket

protein-independent repressive E2Fs have overlapping and perhaps synergistic roles during the

cell cycle. However, the mechanism by which these pocket protein-independent E2Fs mediate

transcriptional repression of E2F-target genes is not known for E2Fs 7-8. Further insights will

likely be established with the creation ofE2f7 and E2/8 knockout mice. Therefore, the

remainder of this discussion will focus on the mechanisms of E2F6-mediated repression as there

is a substantial amount of experimental evidence on this topic.

One mechanism by which E2F6 mediates transcriptional repression is through its

association with polycomb repressive complexes. This association is suggested in vivo by the

phenotype of E2f6- mice; posterior homeotic transformations of the axial skeleton which are

strikingly similar to those observed in mice deficient in a variety of polycomb proteins (Storre et

al. 2002). There are two main types of polycomb repressive complexes: 1) PRC2, which is

thought to be required at the initiating stage of silencing, and 2) PRC1i, which is continuously

required for the stable maintenance of repression once the correct gene expression patterns have

been established. E2F6 has been shown to associate with members of the PRC1 complex

(Trimarchi et al. 2001). This E2F6-PRC1 complex includes the PRC1 complex proteins,

RING1, mel-18, mphl, Bmil, and YY1 binding protein (RYBP), and it is presumed that these

polycomb proteins associate with their target promoters through binding to E2F6. Recently,

another E2F6-polycomb complex (E2F6-GO) was described (Ogawa et al. 2002). This complex,

containing several novel polycomb proteins including Max and HPly, was found to associate

with E2F target promoters in GO but not following re-entry into the cell cycle. This implies a

role for E2F6 in the repression of E2F target genes during GO. However, given that E2F6 is










expressed throughout the cell cycle and remains localized to the nucleus, E2F6 most likely forms

alternate complexes in proliferating cells. Additional studies have revealed that E2F6 is

associated with EPC 1 and EZH2, a component of the PRC2 complex, only in proliferating cells

(Attwooll et al. 2005). Upon overexpression of EPC1, EZH2, and EED, another component of

the PRC2 complex, these three proteins will co-immunoprecipitate. Further, this E2F6

proliferation-specific complex was shown to repress reporter activity but a mutation in E2F6

which renders it unable to bind to EPC1 causes a loss of this repression. Interestingly, the PRC2

component EZH2, which is known for its H3K27 methyltransferase activity, has recently been

shown to recruit DNA methyltransferases to the promoters of target genes (Vire et al. 2006).

This recruitment could serve to 'lock in' the silent state initiated by the polycombs through DNA

methylation. Overall, E2F6 has been shown to associate with components of both the PRC1 and

PRC2 complexes suggesting a role for E2F6 in the establishment and the maintenance of E2F-

target gene repression in both quiescent and proliferating cells.

Other mechanisms for E2F6-mediated repression have been studied but not as thoroughly

as the E2F6-polycomb interactions. These include: 1) E2F6 blocking activating E2Fs from

binding to the promoters of E2F-responsive genes (Oberley et al. 2003), 2) E2F6 recruiting DNA

methyltransferases to induce CpG hypermethylation and lock target promoters in an inactive

state (Pohlers et al. 2005), and 3) E2F6 recruiting histone deacetylases and histone

methyltransferases which infer repressive chromatin marks on histone tails (Storre et al. 2005,

Caretti et al. 2003, Ogawa et al. 2002). For the remainder of this study, we will focus on the

E2F family member, E2F6, and E2F6-mediated repression of a select set of target genes.

Summation

The way by which the human body creates so many different cell types from the same

genomic sequences is inconceivable. However, we now know that such cellular diversity is at









least made possible through the finite control of gene expression. More specifically, the

transcriptional regulation of genes through the binding of transcriptional activators and

repressors has proven to be especially important. Therefore, it was the goal of this work to study

the transcriptional regulation of gene expression. Considering the complexity of such a task, it is

best to simplify the problem into a more manageable solution. Therefore, for the first part of this

study, we chose to focus on the transcriptional regulation of an individual gene and a particular

transcription factor. The gene we selected to analyze was adenine nucleotide translocase-4

(Ant4) because it was recently discovered by our lab (Rodic et al. 2005), and prior to this study

nothing was known about its transcriptional regulation. We chose to investigate the E2F family

of transcription factors, in particular E2F6, for reasons that will become apparent. The biological

significance ofAnt4, along with our findings regarding its transcriptional regulation, is discussed

in Chapter 3. For the second part of this study, we examined the transcriptional regulation of 24

additional genes which were similar in expression to Ant4 because we had reason to believe that

they too were regulated by E2F6. More information regarding the transcriptional regulation of

these 24 genes is given in Chapter 4. Lastly, in Chapter 5, we present data on the expression of

E2F1 and E2F6 in the testis. In summation, the overall goal of this research was to analyze the

transcriptional regulation of gene expression in the context of a specific gene/set of genes.











aciao ..activation surface




L~ TATA
binding site
birdndu Elte for repre~ssor
for act .*ator




I I TATA


A

com8petitive
DNA
binding



B


acTivation
surface


*.~I .. .!. Dtrid~ing site
-- -1 .I I for repressor
Iginlingl Site
banding site for acqivator
for repre~ssr












remdeld nclasoernes


TATAle



....m,,hisori decetlan


C

direct interactiona
with the genera I
transcription fac~to~rs








rersickomplexe










E


recrulitrnant of
Isistene
deacetyleses


Figure 1-1. Five ways in which regulatory proteins can inhibit transcription. The repressing
regulatory proteins can inhibit transcription by: A) competing with activating
regulatory proteins for binding to the same DNA sequence, B) blocking the activity of
the activating regulatory protein even though it may still bind, C) interacting with the
general transcription machinery and blocking it from assembling at the promoter, D)
recruiting repressive chromatin remodeling complexes, and E) recruiting histone
deacetylases and other histone tail and DNA modifying enzymes (from Alberts et al.
2002, Figure 7-49 on page 406).










D~imerization

DNA binding Transactivation
1 Binding to
v" bFaitly~r~l proteinS
I~i _II I MI
I I 11 | MI
| 11 I MI


~


, ,


E2F1
E2F2
E2F~a
2F3b,


|


E2F4
E2ZF5
E2FS
E2F7 ~
E2FS C


| DBD1 | [DBD2
ID~BD1 I: IDRD2 I


Figure 1-2. The E2F family proteins and their core functional domains. E2Fs 1-6 share domains
which mediate DNA binding (light blue) and DP dimerization (pink). E2Fs 7-8 have
two of these DNA binding domains (DBD 1 and DBD2), but lack a DP dimerization
domain. E2Fs 1-5 also have an Rb binding domain (green) within a transactivation
domain that only has transactivating activity in E2Fs 1-5 (modified from DeGregori
and Johnson 2006, Figure 1 on page 740).










GO Quiescence


B Late G1IEarly 8 phase












cycle. A) Dring GO, coplee otiigEF n ocket proteinsbnad




repress 2F targt genes The fuc~tion of activatin E2sisdsbldbyascito






with RB. B) As cells enter the cell cycle, the repressive E2F dissociates from its
pocket protein causing release from target promoters and export to the cytoplasm.
Also, E2F 1 is upregulated and released from RB. It can then replace the repressive
E2F complexes by binding to and activating E2F target genes.









CHAPTER 2
MATERIALS AND METHODS

Cell Culture and Creation of Stable Cell Lines

The cell lines used in this study were murine WT R1 ES cells (a gift from Dr. A Nagy,

Toronto, Canada), HA-E2F6 ES cells, HA-AC-E2F6 ES cells, and NIH3T3 cells (American

Type Culture Collection, Manassas, VA). Both HA-E2F6 and HA-AC-E2F6 ES cell lines were

established by stable transfection of linearized HA-E2F6 and HA-AC-E2F6 expression vectors

(Gaubatz et al. 1998) into parental R1 ES cells. Stable clones were subsequently confirmed to

express E2F6 by western blotting with anti-HA antibody (Cell Signaling Technology, Danvers,

MA), and those with the highest expression were selected and expanded in the presence of

Neomycin (G418) (Calbiochem-EMD Biosciences, San Diego, CA). All ES cells were

maintained in an undifferentiated state on gelatin-coated dishes in Knock-outTM Dulbecco's

Modified Eagle Medium, low glucose (Invitrogen, Carlsbad, CA) containing 10% KnockoutTM

Serum Replacement (Invitrogen), 1% Fetal Bovine Serum (Atlanta Biologicals, Lawrenceville,

GA), 1% Penicillin- Streptomycin-Glutamine (100x) liquid (2mM L-glutamine, 100 U/ml

penicillin, 100 Cpg/ml streptomycin, Invitrogen), 25mM HEPES (Cellgro, Herndon, VA), 300CLM

monothioglycerol (Sigma-Aldrich, St. Louis, MO), and 1,000 U/ml recombinant mouse LIF

(ESGRO) (Millipore, Temecula, CA). NIH3T3 cells were maintained in Dulbecco's Modified

Eagle Medium (Invitrogen) containing 10% Fetal Bovine Serum, 1% Penicillin- Streptomycin-

Glutamine (100x) liquid, and 25mM HEPES.

Gel Mobility Shift Assay

Gel mobility shift assay was performed as described previously (Gaubatz et al. 1998).

Briefly, 5Cl1 of reticulocyte lysates with or without in vitro-translated proteins (see below) were

incubated with 0. 1 picomoles of PAGE purified, y32P-end-labeled, annealed oligonucleotides (5'-









TCAGCGCCCGCTTTCCCGCCAGGGTAAAGCT-3 ') corresponding to the WT E2F6-binding

element (underlined) in the murine Ant4 promoter. Competition experiments were performed

with wild-type and mutant (5' -TCAGCGCCCGCTTTCTTAACAGGGTAAAGCT-3 ')

unlabeled, double-stranded oligonucleotides corresponding to the E2F6 site derived from the

Ant4 promoter, whereby the amount of unlabeled probe relative to the amount of labeled probe

was either 20, 50, or 100 fold in excess. For preparation of in vitro-translated proteins, one

microgram of HA-E2F6 and Myc-DP2 expression vectors (constructed as described previously),

were co-translated in vitro using a coupled transcription/translation reticulocyte lysate system

(Promega, Madison, WI) according to manufacturer' s instructions (Gaubatz et al. 1998).

Chromatin Immunoprecipitation

R1 ES cells were plated on 100-mm plates at a density of 1 x 106 CellS per plate and then

fixed three days later in 1% formaldehyde for 10 minutes at room temperature. Cross-linking

was attenuated by the addition of 125mM glycine. Cells were then collected by scraping with a

cell scraper in cold PBS with the following protease inhibitors (PIs): leupeptin, aprotinin, and

phenylmethylsulfonyl fluoride. Cells were centrifuged at 1200rpm for 5 minutes at 40C and then

resuspended in cell lysis buffer (5mM PIPES pH 8.0, 85mM KCL, and 0.5% NP-40) plus PIs

and incubated for 10 minutes on ice. Nuclei were pelleted at 5000 rpm for 5 minutes at 40C and

then lysed on ice for 10 minutes in nuclei lysis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, and

1% SDS) plus PIs. Next, chromatin was sheared by sonication for 10 repititions of 10 second

bursts with 1 minute rests on ice using a power setting of "4" on a Fisher Scientific Sonicator

Dismembrator Model 100. Lysates were centrifuged for 10 minutes and supernatants were

collected, diluted, and a sample was kept as Input DNA. The supernatant was then pre-cleared

with Protein G agarose (Invitrogen) for twenty minutes and collected again following

centrifugation. Approximately 2Cpg of each antibody; control IgG, (Sigma-Aldrich) and mouse









monoclonal E2f6 (Storre et al. 2002), were added to the supernatant and incubated overnight at

40C. The following day, 60Cl1 of Protein G Agarose-50%/ slurry was added to the reaction and

incubated for 1 hour rocking at 40C. The agarose complex was then collected, washed seven

times with LiCl wash buffer (0.25M LiC1, 0.5% NP-40, 0.5% DOC, ImM EDTA, and 10mM

Tris pH 8.0), and DNA was eluted off with elution buffer (50mM Tris pH 8.0, 1% SDS, and

10mM EDTA). Crosslinking was reversed by incubating the eluted chromatin in 175mM NaCl at

650C overnight followed by 2 hours of Proteinase K treatment at 550C to degrade the proteins.

DNA was recovered using phenol/chloroform extraction followed by PCR Purification (Qiagen,

Valencia, CA). PCR was performed with either semi-quantitative PCR using the Taq DNA

polymerase kit (Eppendorf, Westbury, NY) or with real-time quantitative PCR (as described

below). The primer sequences used for semi-quantitative PCR analysis of Chromatin

Immunoprecipitation (ChIP) are described in Table 2-1. Data from at least three independent

experiments were analyzed by semi-quantitative PCR and expressed as means + standard

deviations (SD).

Real-Time Quantitative PCR

The primers and probes used for real-time PCR were designed with Primer Express

software and synthesized at Applied Biosystems. Each real-time PCR reaction was performed in

a 25C1l reaction mixture containing 50ng of cDNA, 10Cl1 of 2X TaqMan Universal PCR Master

Mix (Applied Biosystems, Foster City, CA), and 1CL1 of 20X TaqMan Gene Expression Assay

Mix (Applied Biosystems) containing the primers and probes. The reaction mixture was

incubated for 10 minutes at 950C and repeated for 40 cycles denaturingg for 15 seconds at 950C

and annealing and extending for 1 minute at 600C) using Applied Biosystems 7900HT Real-

Time PCR System. The TaqMan primers and probes for monitoring transcription levels ofAnt4,

E2fl, E2f6, Dmcl, and Nanog were assay-on-demand gene expression products labeled with









FAM reporter dye (Applied Biosystems). A mixture of mouse B-actin primers with VIC-labeled

B-actin probe was used as endogenous control (Applied Biosystems, catalog number 4352341E).

The primer sequences and probes for quantifying E2f6 enrichment after performing ChlP

analysis were custom ordered: B-actin: sense primer 5'-CGGAGGCTATTCCTGTACATCTG-

3', antisense primer 5 '-CGAGATTGAGGAAGAGGATGAAGAG-3 ', FAM-labeled probe 5'-

CCAGCACCCATCGCC-3'; Ant4: sense primer 5' -GCTGTTCTCCCAGCATCCT-3 ', antisense

primer 5' -GAGAACTGGAAAACCGCTTCAG-3 ', FAM-labeled probe 5'-

CTTTCCCGCCAGGGTAA-3 '; Dmcl: sense primer 5'-GGCCCCGCCCATCAA-3', antisense

primer 5'-CGCCCTGTCGTCGAACA-3', and FAM-labeled probe 5'-CCGCGGCCCTCATT-3'

All samples were tested in triplicate and expressed as means a standard deviations (SD).

Analysis of results was performed using SDS v2.3 software (Applied Biosystems) according to

the manufacturer' s instructions. The comparative CT method (AA~CT) WAS used for quantification

of gene expression.

Plasmid Construction and Site-Directed Mutagenesis

The Ant4 promoter region (from -263bp to +25bp) was PCR-amplified from mouse R1

ES cells' genomic DNA with high fidelity LA-TaqTM (Takara, Otsu, Japan) using the following

Ant4 primers: sense primer 5' -AATCACCGGGTTGGTGTAG-3 ', antisense primer 5'-

GCCACACCAACACTCAAGC-3'. The 288bp fragment was excised with Xhol and HindIII

(New England Biolabs, Ipswich, MA) using a QIAquick gel extraction kit (Qiagen). The Takara

DNA ligation kit was then used to li gate the fragment of the Ant4 promoter into a PGL2-basic

vector (Promega) containing a luciferase reporter gene. Mutations in the Ant4-Luciferase reporter

vector at the location of the E2F6-binding element were generated using the QuikChange@ Site-

Directed Mutagenesis Kit (Stratagene, La Jolla, CA) in combination with the following PAGE

purified primers: Ant4: sense, 5'-









CCCAGCATCCTCAGCGCCCGCGCCAGGGTAAAGCTGAAGCG -3', antisense, 5'-

CC GCTTCAGC TTTACCC TGGC GCGGGCGC TGAGGATGCTGGG -3' (site of mutati on i s

underlined). Reactions consisted of 5Cl1 of 10X reaction buffer, 50ng of vector template, 125ng

each primer, 1CL1 of dNTP mix, and 1CL1 of pfuTurbo@ DNA polymerase up to a final volume of

50Cl1. Reaction conditions consisted of 1 cycle at 950C for 30 seconds, and then 18 cycles of

950C for 30 seconds, 550C for 1 minute, 680C for 9 minutes. Template vector was next degraded

by the addition of 1C1 of DpnI restriction endonuclease and incubated at 370C for 1 hour.

Reactions were transformed into Max Efficiency DH~a chemically competent cells (Invitrogen.),

analyzed by gel electrophoresis, and sequenced.

Transient Transfection and Reporter Assays

NIH3T3 cells were plated at a density of 1 x 105 cells per well in 6-well plates. After 24

hours, FuGENE 6 (Roche) was used to transfect the following according to manufacturer' s

instructions: 0.5Cpg Ant4-Luciferase reporter vector (wild-type or mutant), 0.1Cpg pRL-TK-Renilla

internal control vector (Promega), and 1.0p~g of one of the following vectors: HA-E2F6, HA-AC-

E2F6, HA-E68-E2F6, HA-AN-E2F6 (constructed as we previously described) or pCMVTag2

empty control vector (Stratagene) (Gaubatz et al. 1998). Twenty-four hours after transfection,

the cells were harvested. Firefly and Renilla luciferase activities were measured using a dual-

luciferase reporter assay system (Promega) according to manufacturer' s instructions. The firefly

luciferase data for each sample was normalized based on transfection efficiency as measured by

Renilla luciferase activity. Data from at least three independent experiments were analyzed and

expressed as means + standard deviations (SD). Statistical analysis was performed by Student's t

test and P values of less than 0.05 were considered significant.









Immunoblotting

NIH3T3 cells transiently transfected with one of the following E2F6 expression vectors:

HA-E2F6, HA-AC-E2F6, HA-E68-E2F6, or HA-AN-E2F6 using Fugene 6 (Roche), were lysed

in RIPA buffer (50mM Tris-HCI pH 8.0, 150mM NaC1, 1% NP-40, 0.5% Na-Deoxycholate,

0.1% SDS) plus PIs, and then harvested by scraping with a cell scraper. Cells were incubated for

20 minutes on ice and centrifuged. Supernatants were transferred to fresh tubes and total protein

was normalized by Lowry assay (Bio-Rad, Hercules, CA). Next, 25Cpg of total protein was

separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. After

blocking with 4% bovine serum albumin (BSA) solution in TBST buffer (50mM Tris-HCI pH

8.0, 100mM NaC1, 0. 1% Tween 20) for 1 hour at room temperature, the membrane was

incubated overnight with anti-HA primary antibody (1:250 dilution; Cell Signaling Technology).

After incubation for one hour with anti-mouse secondary antibody conjugated to horseradish

peroxidase (1:5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), chemiluminescent

detection (GE Healthcare, Piscataway, NJ) of horseradish peroxidase activity was performed.

Reverse Transcription-PCR

Total RNA was extracted using an RNAqueous@ kit (Ambion, Austin, TX). The cDNA

was synthesized using a Super Script@ II first-strand synthesis system with oligo(dT)

(Invitrogen) according to manufacturer' s instructions. PCR was performed using Taxq DNA

polymerase (Eppendorf) with the primer sequences listed in Table 2-2. For the samples selected

to be quantified using real-time PCR, an alternative method of reverse transcription was

performed using 1 Clg of total RNA and the High Capacity cDNA Reverse Transcription Kit

(Applied Biosystems) according to manufacturer's instructions. Primer sequences used in real-

time PCR are mentioned above. Regardless of which method of PCR was used, the sense









primers were always designed in different exons than the antisense primers in order to ensure

that the PCR product represented the specific mRNA species and not genomic DNA background.

Computational Analysis

Evolutionarily conserved regions (ECRs) of the mouse and human Ant4 gene and all

conserved E2F6 transcription factor binding sites (TFB S) located within the ECRs were

identified using the r~rista 2.0 software program (Loots & Oveharenko 2004). Additionally, an

interspecies comparison of the Ant4 promoter sequences containing the E2F6 TFBS was carried

out using r~rista. All genomic DNA sequences of meiosis-specific genes were obtained using the

University of California at Santa Cruz (UCSC) Genome Bioinformatics software (Karolchik et

al. 2003). A genome-scale DNA pattern matching algorithm was used to identify all core E2F6-

binding elements (TCCCGC or GCGGGA depending on the direction of binding) within the

proximal promoter regions (-1000bp to +1bp) of all genes in the entire mouse genome (based on

31,113 genes in the database) using Regulatory Sequence Analysis Tools (RSAT) software

(Van-Heldin 2003). The statistical relevance of the rate at which core E2F6 binding elements

appear in the genome was then examined. We considered DNA sequences with specified lengths

of 100bp, 200bp, 500bp, or 1000bp and the probability that a particular subsequence (the E2F6-

binding element) appears exactly n times within these sequences was computed under the

assumption that each of the four nucleotides (A, G, C, or T) appears randomly and with equal

probability (Gentleman & Mullin 1989). This information was used to obtain the probability that

a particular sub sequence appears at least once within a DNA sequence of a given length.

Specifically, the individual probabilities corresponding to each possible outcome of a trial must

sum to one as a consequence of the three axioms upon which probability theory is based. As

such, the probability that a subsequence occurs at least once: P(n > 1) is given by Equation 2-1,









where P(n = 0) represents the probability that the subsequence does not appear at all. The

expression forP(n = 0) is obtained from Gentleman & Mullin 1989.

P(n > 1) = 1- P(n = 0) (2-1)

The present study examines the probability that a bi-directional DNA binding element will

appear in a given sequence. Therefore, consideration is required for the case of multiple

subsequences, eg. One subsequence corresponding to each binding element. If the subsequences

corresponding to each binding element are denoted A and B, respectively, the probability that

either A or B will occur within a given sequence is denoted as P(A u B) and is expressed

approximately as in Equation 2-2. This expression also results directly from the axioms of

probability. The expression shown in Equation 2-2 is actually an upper bound, as the true value

must ideally account for cases where A and B occur simultaneously.

P(A u B) = P(A) + P(B) (2-2)

Bisulfite Sequencing and Combined Bisulfite Restriction Analysis (COBRA)

Genomic DNA from WT and E2f6- MEFs was bisulfite converted using EZ DNA

Methylation Kit (Zymo Research, Orange, CA). Approximately 80ng of PCR purified bisulaite

converted DNA was used as a template for each PCR analysis. Primers used for Ant4 COBRA

and bisulfite sequencing were: sense primer 5'-TTGTTGTGTATTGATTGAGTATG-3 ', and

antisense primer 5'-AAAAAAAACTAAAAAAC-3'. COBRA was performed by digesting the

PCR products with Hhal. After digestion, the COBRA reaction was ran on an 8% PAGE Gel

and then stained for 5 minutes with ethidium bromide. For bisulfite sequencing, undigested PCR

products were cloned into a pCR-2.1 TOPO cloning vector (Invitrogen) as we have previously

described (Rodic et al. 2005). Clones were then screened using blue and white screening, and

white colonies were subsequently sequenced.









Immunostaining

Testes were harvested from 6-week-old wild-type male mice. All mice have been

maintained under standard specific-pathogen-free (SPF) conditions, and the procedures

performed on the mice were reviewed and approved by the University of Florida Institutional

Animal Care and Use Committee (IACUC). The tissues were then fixed in a mild fixative (10%

formalin) overnight with rocking. Following fixation the tissues were dehydrated using an

organic solvent (PBS-Citrasol). The tissues were then imbedded in paraffin and sectioned.

Deparafinized and rehydrated 5Cpm tissue sections were stained with goat polyclonal antibodies

against mouse E2f6, or IgG control. Slides were blocked for endogenous peroxidase activity and

then unmasked in Target Retrieval Solution (Dako, Carpinteria, CA). Antibody was applied at

1:100 for one hour at room temperature prior to identification using the DAB Envision kit

(Dako). Slides were counterstained with hematoxylin.










Table 2-1. Primers used for analyzing ChlP experiments with semi-quantitative PCR


Primer name


Oligonucleotide sequence (5' to 3')


Ant4 sense
Ant4 antisense
Dmc1 sense
Dmc1 antisense
Nanog sense
Nanog antisense
Oct-4 sense
Oct-4 antisense
Smcip sense
Smclp antisense
Spoll sense
Spoll antisense
Stag3 sense
Stag3 antisense
Sycpl sense
Sycpl antisense
Tuba3 sense
Tuba3 antisense
P-Actin sense
P-Actin antisense


ACACGTGTTATGGTCACATGC
GCCTTCTTTGAAGACTGCTTCT
CCACCCCCACAACCTGAG
AGAATTTTCAAGGCGACACC
TCACACTGACATGAGTGTGG
TCTGTGCAGAGCATCTCAGT
ACGCAGAGCCAGCACTTCTC
CCAGTATTTCAGCCCATGTCC
GGAGCAGGGATCCAATTCC
GCCACGACTTGAAATTCTCC
AGCTTCATGTCCTCCCTGAA
AGGGCGTCGAAGAACGAG
AGGAACGTGGTTAGCACGAG
GGCTGAAGAAAGGTCACCAC
ATCCTCCGACAATTTTGAGC
ACACCCTCCACACCCTCAC
TTGTTCCATGCTAAGTTGGATGTC
CCGTTCCTCACCACTGGTCCTCAAC
AAATGCTGCACTGTGCGGCG
AGGCAACTTTCGGAACGGCG










Table 2-2. Primers used for semi-quantitative RT-PCR


Primer name


Oligonucleotide sequence (5' to 3')


Ant4 sense
Ant4 antisense
Dmc1 sense
Dmc1 antisense
E2F6 sense
E2F6 antisense
Nanog sense
Nanog antisense
Oct-4 sense
Oct-4 antisense
Rec8L sense
Rec8L antisense
RecQ1 sense
RecQ1 antisense
RibC2 sense
RibC2 antisense
Smcip sense
Smclp antisense
Spoll sense
Spoll antisense
Stag3 sense
Stag3 antisense
Sycpl sense
Sycpl antisense
Tuba3 sense
Tuba3 antisense
P-Actin sense
P-Actin antisense


TGGAGCAACATCCTTGTGTG
AGAAATGGGGTTTCCTTTGG
AGAATTTTCAAGGCGACACC
CCACCCCCACAACCTGAG
AAGCTGCAGGCAGAACTCTC
TTCACCCACTCGGGATACTC
AGGGTCTGCTACTGAGATGCTCTG
CAACCACTGGTTTTTCTGCCACCG
TGGAGACTTTGCAGCCTGAG
TGAATGCATGGGAGAGCCCA
AGGGCGTCGAAGAACGAG
AGCTTCATGTCCTCCCTGAA
ACTGGAATCCGTAGCCAGTG
AGAGCTGGAAGCATTCAACA
CCATGGAGGTAGCGATGTCT
GTGTTCCGCATGTCATTGTC
GCCACGACTTGAAATTCTCC
GGAGCAGGGATCCAATTCC
GTTGGCCATGGTGAAGAGAG
CTCAAGTTGCCAGCAATCAA
GGCTGAAGAAAGGTCACCAC
AGGAACGTGGTTAGCACGAG
ACACCCTCCACACCCTCAC
ATCCTCCGACAATTTTGAGC
GGACCGGATCCGAAAACTGG
GTCTGGAATTCTGTTAAGTCC
TTCCTTCTTGGGTATGGAAT
GAGCAAT GATC TT GATC TTC










CHAPTER 3
REPRESSION OF ANT4 GENE EXPRESSION BY E2F6

Motivation

Eukaryotic cells require energy for their survival. The maj ority of this energy is

produced through oxidative phosphorylation, a process whereby ATP is synthesized from ADP

and inorganic phosphate. This occurs within the mitochondrial matrix and therefore requires that

ADP and ATP be shuttled across both the inner and outer mitochondrial membranes. Although

nonspecific porins can mediate this transport across the outer membrane, the inner membrane is

highly selective and requires specific carrier proteins for any solutes to get across. The adenine

nucleotide translocases (Ants) are a family of solute carriers that facilitate the exchange of ATP

for ADP across the inner membrane of the mitochondria (Dahout-Gonzalez et al 2006). In this

chapter, the Ant isoforms and their critical roles in cellular respiration, as witnessed by defects

resulting from their mutations, are discussed. In particular, the regulatory mechanisms

governing Ant gene expression are emphasized and new results on the transcriptional repression

of the Ant4 isoform are presented.

Background

Adenine nucleotide translocases are the most abundant proteins of the mitochondrial

inner membrane and are comprised of approximately 300-320 amino acid residues which form

six transmembrane helices. The Ants transport ADP/ATP according to an exchange-diffusion

mechanism with a one-to-one stoichiometry, thereby maintaining the adenine nucleotide pool at

a constant level within the mitochondrial matrix (Dahout-Gonzalez et al. 2006). The Ants are

encoded by nuclear genes whereby the ANT proteins are then translocated into the mitochondrial

inner membrane (Rehling et al. 2004).










Ant genes have been cloned in numerous eukaryotic species including yeast and plants.

Mammalian Ant genes have been cloned in human, rat, bovine, and most recently mouse. All of

these species have multiple Ant isoforms of varying tissue specificity. Until recently, it was

thought that humans only had three members of the ANT family: ANT1 (SLC25A4), which is

specific to heart and skeletal muscle, ANT2 (SLC25A5), which is expressed in rapidly growing

cells and is inducible, and ANT3 (SLC25A6), which is ubiquitously expressed (Stepien et al.

1992, Lunardi et al 1992). In contrast, mice have only two isoforms: Antl, which is expressed

in heart, brain, and skeletal muscle and is located on chromosome 8, and Ant2 which is expressed

ubiquitously with the exception of skeletal muscle and is located on the X-chromosome (Levy et

al. 2000). Mouse Ant2 is the ortholog of human ANT2 and is believed to have the combined

functions of both human ANT2 and ANT3 (Ellison et al. 1996).

Recently, we and others identified a novel member of the Ant family, Ant4, in both mouse

and humans (Dolce et al. 2005, Rodic et al. 2005, Kim et al. 2007). In humans, the Ant4 gene is

located on chromosome 4 whereas in mice it is located on chromosome 3. In contrast to all other

known Ants which only have four exons, Ant4 contains six exons in both mouse and human. The

Ant4 gene is predicted to encode a 320 amino acid protein and a comparison between mouse and

human indicates that the protein is mostly conserved, sharing an 88% overall amino acid identity

(Figure 3-1A) (Rodic et al. 2005). Ant4 also has high amino acid sequence homology with other

previously identified mouse Ant proteins (70.1% and 69.1% overall identity to Ant1 and Ant2,

respectively) (Figure 3-1B). Closer examination of Ant4's amino acid sequence reveals the

presence of a signature sequence common to members of the mitochondrial carrier family, a P-

X-[D/E]-X-X-[K/R] motif found three times at ~100 residues apart (Walker 1992). It is

noteworthy that Ant4 has a unique extension of amino acids at both the N- terminus and the C-









terminus. From the sequence analysis, it appears that Ant4 shares sufficient homology to be

classified as a mitochondrial ADP/ATP transporter but its regions of unique sequence suggest

that it may differ in function or tissue-specificity from the rest of the family. Upon further

analysis, we did in fact find that Ant4 's expression pattern is different from all other Ants in that

its transcripts are detectable only in testis (Brower et al. 2007).

Defects resulting from disruption of Ant gene expression have been reported. In humans,

there is a clinical manifestation associated with ANT1 known as autosomal dominant progressive

external opthalmoplegia (adPEO) (Hirano & DiMauro 2001). This adult-onset disorder is

characterized by the appearance of external opthalmoplegia, ptosis, and progressive skeletal

muscle weakness. Molecularly, adPEO is evidenced by the accumulation of numerous

mitochondrial point mutations including many which are found in ANT1: Al l4P, L98P, A89D,

D104G, and V289M substitutions. Deficiency of this ADP/ATP carrier has also been reported in

striated muscle of patients affected with myopathy and in patients suffering from Sengers

syndrome (Jordens et al 2002). However, in Sengers syndrome, the ANT1 gene itself was not

altered, indicating that the pathology could result from either impairment of the carrier

transcription or from impeded import into the mitochondrial membrane.

Studies have also shown an impairment of ADP/ATP carrier function in heart despite the

existence of high levels of total carrier proteins. Rather than a defect in total protein levels, this

impairment results from a shift in isoform expression. It was shown that an increased amount of

ANT 1 isoform correlates with a decrease in ANT2 isoform in dilated cardiomyopathy (Dorner et

al. 2006). Phenotypes similar to those found in humans were observed in Antl-/- mice, where

the mice were viable but developed mitochondrial myopathy and severe exercise intolerance

along with hypertrophic cardiomyopathy as young adults (Graham et al. 1997).









Genetic disruption of Ant2 in mice presumably results in peri-neonatal lethality but this

observation has not yet been published in a scientific paper

(www.patentdebate. com/PATAPP/20050091 704). ANT2 has been found to be overexpressed in

cancer cells (Chevrollier et al. 2005). It is established that cancer cells display a glycolytic

phenotype resulting from the downregulation of mitochondria-encoded genes and impairment of

mitochondrial function. The ANT2 promoter contains a regulatory element which represses gene

expression in response to oxygen. It is hypothesized that ANT2 imports glycolytic ATP into

mitochondria in exchange for exporting ADP, a process which would sustain basal mitochondrial

activity under the predominant glycolytic status of tumor cell proliferation. There have been no

reports regarding ANT2 or ANT3 mutations in human.

Defects resulting from disruption ofAnt4 gene expression are evidenced by the Ant4

knockout mice generated by our lab (Brower et al. 2007). We observed that males were sterile

and lacked meiotic and post-meiotic cells, thereby establishing that this ADP/ATP mitochondrial

translocase is essential for successful progression of male germ cell meiosis. Furthermore, Ant4-

defieient male mice exhibited increased levels of apoptotic cells within the testis and the maj ority

of these cells were identified as early spermatocytes. Given that Ant2 is encoded on the X

chromosome and the X chromosome is inactivated via meiotic-sex chromosome inactivation

(MSCI) (Turner 2007) at the onset of meiosis, we speculate that Ant4 may have evolved to

compensate for the loss ofAnt2 during MSCI. Thus, it is likely that Ant4 functions as the sole

mitochondrial ADP/ATP carrier during spermatogenesis.

Although the Ants have been studied fairly extensively on a protein level, very little is

known about the regulatory mechanisms governing Ant gene expression. The ANT1 gene is

known to contain classic TATA and CCAAT elements within its promoter. Also found within










the promoter is a positive regulatory element known as OXBOX which is bound by proteins

present only in muscle cells (Li et al. 1990, Chung et al. 1992). Muscle-specifie binding of

transcription factors to OXBOX is thought to account for the induction ofANT1 expression in

muscle. The OXBOX element in the ANT1 promoter is overlapped by REBOX, a potentially

negative regulatory element. Gel shift assays have demonstrated that proteins binding to

REBOX are not muscle-specific, suggesting a potential mechanism for mediating ANT1

repression in non-muscle tissue. However, the mouse Ant1 gene lacks the OXBOX and REBOX

elements identified in the human promoter.

Similar to ANT1, a classic TATA box is present in the ANT2 promoter. ANT2 also

contains three putative Spl control elements near its transcription start site. Two of the Spl

elements, known as the AB box, are located 5' of the TATA box and work synergistically to

activate ANT2 transcription (Li et al. 1996). However, activation via the AB box is modulated

by three separate repressor regions. One of these is another Spl binding element, termed the C

box, and is juxtaposed to the transcription start. When the C box is occupied, ANT2

transcription is decreased. The second repressor region can be found in the distal promoter and

is the site for NF 1 binding (Barath et al. 2004). The third repressor region is located between the

AB activation box and the distal repressor region and is also bound by NF l. Similar to ANT1

and ANT2, the ANT3 gene also has a classical TATA element within its promoter, as well as 16

identified Spl elements. ANT3 has also been shown to be regulated by IL-4 and IFN-y via

STAT-dependent pathways in T cells (Jang & Lee 2003).

In contrast to other ANTs, a TATA box is absent in the ANT4 promoter. We have

previously shown that CpG methylation is required for Ant4 repression (Rodic et al. 2005). We

have also shown a correlation between methylation and expression whereby in testis, Ant4 is










highly expressed and hypomethylated while in all of the somatic tissues examined, Ant4 is

repressed and hypermethylated. In particular, we showed Ant4 derepression in D10 embryoid

bodies (EBs) upon addition of the DNA demethylating agent, 5-aza-dC. Likewise, Ant4 was

derepressed in EBs lacking de novo DNA methyltransferases (Dnmts). In this study, we have

investigated the transcriptional regulation ofAnt4 with the aim of determining how Ant4 is

repressed in all somatic cells. We believe that understanding Ant4 gene regulation is important

given its unique meiosis-specific expression pattern and its essential role in spermatogenesis.

Results

After our recent identification of the novel Ant4 isoform and further characterization of

the Ant4-/- mouse, we next examined the promoter region ofAnt4 in search of potential

transcription factor binding sites (TFB S). Given that other Ant isoforms contain multiple Spl

TFB S in their promoters which have been previously shown to play a role in their regulation (Li

et al. 1996), we searched the mouse and rat Ant4 promoters for Spl TFBS. We compared the

sequences of mouse and rat Ant4 (Slc25a31) genes and surrounding non-coding regions using

rVISTA sequence analysis software (Loots & Oveharenko 2004). The rVISTA program

identified several evolutionary conserved regions (ECRs) of sequence similarity and located

conserved Spl binding sites within the proximal promoter region (Figure 3-2A). However, when

we compared the mouse Ant4 sequence to human Ant4, no conserved Spl binding sites were

found (Figure 3-2B). We then ran a search for all conserved TFBS between mouse and human

Ant4 and found that there was only a handful of conserved TFBS in the proximal promoter

region (data not shown). One of these was an E2F binding site with the core E2F6 sequence,

TCCCGC (Figure 3-3A). This potential E2F TFBS was located 36bp upstream of the

transcription initiation site and an interspecies sequence comparison indicated that the E2F6

TFBS and surrounding flanking regions were mostly conserved (Figure 3-3B). We chose to









investigate this E2F6 site further due to previous literature implicating E2F6 in the repression of

meiosis-specific genes (Storre et al. 2005, Pohlers et al. 2005).

Next, we investigated the functional significance of the E2F6 TFB S in the murine Ant4

promoter by analyzing the ability of E2F6 to interact with this site. To address this question, we

initially performed a gel retardation assay to determine whether E2F6 would bind in vitro to a

radiolabeled oligonucleotide encompassing the E2F6 TFBS in the Ant4 promoter (-54bp to -

24bp). In order to prepare E2F6 protein and its binding co-factor DP2, we used a rabbit

reticulocyte lysate in vitro translation (IVT) system to co-express HA-tagged E2F6 (HA-E2F6)

and Myc-tagged DP2 (Myc-DP2). As shown in Figure 3-4, addition of the E2F6/DP2 protein

complex to radio-labeled Ant4 probe generated an additional retarded band. The specifieity of

this interaction was verified by competition with wild type (wt) and mutant (mut) cold

competitors in 20, 50, or 100 fold excess amounts over radiolabeled Ant4 probe, whereby only

the wt competitor was able to efficiently compete for binding to the E2F6 TFB S (filled arrow,

Figure 3-4). It should be noted that the reticulocyte lysate used for IVT contains an endogenous

protein which interacts with the Ant4 probe. This interaction is evidenced by the presence of a

retarded band when the probe was incubated with control rabbit reticulocyte lysate in the absence

of IVT (open arrow, Figure 3-4). However, addition of specific antibodies against HA and Myc

into the protein/probe mixture selectively reduced the intensity of the bands indicated by the

filled arrow as we previously demonstrated (Gaubatz et al. 1998) (data not shown).

Subsequently, we analyzed the occupancy of this E2F6 TFB S in vivo using Chromatin

immunoprecipitation (ChIP). Here we show that endogenous E2f6 binds to the proximal

promoter ofAnt4 but not to B-actin in undifferentiated R1 ES cells (Figure 3-5). Both semi-

quantitative PCR (Figure 3-5A) and quantitative real-time PCR (Figure 3-5B) analysis of ChlP









resulted in the same conclusion. Collectively, these observations show that Ant4 contains a

conserved E2F6 TFB S that binds E2F6 both in vitro and in vivo.

To clarify the role that E2F6 plays in the regulation of Ant4 transcription, we performed

luciferase assays using an Ant4 reporter vector in combination with various E2F6 expression

vectors. The Ant4-Luciferase reporter contains a 288bp region of the Ant4 promoter (-263bp to

+25bp) which includes the E2F6 TFB S. For transient transfections, we used NIH3T3 cells due

to their high transfection efficiency and their apparent absence of endogenous E2f6 expression

(data not shown). The activity of the wt Ant4-Luciferase reporter significantly decreased upon

co-transfection with an HA-E2F6 expression vector (Figure 3-6). However, when the E2F6

TFB S in the Ant4-Luciferase reporter was mutated using site-directed mutagenesis, co-

transfection with HA-E2F6 failed to repress Ant4 transcription (Figure 3-6). Similarly, when the

wt Ant4 reporter vector was co-transfected with HA-E2F6 expression vectors in which either the

DNA binding domain was mutated (HA-E68-E2F6) or the repression domain was mutated (HA-

AC-E2F6), reporter activity was not repressed (Figure 3-7A). E2F6 proteins containing a

mutation in the N-terminal domain (HA-AN-E2F6), which have been shown to retain their

repressor activity, had similar repressive effects to that of WT HA-E2F6 (Gaubatz et al. 1998).

Western blotting confirmed that the E2F6 proteins were expressed at comparable levels when

equivalent amounts of DNA were transfected (Figure 3-7B). These reporter assays indicate that

the E2F6 protein can repress Ant4, providing its DNA binding and repressor domains are intact,

and that this repression depends on sequence-specific DNA binding.

To further elucidate the potential role of E2F6 in the repression of the Ant4 promoter, we

next created stable ES cell lines overexpressing either HA-E2F6 or HA-AC-E2F6 expression

vectors. In contrast to somatic cells, many germ-cell-specific genes including Ant4 are









considered to be transcriptionally permissive in ES cells and therefore have detectable mRNA

transcripts (Rodic et al. 2005, Geij sen et al. 2004). The CpG island of the Ant4 gene promoter is

hypomethylated in ES cells and a low level of Ant4 transcription is detectable. Using both semi-

quantitative RT-PCR (Figure 3-8A) and quantitative real-time PCR (Figure 3-8B), we show that

overexpression of HA-E2F6 but not mutant HA-AC-E2F6 is able to reduce Ant4 transcription

relative to parental R1 ES cells. This indicates that increasing the amount of E2F6 protein

present in a cell is sufficient to trigger E2F6-mediated repression. Based on this result, it is

reasonable to believe that the opposite scenario, in which E2F6 protein has been depleted from a

cell, could potentially result in the derepression ofAnt4 transcription. To this end, we examined

the expression ofAnt4 in mouse embryonic fibroblast (MEF) cell lines derived from both wild

type and E2f6 null mice. Using semi-quantitative PCR, we demonstrate that Ant4 is in fact

aberrantly expressed in E2f6-/- MEFs (Figure 3-9). Taken together, these findings indicate that

E2F6 is an essential repressor for the Ant4 gene in somatic cells.

Given that E2F6 has been shown to be associated with polycomb proteins (discussed in

Chapter 1), we analyzed the binding of the polycomb proteins, Eed and EZH2, to R1 ES cells at

DO and D6 of differentiation (Figure 3-10). Interestingly, we observed an increase in binding of

Eed and EZH2 to the Ant4 promoter at D6 but not DO of ES cell differentiation. The polycomb

binding at D6 coincides with Ant4 repression by D6. However, it is a bit concerning that we still

detected acetylated histone 3 (AH3), a marker of active chromatin, on D6 when Ant4 expression

should be silenced. Because the ChlP primers were designed around the E2F6 binding site in the

Ant4 promoter, and both Eed and EZH2 cannot themselves bind to DNA, we speculate that these

proteins maybe using E2F6 as a binding platform. Upon binding to E2F6, they would then be

able to exert their repressive effects on the Ant4 promoter.









An alternative mechanism of E2F6-mediated repression of Ant4 may be through the

recruitment of DNA methyltransferases. It was previously shown by our lab that DNA

methylation is required for Ant4 repression during embryonic stem cell differentiation (Rodic et

al. 2005). We first wanted to confirm that Ant4 still required DNA methylation to remain

repressed in somatic cells and not just initially during ES cell differentiation. Therefore, we

treated NIH-3T3 cells with the DNA demethylating agent, 5-Aza-Deoxycytidine (5-AZA-DC)

(Figure 3-11). We found that DNA methylation was indeed required for the repression ofAnt4

in somatic cells indicating the continued importance of DNA methylation in Ant4 repression.

Additionally, we examined the effects of the HDAC inihibitor, Trichostatin-A (TSA), on Ant4

expression. We found that in contrast to 5-AZA-DC, TSA had no affect on Ant4 expression

indicating that histone acetylation was most likely not the mechanism of E2F6-mediated

repression. Perhaps Ant4 repression not being effected by histone acetylation may help to

explain how Ant4 can be repressed at D6 of ES cell differentiation despite the detection of AH3

(Figure 3-10). We next compared the methylation status of Ant4 in WT and E2f6-/- MEFs using

both COBRA and Bisulfite Sequencing (Figure 3-12). Interestingly, we observed a reduction in

DNA methylation at the Ant4 promoter in E2f6-/- MEFs. However, this decrease, from 96%

methylation in WT MEFs to 80% methylation in E2f6-/- MEFs was minor. This is in contrast to

the high percentage of demethylation found in the promoter region of the germ-cell-specific

gene, Tuba3 (Figure 3-13). Findings regarding Tuba3 demethylation in E2f6-/- MEFs were

previously published (Pohlers et al. 2005), and the Tuba3 data presented in Figure 3-13 was

generated in collaboration with Emily Smith and Dr. Resnick. Next, we checked for the binding

of Dnmt3 -containing complexes to the Ant4 promoter using ChlP assay but the results appeared

negative (Figure 3-10A). To further explore the potential involvement of E2F6 in Ant4










methylation, we compared our HAE2F6 ES cell line to WT R1 ES cells at DO and D6 of ES cell

differentiation. If E2F6 was repressing Ant4 by recruiting DNA methyltransferases, then we

would expect to observe an increased rate of DNA methylation in those cells overexpressing

E2F6. As shown in Figure 3-14, there was no such increase in DNA methylation leading us to

believe that DNA methylation is not the primary mechanism of E2F6-mediated repression.

Discussion

In this study, we first demonstrated that the meiosis-specific gene, Ant4, contains an

evolutionarily conserved E2F6 binding site in its proximal promoter region. This indicates that

E2F6-mediated regulation of Ant4 is likely important both within and between species. The

importance of conserving such a binding site is highlighted by the fact that two-thirds of the

sequence conserved among mammals is not protein-coding (Adams 2005). Therefore, the

noncoding regulatory regions upon which transcription factors bind must play a large part in

controlling the expression of protein-coding genes. From this knowledge, it is tempting to

speculate that an in vivo disruption of the E2F6 binding site located at the Ant4 promoter could

result in: 1) defects in fertility similar to those observed in the Ant4 knockout mouse, or 2)

aberrant expression of Ant4 in somatic tissues. The data presented here are in support of the

latter effect, although both possibilities could be true. Additional studies whereby mice are

created from mutations knocked-in to the E2F6 binding site will elucidate if such a binding site

is required for Ant4 expression during meiosis and consequently fertility. For the remainder of

this discussion, we will focus on the mechanisms ofAnt4 repression in somatic tissues.

From these experiments, it is clear that there are multiple players involved in the

repression ofAnt4 gene expression. The three key players that we found to be involved are

E2F6, polycomb proteins, and DNA methylation. Examination of these factors on an individual

basis is what led us to the conclusion that they were important to Ant4 repression. For E2F6,









sufficient evidence came from the combined findings that E2F6 binds to the Ant4 promoter

(Figures 3-4 and 3-5), that overexpression of E2F6 induces Ant4 repression (Figures 3-6, 3-7,

and 3-8), and that Ant4 derepression occurs in E2f6-/- MEFs (Figure 3-9). For polycomb

proteins, proof came from the binding of Eed and Ezh2 to the Ant4 promoter in D6 EBs (Figure

3-10). Based on previous studies in our lab, we already knew that DNA methylation was

essential to Ant4 repression (Rodic et al. 2005). These experiments showed that Ant4 was

derepressed in both ES cells following treatment with 5-AZA-DC and in Dnmt3a-Dnmt3b

double null ES cells. Additionally, we demonstrated the importance of DNA methylation in

Ant4 repression by verifying that Ant4 was derepressed in somatic cells (NIH-3 T3 s) after

treatment with 5-AZA-DC (Figure 3-11). These findings support a role for E2F6, polycomb

proteins, and DNA methylation in the repression ofAnt4, but fail to explain how these key

components coordinate with one another to regulate Ant4 gene expression.

A relationship between E2F6 and polycomb proteins has been observed in multiple

studies (Ogawa et al. 2002, Trimarchi et al. 2001, Attwooll et al. 2005). As discussed in Chapter

1, E2F6 associates with components of both the PRC2 and the PRC 1 polycomb repressive

complexes. Given that these polycomb proteins do not bind to DNA directly, E2F6 has been

suggested to serve as a platform on which polycombs can bind. Binding to E2F6 would then

enable these polycomb proteins to exert their repressive effects on DNA. In agreement with this

concept is our finding that the PRC2 components, Eed and Ezh2, appear to be associated with the

Ant4 promoter in the same region that E2F6 is bound (Figure 3-10). Further, we demonstrate

that only the endogenous E2F6 platform binds at DO when Ant4 is expressed, whereas the Eed

and Ezh2 polycomb proteins do not bind and assemble until just after Ant4 is repressed at around

D6 of differentiation. This suggests that although a docking platform for the polycomb proteins










may bind to polycomb-target promoters prior to polycomb complex assembly, gene repression

will not occur until the full polycomb repressive complex has arrived and assembled on such a

platform. Further studies will need to be performed to confirm these findings and to establish

when and where the protein-protein interactions involved in such a repressive complex are

occurring.

An association between polycomb proteins and DNA methylation has also been reported

in literature. Recently, EZH2, a component of the PRC2 complex that is known for its ability to

methylate histone H3K27, was shown to physically interact with DNA methyltransferases (Vire

et al. 2006). This 2006 report was the first time that any evidence suggesting a cross-talk

between these two silencing pathways had ever been presented. The study showed that

knockdown of Dnmtl, 3a, or 3b led to derepression of EZH2-target genes. These target genes

were also found to bind both EZH2 and the DNA methyltransferases. Additionally, EZH2

overexpression increased the CpG methylation at EZH2 target promoters, whereas EZH2

knockdown decreased CpG methylation. Moreover, EZH2 was needed to bring Dnmts to the

promoters of EZH2-target genes. Depletion of EZH2 disturbed this recruitment, and enabled the

aberrant expression of target genes.

Out of all the possible interactions between the key players involved in Ant4 repression,

we focused our efforts on elucidating the connection between DNA methylation and E2F6.

Interestingly, a recent study found that the germ cell-specific gene, Tuba3, was derepressed in

E2f6-/- MEFs (Pohlers et al. 2005). This derepression was correlated with a maj or reduction in

DNA methylation at the Tuba3 promoter, suggesting that E2F6-mediated repression involves the

recruitment of Dnmts to target promoters which then locks them in an inactive state. Given that

Ant4 is also a germ cell-specific gene, and that DNA methylation and E2F6 are both essential for









Ant4 repression, we predicted that these key players would behave similarly in mediating the

repression of Ant4. Surprisingly, Ant4 methylation in E2f6-/- MEFs was only slightly reduced

(~16%) (Figure 3-12) in comparison to the massive reduction reported for Tuba3 methylation

(~69%) (Pohlers et at. 2005). We have proposed the following theories in an effort to explain

why the Ant4 promoter remains mostly methylated in E2f6-/- MEFs.

The first theory to explain why there is only a slight reduction in Ant4 methylation is that

there is heterogeneity among cultured MEF cells where some cells are more differentiated than

others. Recent studies have demonstrated that cell populations such as ES cells, which were

once believed to be homogeneous, are in fact rather heterogeneous (Singh et at. 2007).

Additionally, the methylation status of the germ line and pluripotent cell marker, Oct4, was

shown to be heterogeneous among populations of somatic cells indicating that somatic cells may

even exhibit some degree of heterogeneity (Marikawa et at. 2005). It is feasible that within the

mixture of cultured MEF cells, some cells may be more dependent on E2F6 for DNA

methylation than others depending on their differentiation status. However, if this were the case,

one would predict that the Tuba3 promoter would show a similar distribution of methylation

among these mixed cell types as Ant4. We examined the methylation status of the Tuba3

promoter in E2f6-/- MEFs using COBRA (data not shown), and found that Tuba3 was indeed

less methylated than Ant4. This finding eliminates the possibility that variations in culture

conditions between our experiments and those from the previous report on Tuba3 are responsible

for this difference in methylation. Thus, it is unlikely that cellular heterogeneity can account for

the slightly reduced Ant4 methylation in E2f6-/- MEFs.

An alternative theory for why there is very little demethylation of Ant4 in E2f6-/- MEFs

is that in these cells, Ant4 expression is leaky. The term "leaky" generally refers to genes that









are considered to be specific to one tissue type but then become expressed in another tissue type

at much lower levels. Instead of looking at the total percentage of methylation that is occurring

on the Ant4 promoter in E2f6-/- MEFs, examining the spatial arrangement of DNA methylation

may provide more clues regarding whether Ant4 expression is indeed "leaky." As shown in

Figure 3-12, several of the Ant4 clones analyzed during bisulfite sequencing are entirely

demethylated. Thus, rather than thinking of an 80% overall methylation rate, let us divide the

multiple clones ofAnt4 from bisulfite sequencing into two populations: one group which has 0%

methylation (3 of the 18 clones) and another group with methylation rates nearly equivalent to

that found in WT MEFs (15 out of the 18 clones). Interestingly, we noticed this same "All" OR

"None" distribution of methylation for Tuba3 clones from a previous publication reporting

methylation of the Tuba3 promoter in E2f6-/- MEFs (Pohlers et al. 2005). We realized that 7 out

of 10 of these clones were 0% methylated and 3 out of 10 of the clones had high methylation

rates equivalent to WT MEFs. Although the percentage of Tuba3 clones which were fully

demethylated in E2f6-/- MEFs was much greater than the percentage demethylated for Ant4, the

conserved "All" OR "None" pattern implies biological relevance. In collaboration with Dr.

Resnick' s lab, we further showed that this same "All" OR "None" pattern exists at the Tuba3

promoter in E2f6-/- mouse tail tissue (Figure 3-13) indicating that the heterogeneity of cultured

MEF cells is most likely not responsible for these two different populations. Thus, if the

difference in methylation between the "All" and "None" populations is not a result of varying

degrees of differentiation among MEFs, but instead occurs in a rather homogeneous population

of cells, then there must be some other existing phenomenon to explain these findings.

This "All" OR "None" effect may be a result of an incomplete functional redundancy.

Although the body has developed backup mechanisms to ensure that processes which are









essential to genome integrity be protected, oftentimes these backup mechanisms are not as

efficient as the original. One such process that may exemplify this point is DNA methylation.

For successful DNA methylation to occur there needs to be functional DNA methyltransferases

(Dnmts) as well as proteins which can recruit these Dnmts to target gene promoters. From the

previous publication on Tuba3 expression in E2f6- MEFs, it was suggested that E2F6-mediated

repression occurred through E2F6 recruitment of Dnmts (Pohlers et at. 2005). Previous studies

have also shown that E2F family members often compensate for each other when another E2F

family member is lost (Kong et at. 2007). More specifically, E2F4 has been shown to

compensate for E2F6 by repressing E2F6-target genes when E2F6 is absent (Giagrande et at.

2004). Further, E2F4 has been found in a complex with Dnmt1 (Macaluso et al. 2003).

Therefore, we propose a model whereby under normal conditions, E2F4 and E2F6 may

both have roles in recruiting Dnmts to E2F-target genes (such as Tuba3 and Ant4) but that in

E2f6- MEFs, E2F4 must attempt to compensate for the loss of E2F6 by becoming the sole

recruiter of Dnmts (Figure 3-15). It should be noted here that many other proteins may be

involved in the recruitment of Dnmts to promoters but for the purposes of this discussion, we are

specifically focusing on the recruitment of Dnmts to E2F-target genes. Also, here we imply that

E2F4 and E2F6 are the only Dnmt recruiters for E2F-target genes, but additional studies are

needed to confirm such a theory. However, if this theory holds true, then E2F4-mediated

recruitment of Dnmts is serving as a functionally redundant backup for E2F6 in E2f6- MEFs.

We hypothesize that although E2F4 can mostly compensate for the loss of E2F6, it is likely that

the efficiency of Dnmt recruitment to target promoters may be lower than under normal

conditions. This reduction in efficiency is due to the fact that E2F4 now has double the

workload. If E2F4 is successful in recruiting Dnmts to a specific promoter, then the methylation










pattern at that promoter will be nearly identical to WT (Figure 3-15). However, when E2F4 fails

to recruit Dnmt to a specific promoter because it is now overwhelmed with having to recruit

Dnmts to so many additional promoters, then this promoter will show 0% methylation (Figure 3-

15). This theory could explain why in some cells, methylation of Ant4 and Tuba3 promoters is

completely "missed."

Simply stated, we believe that "leaky" expression of Ant4 is a direct consequence of

incomplete functional redundancy. The extent of leaky expression is determined by the number

of times in which E2F4 fails to recruit Dnmts to the Ant4 promoter in the absence of E2F6 (the

clones having 0% methylation). A possible explanation for why a higher percentage of Tuba3

promoters are demethylated in E2f6-/- MEFs could be that Tuba3 primarily relies more on E2F6

for Dnmt recruitment whereas Ant4 depends more on E2F4. Therefore, we predict that in an

E2f4-/- cell line we would see the opposite effect where Ant4 promoters are more demethylated

than Tuba3 promoters. Additionally, if this functional redundancy model holds true, in a double

E2f4-/- E2f6-/- cell line we would expect to see a nearly 100% overall demethylation rate for

both Ant4 and Tuba3 promoters. Taken together, we predict that future experiments such as

these will help to further support the theory that Ant4 expression is leaky in E2f6-/- MEFs due to

incomplete functional redundancy.

Additional experiments will be necessary to validate that E2F4 and E2F6 are indeed

recruiters of Dnmts. We did not present the data here in this study, but attempts were made to

demonstrate a physical interaction between Dnmt3 and E2F6. We transiently transfected tagged

E2F6, Dnmt3a and Dnmt3b expression vectors in NIH-3T3 cells and then performed co-

immunoprecipitation but saw no interaction between E2F6 and Dnmt3. Similarly, we tried to

coimmunoprecipitate endogenous Dnmt3 with exogenous HAE2F6 in HAE2F6 ES cell lines but









to no avail. It should be noted here that those studies were preliminary and several repetitions as

well as optimization studies would be needed before considering such results as reliable.

Given the possibility that E2F6 and Dnmt3 may not physically interact with each other

but could still be part of the same repressor complex, we performed ChlP. As shown in figure 3-

10, we were unable to find Dnmt3 association with the Ant4 promoter region despite the

presence of prominent E2F6 binding. However, the possibility that the Dnmt3 antibody quality

was insufficient for ChlP assays cannot be ruled out. Additionally, because E2F6

overexpression was unable to enhance DNA methylation at the Ant4 promoter (Figure 3-14) we

cannot draw any conclusions to suggest a relationship between E2F6 and DNA methylation at

this time. However, according to our "leaky" expression theory in combination with the

previous publication on Tuba3 (Pohlers et al. 2005), E2F6 should recruit Dnmts to E2F-target

promoters. The validity of this theory is pending further results which demonstrate an

interaction, either direct or indirect, between E2F6 and Dnmts. Moreover, these experiments

have attempted to elucidate the regulatory mechanisms governing Ant4 repression in somatic

cells by identifying E2F6 as an essential repressor. In the next chapter, we will further explore

the relationship between E2F6 and other genes which are similar to Ant4.



















.ARSIC ICCl\~CYI~1:rC~
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SSSGD


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-------------MTDA mjlAe~j~,~l~I~J~kF~j~mm~kF~i~i
MsNEssKKQssKKALFD~~aII~CICI~~IY~\rl~~3


SI:IYII~,IYT~F~J
~I~V~Y~J
EI:I~C~I[*,ICIMIC1WC~L~


hANT4
mAnt4

hANT4
mAnt4

hANT4
mAnt 4

hANT 4
mAnt 4

hANT 4
mAnt4

hANT4
mAnt4



B



Ant1
Ant2
Ant4

Anti


Ant1
Ant2
Ant 4
Ant1


Ant2
Ant4


,NIDVGGSSSGD


Figure 3-1. Ant4 encodes a novel isoform of adenine nucleotide translocase. A) Deduced amino
acid sequence of the mouse Ant4 gene is aligned with previously identified mouse
Ant proteins. B) Deduced amino acid sequence of the mouse Ant4 gene is aligned
with ANT4 human orthologue (from Rodic et. al 2005, Figure 2 on page 1318 with
permission).


~i~mLlrlrr~.Ycrcr~r~rCr~u~l~LurrWr'r31


S~


KC I P
KC I P,,~


IA'IC1IC1DI ~i~ml
IA'IC1IC1DI ~i~m:


I~~~ I m m imma


~Y'13YIYIl:1:130101~~Ic~l
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L


































Human
VS.
Mouse

25.0kb


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Sc25sa31


Sc2fia31


CONSER\~ED


slrZI
arrcl
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6.2kb


Figure 3-2. The conservation of Spl binding sites in the Ant4 promoter. Evolutionary conserved
regions (ECRs) between A) rat and mouse and B) human and mouse Ant4 (Slc25a31)
genomic regions are indicated as peaks on the graph and are color-coded according to
the type of genomic DNA in each ECR. Coding exons are in blue, untranslated
regions are in yellow, intronic noncoding ECRs are in pink, and intergenic ECRs are
in red. The Y-axis represents the percentage of conservation between the species
being compared. Spl transcription factor binding sites (TFBS) which are conserved
between mouse and rat but not between mouse and human are located within an
intergenic ECR and an intron as indicated by vertical tick marks above the graph in
A).


ii ~U~ ni~ TJJTlr~n~nnbnmE


IVVO
Rat
vs.
Mouse





















IE2FS ~
Mouse GATGCGCTGTTcTCCCAG~'ACTCCTCA~GCGCCjC G T AGGGTAAACTG AAGcGGTTCCAGTTCTCCT
Rat GATOCGCTGTTCTC~AC'AGTTCCTCAT'CGc"' OCTT1 AGO3ATAAACTOAAUOCOGTTTTCCGOTCCTECTI
Dog TcGCGcGOnTGATCTcCCA TcC~ccCTG ~CGCG' "AGG A.CGcAC GCC GAAGCGG~TTITECGC TCCCTTC
Hurnan GjGCGCGiCGGCTI~CTCTCAGCGTrCC wntccAGAC IICA~ CGTCG TGc~TGCAGCGGTTTCCGJ~GTTECCG C

Figure 3-3. The proximal promoter region of the Ant4 gene has a conserved E2F6 binding site.
A) Evolutionary conserved regions (ECRs) of mouse and human Ant4 (Slc25a31)
genomic regions are indicated as peaks on the graph and are color-coded according to
the type of genomic DNA in each ECR. Coding exons are in blue, untranslated
regions are in yellow, intronic noncoding ECRs are in pink, and intergenic ECRs are
in red. A scale is shown to indicate the width of the ECRs in kilobases (kb) as
measured along the X-axis. The Y-axis represents the percentage of conservation
between mouse and human. The position of a single conserved E2F transcription
factor binding site (TFBS) with the preferred E2F6 binding sequence (TCCCGC) was
found within an intergenic ECR and is indicated by a vertical gray tick mark above
the graph. B) The E2F6 TFBS is located at 36 base pairs (bp) upstream of the
transcription initiation site. The intergenic ECR containing the site is approximately
149bp in length and is conserved between species. The E2F6 binding site (dark gray)
and conserved flanking regions (light gray) are shown for mouse, rat, dog, and human
Ant4.
























12 3 4 5 6 78 9


Figure 3-4. Gel mobility shift assay of E2F6 binding to the Ant4 promoter. A labeled
oligonucleotide corresponding to the E2F6 TFBS in the mouse Ant4 promoter (lane
1) was incubated with control rabbit reticulocyte lysate alone (lane 2) or in vitro co-
translated HA-E2F6 and myc-DP2 proteins (lanes 3-9). Unlabeled Ant4 wild type
(wt) (lanes 4-6) or mutated (mut) (lanes 7-9) oligonucleotides were added in excess
amounts over the amount of labeled probe as indicated. Filled arrow: position of the
specific E2F6/DP2 complex, open arrow: endogenous binding activity in the
reticulocyte lysate.


rCII iir~













Ant4

p-actin

B








Input IgG E2f6



Figure 3-5. The proximal promoter region of Ant4 is bound by endogenous E2f6 in
undifferentiated WT R1 ES cells. Chromatin immunoprecipitation (ChIP) was
performed using mouse-anti E2f6 antibody. Primers amplifying the Ant4 proximal
promoter region containing the E2F6 TFBS and p-actin control primers lacking the
E2F6 TFB S were used for both A) semi-quantitative PCR and B) quantitative Real-
Time PCR analysis of ChlP. Samples from each primer set were precipitated with
IgG to control for nonspecific enrichment. Enrichment of E2f6 binding is expressed
as a percentage of the input chromatin. Input samples represent 1% of the starting
amount of chromatin and were analyzed to confirm that the different chromatin
preparations contain equal amounts of DNA. All samples were tested in triplicate and
expressed as means + standard deviations.











we CGrCTTTCCCGrCCAGr

mut CGrC GCCAG



140






Reporter E--~ MMe mut-
E2FS +

Figure 3-6. E2F6-mediated repression of Ant4 requires an intact transcription factor binding
site. Ant4 promoter (-263 to +25bp) luciferase reporter plasmids containing wild type
(wt) or mutated (mut) E2F6 TFBS were transiently co-transfected with an empty
vector (-) or an E2F6 expression vector (+) into NIH-3T3 cells. Luciferase activity
was measured relative to the Renilla internal control vector as described in Materials
and Methods Chapter 2. Sequences for both wt and mut E2F6 TFBS reporters are
shown above the bar graph. *P<0.05 and data from at least three independent
experiments were analyzed and expressed as means a standard deviations.














E2F6 WT

E2F6 E68

E2F6AN

E2F6AC


1 281

1 68 281

59 281

1 220


80




40 -0


E2F6 VT E68 AN AC
I Ant4 wt Reporter


Figure 3-7. E2F6-mediated repression of Ant4 requires an intact C-terminal and DNA binding
domain. A) Luciferase reporter assay in NIH-3T3 cells transiently co-transfected with
either empty vector (-), WT E2F6, or mutant (E68, AN, or AC) HA-E2F6 expression
vectors and the Ant4 wt reporter vector. A schematic representation of the HA-tagged
E2F6 vectors is shown above the graph; WT: intact E2F6, E68: a DNA binding
domain point mutation, AN: an N-terminus deletion mutant, AC: a C-terminus
deletion mutant, dark gray boxes: DNA binding domains, light gray boxes:
dimerization domains, black boxes: marked boxes, and black box inside dark gray
box: site of DNA binding domain mutation. B) Western blot analysis using anti-HA
antibody, showing that all the E2F6 expression vectors transiently transfected for
luciferase reporter assays in A) expressed their respective E2F6 proteins at
comparable levels. For A), *P<0.05 and data from at least three independent
experiments were analyzed and expressed as means + standard deviations.


37KDe HEF

25KDa












A B





)06

Ri HA AC

Figure 3-8. Ant4 transcription is repressed by stable E2F6 overexpression in ES cells. A) Semi-
quantitative RT-PCR analysis of Ant4 and f3-actin expression in parental R1, HA-
E2F6, and HA-AC-E2F6 ES cell lines. B) Quantitative Real-Time PCR analysis of
samples shown in A). All samples were tested in triplicate and expressed as means +
standard deviations.
























Figure 3-9. Ant4 transcription is derepressed in E2f6- MEFs. Semi-quantitative RT-PCR
analysis is shown for E2f6, Ant4, and B-actin expression in WT MEFs and E2f6- -
MEFs. Reverse transcriptase (RT) minus samples were also amplified to eliminate
the possibility of genomic DNA contamination.












,09"~ ~c~


~"' ,~c s ,~?c" ~"_~~l;i~a


Ant4
Actin


Ant4
Acting


,ods~'l~j ~~ C~C~cb


Ant4~

Actinz


Figure 3-10. The polycomb proteins Eed and Ezh2 bind to the Ant4 promoter in D6 R1 EBs.
Chromatin immunoprecipitation (ChIP) was performed using anti- E2f6, Eed, Ezh2,
Dnmt3, Suzl2, and acetylated histone H13 (AH3) antibodies. Primers amplifying the
Ant4 proximal promoter region containing the E2F6 TFB S and B-actin control
primers lacking the E2F6 TFB S were used for semi-quantitative PCR for A)
undifferentiated ES cells (DO) and B) differentiated EBs (D6). Samples from each
primer set were precipitated with IgG to control for nonspecific enrichment. Input
samples represent 1% of the starting amount of chromatin and were analyzed to
confirm that the different chromatin preparations contain equal amounts of DNA.


Day 0


Day 6











F;


4
o~~,~g
~O c~


Ant4

fl-actinz


Figure 3-11. Ant4 is derepressed in NIH-3T3s after treatment with 5-AZA-DC but not TSA. 5CLM
or 10 CLM of AZA-DC were added either alone or in combination with 200nm TSA
for 65 hours in NIH-3T3s. RNA was harvested and Ant4 and B-actin primers were
used for semi-quantitative RT-PCR.






















WT


E2f6-/-


,"'"'~
~! _G6
i' c-~' ~` cs~'


Undigested


Hhal


Ant4


Figure 3-12. CpG methylation at the Ant4 promoter is partially reduced in E2f6- MEFs. A)
COBRA analysis of the methylation status of Ant4 in various tissue types. Top band
is undigested PCR product prior to COBRA digestion with Hhal. Upon digestion
there is three bands, the single band marked as U represents unmethylated DNA; the
two bands labeled M represent the methylated DNA. B) Bisulfite Sequencing analysis
of the Ant4 promoter in WT MEFs (left) and E2f6- MEFs (right) showing a decrease
from 96% methylation in WT to 80% methylation in E2f6- -.












WT Tail DNA







E-f-II- TilII DNA-




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analysis~--~lC ofteBb3pootri Tmuetil (o)adEf-mueti
(bottom)~------ shwn eres rm9% ehlto n Tt 75 mtyaini
E ~ ~ ~ ~-f64- ~ -C (wt emiso rm ml mihadD. enc)















" """"""


~


__' ""


TIFIII
+212


TT T '
+212 50
0


-500 ,, 1111111 1 r 1 1 +211 1 2 LL







Day 6- WT R1 Differentiated


-500 ,, 1 11 111 rf i1 111lol +21 2







Day 6- HA-E2F6 Differentiated


Figure 3-14. E2F6 overexpression does not increase CpG methylation at the Ant4 promoter.
Bisulfite Sequencing analysis comparing the methylation status of the Ant4 promoter
between WT R1 ES cells and HAE2F6 ES cells at A) DO and B) D6 of ES cell
differentiation. The Ant4 promoter region is shown by a horizontal line with an arrow
indicating the transcription initiation site. Each vertical tick mark represents the
location of a CpG site with 16 CpG sites residing within the sequenced region.


TF IfT il I l i I II I II II I I I 1
-500







Day 0- WT R1 ES cells


F I O FT II I 1 1 I li l li ll I I I I | | | | ||1 | ill||||








Day 0- HA-E2F6 ES cells








































Figure 3-15. The "All" OR "None" model of E2F4 compensation. In WT somatic cell types both
E2F4 and E2F6 play a role in the methylation of Tuba3 and Ant4 promoters. Then, in
E2f6-/- tissues one of two things will happen. In the "All" model, E2F4 will
compensate for the loss of E2F6 and completely methylate the Tuba3 and Ant4
promoters, thereby contributing to transcriptional repression. In the "None" model,
E2F4 is unable to compensate for the loss of E2F6 and will not methylate the Ant4
and Tuba3 promoters, thereby resulting in "leaky" expression.









CHAPTER 4
REPRESSION OF ADDITIONAL MEIOSIS-SPECIFIC GENES BY E2F6

Motivation

The mechanisms controlling germ-cell-specific gene expression are diverse (Dej ong 2006

Eddy 2002, MacLean & Wilkinson 2005). Studies have found these genes to be regulated

extrinsically by hormones secreted from the endocrine system, interactively by factors released

from neighboring supportive cells in the gonads, and intrinsically by factors affecting

transcription, translation, DNA methylation, and histone modifications. Among such germ-cell-

specific genes, it is especially important that those genes which are highly expressed and critical

during the meiotic phase of gametogenesis be appropriately regulated. Aberrant expression of

these genes in somatic cells is presumed to be associated with disruptions in the mitotic cell

cycle and may lead to dire consequences such as oncogenic transformation (Sagata 1997,

Simpson et al. 2005). Many of these critical genes are germ-cell-specific and are therefore

repressed in all somatic cells in the body. This chapter focuses on the somatic repression of a

group of germ-cell-specific genes which are similar to Ant4 in that their protein products

function specifically during meiosis. Results suggesting the existence of a common

transcriptional repressor of these meiosis-specific genes are presented herein.

Background

Much of our current understanding regarding the transcriptional regulation of genes

involved in meiosis can be credited to the use of transgenic mouse models. Studies have

demonstrated that short proximal promoter regions are sufficient to specify somatic silencing and

germ cell activation for meiotic genes such as: Sycpl, CyclinA1, Pgk2, HistoneH~t, AlfJ and

Pdha2 (Bartell et al. 1996, Han et al. 2004, lannello et al. 1997, Lele & Wolgemuth 2004,

Robinson et al. 1989, Sage et al. 1999). A more detailed analysis of their minimal promoters










revealed DNA regulatory elements harboring transcription factor binding sites. Further,

incubation of these sequences with nuclear extracts from either somatic or testicular tissue

indicated that nuclear proteins were indeed binding to these elements. Variations in binding

between somatic and testicular nuclear extracts imply that these proteins mediate testis-specific

expression and somatic cell repression. For instance, M~os, a gene found to contain a negative

regulatory element in its proximal promoter, is bound by a protein present only in nuclear

extracts from somatic cells and not from pachytene spermatocytes (Xu & Cooper 1995). This

observation, coupled with the absence of expression of2~os in somatic cells, infers that this

protein may be serving as a transcriptional repressor. Further studies revealed that this protein

was COUP-TF (Lin et al. 1999). Additional transcription factors which bind to various germ-

cell-specific genes expressed during meiosis are beginning to be uncovered and include Spl,

Sp3, Ctfl, Rfxl, Rfx2, Ctcf, and Bmyb (Bartusel et al. 2005, Gebara & McCarrey 1992, Horvath

et al. 2004, Kim et al. 2006, Wilkerson et al. 2002). However, the existence and identity of a

master regulatory protein or protein family which binds to the proximal promoters and

coordinately regulates the expression of multiple meiosis-specific genes uniformly as a group

remains to be elucidated.

Recently, the E2F6 transcription factor was shown to be required for the repression of a

subset of germ-cell-specific-genes in somatic cells. These six genes, Tuba3, Tuba7, XV1l96054,

Texl2, Stag3, and Smclf, are aberrantly expressed in E2f6 null mouse embryonic fibroblasts

(MEFs) and/or somatic organs (Pohlers et al. 2005, Storre et al. 2005). All of these germ-cell-

specific genes contain the core E2F6-binding element, TCCCGC, within their proximal promoter

regions (Cartwright et al. 1998). Although the expression patterns of these genes are similar in

that they are all germ-cell-specific, their expression patterns and functions within germ cells are









diverse. Tuba3 and Tuba7 are a-tubulins which heterodimerize with P-tubulins to form

microtubules that constitute the primary structural components of mitotic and meiotic spindles

(Hecht et al. 1988). Tuba3 and Tuba7 are encoded by separate genes but have identical protein

products which are exclusively expressed in male gonads. More specifically, Tuba3 and Tuba7

are highly expressed in the metaphase spindles of spermatogenic cells undergoing meiosis and in

the manchettes of elongated spermatids. Indeed, the Tuba3 gene initiates significant levels of

expression at the onset of meiosis in primary spermatocytes (Wang et al. 2005). Little is known

about XVI 196054 other than that it is a gene encoding a novel protein of unknown function with

testis-specific expression (Storre et al. 2005). Texl2 is a male-meiosis-specific component of

the synaptonemal complex which is a group of proteins involved in the alignment and pairing of

homologous chromosomes during prophase one of meiosis (Hamer et al. 2006). Stag3 and

Smc Ip are also meiosis-specific proteins, but are components of the cohesion complex which is

a group of proteins that act as a molecular glue to hold sister chromatids together during

prophase one of meiosis (Revenkova & Jessberger 2005). Stag3 is restricted to male meiotic

germ cells whereas Smclp can be found in both male and female meiotic germ cells (Pezzi et al.

2000, Remelpva et al. 2001).

In the last chapter (Chapter 3), we presented evidence that E2F6 is required for the

repression of another germ-cell-specific gene, Ant4, in somatic cells. Ant4 contains the core

E2F6-binding element within its proximal promoter region and, similar to Texl2, Stag3, and

Smc Ip, its protein product is selectively expressed in meiotic cells (Brower et al. 2007). This

discovery prompted us to investigate whether additional germ-cell-specific genes require E2F6

for their repression. In particular, we focused on a subpopulation of germ-cell-specific genes

whose expression is not only high in meiotic cells but is also claimed to be restricted to the









meiotic phase of germ cell development. Here we show that most of the 24 meiosis-specific

genes examined in this study have the core E2F6-binding element within 200 basepairs (bp)

upstream of their transcription initiation sites. Moreover, using murine embryonic stem (ES) cell

culture, we demonstrate that E2F6 indeed plays a broad role in the transcriptional repression of

these meiosis-specific genes by binding to their proximal promoter regions.

Results

The discovery that another meiosis-specific gene is regulated by E2F6 prompted us to

investigate whether additional meiosis-specific genes require E2F6 for their repression. After an

extensive literature search and without bias, we compiled a list of 24 genes which were

previously reported to have meiosis-specific expression (Table 4-1). It should be noted here that

we excluded genes from the list which continue to have predominant levels of expression post-

meiotically. For each of these meiosis-specific genes, we screened a genomic region spanning

1kb upstream of their transcription initiation sites for the presence of E2F6-binding elements

(TCCCGC or GCGGGA, depending on the direction of binding) using UCSC Genome Browser

(Karolchik et al. 2003). As shown in Table 4-1, potential E2F6-binding elements are

accumulated within the proximal promoter (~200bp) regions of these meiosis-specific genes. In

total, 19 out of the 24 meiosis-specific genes (79.2%) contain at least one E2F6-binding element

within 200bp upstream of their transcription initiation sites (white bars, Figure 4-1). The

relevance of this finding was examined through the application of a genome-scale DNA pattern

matching software tool, known as Regulatory Sequence Analysis Tool (RSAT) (Van-Heldin

2003). The RSAT software was used to identify the E2F6-binding elements within promoter

regions (-1000bp to +1bp) of all genes in the entire mouse genome (based on 3 1,1 13 genes in the

Ensembl database) (gray bars, Figure 4-1). A comparison between the meiosis-specific genes









and all genes in the mouse genome indicates that the proximal promoter regions of these

meiosis-specific genes are indeed enriched with E2F6-binding elements.

As an additional measure of enrichment, the probability that an E2F6-binding element

would randomly occur within a nucleotide sequence of a specified length was calculated. This

calculation was made under the assumption that the four nucleotide base pairs (A, G, C, or T)

occur at random and with equal probability at each nucleotide position (Gentleman & Mullin

1989). The resulting value can be considered as the expected rate of random occurrence with

which the E2F6-binding element appears throughout a population of actual DNA sequences, and

is used here as a reference point for statistical significance (black bars, Figure 4-1). A

comparison between this expected rate of random occurrence and the actual frequency of E2F6-

binding element appearance indicates that the E2F6-binding element is specifically enriched

towards the proximal, but not the distal, promoter regions of all genes (gray bars, Figure 4-1).

The rate of occurrence is shown to be appreciably elevated for meiosis-specific genes (white

bars, Figure 4-1). The selectivity of the E2F6-binding element to the proximal promoter, a

genomic region which is known to harbor binding sites of critical transcriptional regulators,

suggests that this E2F6-binding element possesses a high degree of functional significance.

Next, we examined whether E2F6 binds to the E2F6-binding elements found in the

proximal promoter regions of meiosis-specific genes. We performed ChlP analysis using

undifferentiated ES cells and observed endogenous E2f6 binding to several meiosis-specific

genes (Figure 4-2). Figure 4-2A shows E2f6 binding to the promoters of genes which are known

to be derepressed in E2f6 MEFs. To clarify, Tuba3 is one of these derepressed genes but its

expression is not restricted to meiotic cells and was therefore not included in Table 1. Figure 4-

2B suggests that several of the newly identified E2F6-binding elements in the proximal










promoters of meiosis-specific genes (see Table 4-1) are in fact occupied by E2f6. Figure 4-2C

verifies the specificity of E2F6 binding as evidenced by the absence of E2f6 binding in both

housekeeping and ES cell-pluripotency genes. Figure 4-2D is a quantitative analysis from a

sampling of genes in Figures 4-2A, 4-2B, and 4-2C.

Next, we looked to see whether exogenous E2F6 overexpression was capable of

repressing meiosis-specific genes. Using HA-E2F6 and HA-AC-E2F6 ES cells, we demonstrate

that meiosis-specific transcripts are specifically reduced in HA-E2F6 but not in mutant HA-AC-

E2F6 ES cells relative to parental R1 ES cells (Figure 4-3). This observation brought about the

reverse question of whether the absence of E2F6 would result in derepression of additional

meiosis-specific genes beyond that of Stag3, Smclf, Texl2, and Ant4. We examined the

expression status of meiosis-specific genes in E2f6- MEFs for the presence of aberrant gene

expression, but found that these additional meiosis-specific genes remain repressed (Figure 4-4).

Overall, these observations suggest that E2F6 is not required for somatic cell repression of most

meiotic genes and supports the possibility that a functional redundancy exists whereby other

safeguards are in place to ensure their repression in the absence of E2F6.

Discussion

During embryonic development, there is a coordinated regulation of gene expression

whereby genes whose protein products function in the same physiological processes are

concomitantly expressed. Such coordinate regulation is thought to be controlled, at least in part,

by the presence of binding sites for the same transcription factors in the promoter regions of

these genes. Several studies have revealed common transcription factor binding sites in groups

of genes having similar tissue-specific expression patterns or physiological roles (Kel et al. 2001,

Tronche et al. 1997, Wasserman & Fickett 1998). For instance, previous studies have screened

DNA sequences from the EMBL and Genbank data banks and found that liver-specific gene










promoters harbor binding sites for the HNF-1 transcription factor 2.5 times more frequently than

do other genes (Tronche et al. 1997). Likewise, NFAT/AP-1 binding sites are present at rates of

10 times higher in the promoters of immune response genes than in random sequences pulled

from EPD and Genbank databases (Kel et al. 2001). Further, the promoter regions of genes

whose protein products are known to play a role in cell cycle progression were found to have a

high frequency of E2F binding site (the traditional TCGCGC site common to E2Fs 1-5 rather

than the E2F6-preferred TCCCGC site) occurrence in comparison to the promoters of

functionally different genes (Kel et al. 2001). In the present study, our examination of the

promoter regions of a group of 24 meiosis-specific genes revealed a common E2F6 transcription

factor binding site.

Kel et al., defined a set of rules to predict whether a transcription factor binding site could

contribute to the shared pattern of gene expression observed in sets of functionally related genes

(Kel et al. 2001). If a binding site met these criteria, it was termed a "promoter-defining site."

Several features of the common E2F6 binding site investigated in this report suggest that it is

indeed a "promoter-defining site." First, the site occurs in the meiosis-specific gene promoters at

a frequency significantly higher than in random sequences (Figure 4-1). Second, the frequency

of E2F6 binding site occurrence significantly differs between the promoters of genes belonging

to various functional groups. In this case, the occurrence of E2F6 binding sites in the promoters

of the meiosis-specific genes examined in this study was drastically elevated in comparison to

the frequency of binding site occurrence in the promoters of all genes in the mouse genome

(Figure 4-1). Third, the E2F6 binding site is located in close proximity to the transcription

initiation site (Table 4-1). According to Kel et al., in order to be considered a "promoter-

defining site," the candidate site should occur within approximately 300bp upstream of the









transcription initiation site (Kel et al. 2001). The observation that 19 (79.2%) of the 24 meiosis-

specific genes examined here had an E2F6 binding site within 200bp upstream of their

transcription initiation sites is a convincing indicator that this E2F6 site should be considered

"promoter-defining."

One of the fundamental questions raised by our results pertains to why almost every

meiosis-specific gene promoter examined in this study contains an E2F6 binding site which

binds E2f6 in vivo, yet very few of these genes actually require E2F6 for their repression in

somatic cells. This discrepancy does not eliminate the potential role of E2F6 as a broad

repressor of meiosis-specific genes, but rather suggests the existence of a functional redundancy

whereby even in the absence of E2F6, another repressor can compensate. Previous studies have

shown that E2f4 can compensate for the loss of E2f6 by binding to the promoters of Gl/S-

regulated genes (which under normal circumstances are bound by E2f6) in the absence of E2f6

(Giagrande et al. 2004). E2f4-knockdown and E2f6-null MEFs showed no derepression of these

genes individually, but when both E2f4 and E2f6 were simultaneously inhibited using E2f4-

knockdown-E2f6-null MEFs, specific derepression of Gl/S-regulated genes was observed.

Other studies have shown that E2F4 and E2F6 are concurrently bound to the promoters of

numerous genes including: Cyclin A2, Cyclin B2, Cdc2, Art-2 7, Hpla, Rpab48, and Ctip (Caretti

et al. 2003, Oberley et al. 2003). Interestingly, ChlP analysis of the Stag3 promoter shows

binding of E2f6 and the activating E2f3 but not E2f4 in WT MEFs (Storre et al. 2005). Perhaps

this lack of redundancy with E2F4 could explain why Stag3 is aberrantly expressed in E2f6-/-

MEFs. Conversely, Tuba3 and Tuba7 germ-cell-specific genes are also aberrantly expressed in

E2f6-/- MEFs but do in fact bind E2F4 according to affinity chromatography and gel mobility

shift assays (Pohlers et al. 2005). This finding implies that the presence or absence of E2F4









binding is not a sole criterion by which to decipher the occurrence of E2F6 functional

redundancies. It should be noted that the E2F4 binding status of Tuba3 and Tuba7 promoters

was not determined in WT MEFs, leaving open the possibility that the discrepancies in binding

between these promoters and Stag3 could be owed to differences in cell type.

It would be naive to infer that the only transcription factors capable of compensating for

the loss of E2F6 are other E2F family members. Transcriptional regulation is a combinatorial

process whereby each gene is under the control of a multiplicity of elements, some of which can

be dispersed over several kilobases. One example of a redundancy that most likely does not

involve other E2F family members may be Ribc2. Ribc2 is one of the many meiosis-specific

genes that remain repressed in E2f6-/- MEFs (Figure 4-4). This observation is noteworthy given

that Ribc2 and SmcrB genes overlap on chromosome 15 and are transcribed in opposite

orientations with their promoter regions embedded within each other (Arango et al. 2004).

These genes are not only transcribed at similar times during meiosis, but they share an E2F6-

binding element in their overlapping promoter region which we have shown binds E2f6 in vivo

(shown as Smclp, Figure 4-2). There are only 261bp of sequence in between the transcription

initiation sites of Ribc2 and SmcrB. Within this narrow region, the shared E2F6 site is located at

-175bp to -170bp relative to Ribc2 and -47bp to -42bp relative to SmcrB transcription initiation

sites. An explanation regarding why SmcrB is derepressed in E2f6-/- MEFs but not Ribc2 could

be partially due to a distance effect whereby the proximity of the E2F6-binding element to the

transcription initiation site could determine the essentialness of E2F6 binding. Alternatively,

DNA elements existing within introns and 3' regions specific to each gene may be accountable

for the differential regulation. Although, the mechanisms responsible for this distinction in gene










regulation remain unclear, a redundancy of an E2F family member at the location of this shared

E2F6 binding site would most likely not account for such a phenomenon.

The discovery that only a handful of meiosis-specific genes were aberrantly expressed in

somatic cells in the absence of E2F6 is in agreement with previous studies. Prior reports have

described cDNA microarray experiments with mRNA from WT and E2f6-/- MEFs and shown

that very few germ-cell-specific genes were upregulated in E2f6-/- MEFs (Pohlers et al. 2005,

Storre et al. 2005). Further, our findings are consistent with the mild phenotype observed in

E2f6-/- mice. Although these mice display homeotic transformations of the axial skeleton, they

are viable, fertile, and display no signs of tumor formation (Storre et al. 2002). A higher

incidence of abnormally regulated meiosis-specific genes would likely result in a more severe

phenotype. Evidence supporting this speculation is seen by the aberrant expression of germline

genes in cancers whereby expression reflects the acquisition of the silenced gametogenic

program in somatic cells (Simpson et al. 2005). This acquisition is thought to be one of the

driving forces in tumorigenesis. The proteins that become aberrantly expressed as a result of this

phenomenon are termed cancer/testis antigens. Two examples of known cancer/testis antigens

which have meiosis-specific expression in normal tissues are Spoll and Sycpl (Simpson et al.

2005, Tureci et al. 1998). They are both components of the synaptonemal protein complex but

have different functions. Spol l causes double-stranded-breaks which are thought to initiate

recombination events during meiosis (Keeney et al. 1997). Sycp l makes up the transverse

filaments of the synaptonemal complex (Pousette et al. 1997). It is theorized that aberrant

expression of either of these meiosis-specific proteins in mitotic cells leads to abnormal

chromosome segregation and aneuploidy, the hallmarks of cancer cells (Simpson et al. 2005).

The finding that overexpression of Sycpl1 in COS cells leads to the formation of synaptonemal










complexes supports this theory (Ollinger et al. 2005). In agreement with the absence of tumor

formation in the E2f6-/- mice; we find here that although E2F6 binds to the promoters of both

Spoll and Sycpl (Figure 4-2), they remain repressed in E2f6-/- MEFs (Figure 4-4).

Even though very few meiosis-specific genes examined in this study require E2F6 for

their repression in somatic cells, all of the genes analyzed here were at least partially repressed

by E2F6 overexpression (Figure 4-3). The obvious question raised by this observation concerns

the mechanisms by which E2F6-mediated repression occurs. As discussed in Chapter 1, there

are several ways that E2F6 can repress its target genes. Whether E2F6-mediated repression

occurs via the same mechanism for all the genes we analyzed or whether each individual gene

utilizes a different mode of E2F6-induced repression remains to be determined. In particular, it

will be important to distinguish whether those genes which are derepressed in E2f6-/- IVEFs

utilize a different mechanism of E2F6-mediated repression than those which remain repressed.

We predict that only those genes which are derepressed in E2f6-/-1VIEFs will have repression

mechanisms similar to those found for Ant4 (discussed in Chapter 3). Further studies analyzing

the methylation status as well as polycomb protein occupancy at these meiosis-specific gene

promoters are needed before such conclusions can be drawn. Another interesting point is that the

repression of Stag3 and Smclf can be restored in E2f6-/- IVEFs reconstituted with E2F6 (Storre

et al. 2005). This observation brings into question, the role that E2F6 plays in initiating somatic

cell repression versus maintaining repression.

Overall, the present study has provided evidence suggesting that E2F6 may play a broad

role in the repression of meiosis-specific genes. However, this role is most likely masked in vivo

by the existence of functional redundancies where other repressive mechanisms are in place to

ensure the repression of most meiosis-specific genes in somatic cells. Given the catastrophic










outcome that can result from aberrant expression of meiosis-specific genes (Sagata 1997,

Simpson et al. 2005); it is not surprising that organisms have developed safeguards to guarantee

their repression.












Table 4-1. Locations of E2F6 TFBS (TCCCGC) within upstream promoter regions of meiosis-
specific genes relative to their transcription initiation sites

Gene Name Upstream Promoter Regions in Basepairs (bp) References

+1 to -100 -100to -200 -200to-300 -300to -400 -400to -500
Ant4 (S/c25a31) i (Brower et al 2007)
Boule* (Eberhart et al 1996, Xu et al 2001)
Cyclin Al (Raynick & Wolgemuth 1999, Sweeney et al 1996)
Dmc1 (Liml5h) i i (Habu et al 1996)
Lamin C2* (Alshelmer et al 1996)
Meg1 (Calmegin) i (Don & Wolgemuth 1992, Watanabe et al 1994)
Mnd1 i (Petukhova et al 2005, Pezza et al 2006)
Mns1 i (Furukawa et al 1994)
Mzf~d i (Looman et al 2003)
Prdm9(Meisetz)* i (Hayashl et al 2005)
Psmc3ip (Hop2)* i (Petukhova et al 2005, Pezza et al 2006)
Rec8 i (Lee et al 2003)
RecQL (isoform beta) i (Wang et al 1998)
Ribc2 (Tnb) i (Arango et al 2004)
Smclp i (Renekova et al 2001)
Spoll i (Shannonetal 1999)
Stag3 i (Pezzi et al 2000, Prleto et al 2001)
Sycel (Costa et al 2005)
Syce2 (Cesc1) i (Costa et al 2005)
Sycpl (Scpl) i (Meuwissen et al 1992, Yuan et al 1996)
Sycp2 (Offenberg et al 1998)
Sycp3 i (DI Carlo et al 2000, Lammers et al 1994)
Tcte2* i i (Braldotti et al 1997)
Tex12 i (Hamer et al 2006)

*These genes have an E2F6 TFBS within their 5' UTRs
Ij Denotes the presence of an E2F6 TFBS within the upstream region of the corresponding to the column











83.3%


83.3%


58.3%


80 -

b: 70



S50 -


a. 30 -
~ 0-


20

-


"100bp


200bp 500bp
Basepairs (bp) Upstream


1000bp


Figure 4-1. Frequency of appearance of E2F6 TFB S within upstream regions of genes relative to
their transcription initiation sites. Bar graph indicates the occurrence rate of E2F6
TFB S for the following categories: Random: the probability that an E2F6 TFBS
would randomly appear at least once within a sequence of a given length as described
in Methods, RSAT: the actual frequency that the E2F6 TFBS occurs at least once
within the proximal promoter regions of genes (3 1,1 13 genes) in the mouse genome,
Meiosis-specific: the actual frequency that the E2F6 TFBS occurs at least once within
a population of meiosis-specific genes (those listed in Table 1). The X-axis indicates
the location of the E2F6 TFB S to be within 100bp, 200bp, 500bp, or 1000bp
upstream of the transcription initiation site; the Y-axis denotes the percentage of
genes from each category (see figure legend) that have at least one E2F6 TFB S within
a given upstream region.


90


M Random
O RSAT
O Meiosis-specific











A B C


Ant4 D c a a



Smc8 ~1Sporl f 8-CI

Tuba3









D g0200





Ant4 Dmc1 Actin

Figure 4-2. E2f6 binds to the promoters of meiosis-specific genes. Chromatin
immunoprecipitation (ChIP) of endogenous E2f6 binding activity in WT R1 ES cells.
ChlP was performed using mouse-anti-E2f6 antibody and analyzed by semi-
quantitative PCR. Input and IgG are shown as controls. E2f6 binding activity on: A)
the promoters of meiotic genes which are known to be derepressed in E2f6-/ -1VEFs,
B) the promoters of other meiosis-specific genes, and C) the promoters of non-
meiotic ES cell pluripotency and f-actin genes. D) Real-time PCR quantification of
binding as determined by the ratio of specific ChlP/IgG ChlP relative to input. All
samples were tested in triplicate and expressed as means + standard deviations.





















Rec8L

RecQf


s
u;
~
~
o- k


n4

S a3

Smc1/3

Tuba3


Oct4
H Ant4
O Sycpl


1 --


1.2

1 -

e 0.8-

-a 0.6-

0.4-

0.2-

0-


T ,


DeltaC ES


R1 ES


Figure 4-3. Many meiosis-specific genes are repressed by E2F6. Semi-quantitative RT-PCR
analysis of meiosis-specific gene expression in R1, HA-E2F6, and HA-AC-E2F6 ES
cells. A) Genes derepressed in E2f6- MEFs. B) Other meiosis-specific genes. C)
Non-meiotic ES cell pluripotency and f3-actin genes. D) Quantitative Real-Time PCR
analysis of a representative group of samples from A), B), and C). All samples were
tested in triplicate and expressed as means + standard deviations.


Nanog ,



73-actin-- -r


HAE2F6 ES












~c?;;~:k~~t~tG~~i~~


~oc~


~L"
~V~:


Sycpl

Spo11
Rec8L

Ribc2

RecQ1


E2fB
An t4

Stsg3

smcal

Turba3


Nanog

Oct4

13-actin


r T


~CCII


R1 ES WVT M EF E2F6-1- MEF


Figure 4-4. Limited meiosis-specific genes are derepressed in E2f6- MEFs. Semi-quantitative
RT-PCR analysis of meiosis-specific gene expression in WT testis from 6-week old
mice, R1 ES cells, WT MEFs, and E2f6- MEFs. A) E2f6 and genes showing
derepression in E2f6- MEFs. B) Other meiosis-specific genes. C) Non-meiotic ES
cell pluripotency genes and f3-actin genes. D) Quantitative Real-Time PCR analysis
of a representative group of samples from A), B), and C). All samples were tested in
triplicate and expressed as means + standard deviations.


E2f6
H Ant4
O Dmc1
0 Nanog









CHAPTER 5
THE ROLE OF E2F6 DURING MALE MEIOSIS AND SPERMATOGENESIS

Motivation

From the data presented in Chapters 3 and 4, we can infer that E2F6 is involved in the

repression of several meiosis-specific genes in somatic tissues. One of the remaining questions

pertains to whether E2F6 also plays a role in regulating meiosis-specific gene expression in non-

somatic tissues. During male germ cell development, there is a rapid transition in the expression

of meiosis-specific genes from a repressed state in pre-meiotic spermatogonia, to a highly active

state in meiotic spermatocytes, to a restored repressive state in post-meiotic round spermatids.

Therefore, we have analyzed the expression pattern of E2F6 in mouse testis with the expectation

of Ending that, in agreement with E2F6's role as a transcriptional repressor, there would be high

E2F6 expression in spermatogonia, followed by low expression in spermatocytes, and then high

expression again in spermatids. Surprisingly, the data presented here contradicts our prediction.

Such results have convinced us to explore the possibility that during meiosis, E2F6 may instead

be serving as a transcriptional activator of meiosis-specific genes. Herein, we discuss what is

currently known about meiosis-specific gene activation in male germ cells, as well as speculate

how our new findings on E2F6 expression may fit into these already established paradigms.

Background

There are several ways by which meiosis-specific genes may become activated at the

onset of meiosis. These include: 1) activation by testis-specific transcription factors, 2)

activation by general transcription factors, 3) derepression by removal of repressing factors, and

4) epigenetic alterations in chromatin structure which then promote transcriptional activation.

One example of a testis-specific transcription factor that may activate meiotic genes is A-Myb.

The murine A-myb gene arises from alternatively spliced mRNA and is expressed predominantly









in the testis (Latham et al. 1996). In situ hybridization analysis of mouse testis shows that A-myb

expression increases at post-natal day 10, when primary spermatocytes first appear. In the adult,

A-myb mRNA is highly expressed in a sub-population of spermatogonia and in primary

spermatocytes, but is not detectable in spermatids. This expression pattern suggests that A-myb

may play a role in activating meiosis-specific gene expression. Additionally, in A-myb-/- male

mice, germ cells enter meiotic prophase and arrest at pachytene, confirming that A-myb is

required for the proper progression of meiosis (Toscani et al. 1997). Another myb family

member, B-myb, is not testis-specific but is highly expressed in spermatogonia and early

spermatocytes (Latham et al. 1996). B-myb expression then decreases by the late pachytene

spermatocyte stage. Further studies comparing the differences between protein occupancy at

meiosis-specific gene promoters incubated with either testis or somatic tissue nuclear extracts

may reveal additional testis-specific activator proteins.

Meiosis-specific genes have also been shown to be activated by general transcription

factors which are ubiquitously expressed. For example, the widely expressed Spl protein

activates transcription of the meiosis-specific gene, Cyclin Al (Bartusel et al. 2005). Cyclin Al

contains Spl binding sites in its promoter region and it is suggested that Spl's ability to activate

Cyclin Al is a result of Spl1 interactions with B-myb. Therefore, it is speculated that Spl1 and B-

myb are binding together as an "activation complex" at these Spl sites. Mutation of the Spl

binding sites prevents Cyclin Al transcription. Likewise, mutation of the C-terminal domain of

B-myb inhibits Cyclin Al activation, suggesting that this domain is required for B-myb

interaction with Spl.

Spl has also been shown to serve as a transcriptional repressor, but depending on the

factors with which it associates, or alternatively, depending upon how it is spliced, Spl may









switch between being an activator and being a repressor (Thomas et at. 2007). Additionally, Spl

has been found to associate with E2F family members. When these two proteins associate, they

too serve as an "activation complex." Such is the case at the murine thymidine kinase gene

promoter (Rotheneder et at. 1999). Spl also interacts with the pl07 pocket protein suggesting

that Spl may change roles during the cell cycle (Datta et at. 1995). Other studies indicate that

Spl can interact with components of the general transcription machinery including TBP-

associated factors (TAFs). The TAFs associate with histone modifying enzymes and chromatin

remodeling factors to establish open chromatin during spermatogenesis (Thomas et at. 2007).

An alternative way in which meiosis-specific gene transcription may be activated, is

through the removal of repressive factors which reside at the promoters of these meiosis-specific

genes. For example, polycomb proteins have been shown to repress genes which are involved in

promoting differentiation. Interestingly, a recent paper using Drosophila as a model organism

showed that these polycomb proteins are removed from gene promoters at the onset of meiosis

(Chen et at. 2005). Five testis-specific TAFs (tTAFs), which associate with the TBP portion of

the general transcription factor TFIID and have been shown to be required for meiotic cell cycle

progression, are responsible for counteracting these polycomb proteins. Chen and colleagues

found that these tTAF proteins are concentrated in a particular subcompartment of the nucleolus.

They then become expressed at the initiation of spermatocyte differentiation and persist

throughout the remainder of the primary spermatocyte stage, disappearing as cells enter the first

meiotic division. This tTAF expression coincides with both the localization of PRC 1

components to the nucleolus and the activation of tTAF-target genes. This is in agreement with

the finding that polycomb proteins are strongly expressed in all cells of the testis until the onset

of meiosis (Ringrose et at 2006).









Further, mutation of tTAFs results in the inability of PRC 1 components to localize to the

nucleolus, suggesting that tTAFs may be involved in the recruitment of PRC 1 components away

from target promoters and towards the nucleolus (Chen et at. 2005). ChlP analysis revealed that

these tTAFs also bound to target promoters just upstream of their transcription start sites,

reduced polycomb binding, and promoted local accumulation of H3K4me3, a mark of Trithorax

action. Trithorax proteins have been shown to activate transcription by opposing the repressive

action of polycomb proteins (Ringrose et at. 2006). In tTAF mutant testis, PRC1 components

continued to bind to tTAF-dependent target genes. These findings suggest that tTAFs activate

germ-cell-specific gene expression by counteracting repression by polycomb proteins. Such

transcriptional derepression by sequestration of polycomb proteins has also been observed during

HIV-1 infection when the viral Nef protein recruits the PRC2 component, Eed, to the plasma

membrane (Witte et at. 2004). From these findings, it appears that polycomb proteins may be

blocking the expression of meiosis-specific genes. Upon entry into meiosis, polycomb

repressive complexes are then disabled through tTAFs and these meiosis-specific genes become

expressed. Whether the interactions between tTAFs and polycomb proteins are direct or indirect,

remains to be elucidated.

Meiosis-specific gene transcription may also be activated during meiosis as a result of

reorganization in chromatin structure towards a conformation that is more accessible to RNA

polymerase II. This restructuring often involves a switch from somatic isoforms to unique germ-

cell-specific isoforms of various histones and histone modifying enzymes. For example, during

male gametogenesis several of the core histones (H2A, H2B, H13, and H14), along with linker

histone H1, are partially or completely replaced by testis-specific histone isoforms such as Hit,

TH2A, TH2B, and H3t (DeJong et al. 2006). These germ-cell-specific histones appear in










spermatogonia, and later in spermatids, but the maj ority are synthesized and incorporated into

chromatin during meiosis. Methylation of somatic histone H1 by the H3K27/H1K26 HMT and

polycomb protein, Ezh2, seems to be important for transcriptional repression. However, testis

H1 variants have glycine or alanine residues in place of lysine which makes them unlikely to be

targeted by Ezh2, and suggests that testis-specific H1 variants may represent a more active

chromatin status than their somatic counterparts.

In addition to unique histone isoforms, there are also differences in histone

methyltransferases (HMTs) between somatic cells and germ cells. Many of these HMTs have

roles in transcriptional repression, but one in particular may aid in the activation of meiosis-

specific genes. This meiosis-specific HMT, known as Meisetz (Prdm9), trimethylates histone

H3K4. H3K4 trimethylation has been shown to be associated with active chromatin. Meisetz is

specifically expressed in early meiotic germ cells in both testis and ovary (Matsui & Hayashi

2007). Interestingly, there is an E2F6 binding site in the proximal promoter region of this HMT

(see Table 4-1).

It is suggested that Meisetz may trimethylate H3K4 around a set of genes essential for

meiosis, thereby activating their expression. A role for Meisetz in activating meiosis-specific

genes is observed in M~eisetz null mice which have specific abnormalities in meiotic prophase

and fail to activate meiosis-specific genes (Hayashi et al. 2005). Also, under normal conditions

H3K4 trimethylation is upregulated in pachytene spermatocytes, further supporting the

involvement of2~eisetz in transcriptional activation. It should be noted here that although DNA

demethylation may be an additional mechanism whereby meiosis-specific genes are derepressed

at the onset of meiosis, it was not discussed here because to date, no DNA demethylases have

been identified.









Results

After demonstrating that E2F6 indeed plays a role in the repression of meiosis-specific

genes, we were curious to see if the expression pattern of E2F6 in the testis could help to explain

our Eindings. As mentioned in the motivation portion of this chapter, we postulated that E2F6

expression would be high in spermatogonia and then suddenly drop off upon entry into meiosis,

thereby releasing the repression of meiosis-specific genes. At the completion of meiosis, we

predicted that E2F6 expression levels would peak again in order to silence the expression of

meiosis-specific genes in post-meiotic cells such as round spermatids and sperm. We also

predicted that the activating E2F, E2F l, would increase upon entry into meiosis in order to

activate meiosis-specific gene expression.

Surprisingly, when we used real-time PCR to examine the expression patterns of both

E2fl and E2f6 in purified spermatogenic cells harvested at various stages of spermatogenesis

(cells were a kind gift from JR McCarrey), we found expression patterns which were almost

completely opposite to what we had predicted (Figure 5-1). According to these data, E2f6

expression was highest in those cell types which were undergoing meiosis whereas E2fl

expression was drastically reduced at this stage. Additionally, both E2fl and E2f6 expression

was low in the somatic population of cells in the testis (sertoli cells). Further confirmation of

this expression pattern came from the analysis of E2f6 protein levels in testis from 6 week old

mice using immunohistochemistry (Figure 5-2). Staining revealed that E2f6 expression was

highest in meiotic cells. The classification of cells as meiotic was determined by cellular

morphology and cellular location within the seminiferous tubules. Staining appeared specific

when compared to staining using IgG as a negative control. Further immunohistochemistry will

be needed to confirm these results.









Discussion

There has only been one published study which describes the expression pattern of E2F

family members in the testis (El-Darwish et al. 2006). In this report it was found that the E2F 1

protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of

mice while strong staining of E2F 1 in some cells close to the basal lamina of rat tubules suggest

that it may also be expressed in undifferentiated spermatogonia. These findings are in contrast to

the expression pattern that we observed for E2Fl1. Given the inherent nature of high

backgrounds during immunohistochemical staining, we are tempted to trust our data more as we

have quantitatively shown mRNA expression rather than subj ectively characterizing expression.

Further, this study showed discrepancies in E2F 1 expression patterns between mouse and rat

which seems highly unlikely considering the high homology of E2F 1 between the two species.

Additionally, this study did not examine E2F6 expression in testis. Therefore, for the remainder

of this discussion, we will assume that the expression patterns found from our research are

correct.

One possible explanation for this unexpected expression pattern is that E2F6 has dual

roles as both an activator and a repressor during different stages of the cell cycle. These roles

may be determined by differences in the complexes by which E2F6 is bound. For instance, if

E2F6 represses transcription by recruiting either Dnmts or polycomb protein complexes to the

promoter, then the actual repression activity is coming from the proteins which were recruited by

E2F6 rather than from E2F6 itself. Therefore, it is possible that in meiotic cells, E2F6 maybe

recruiting an activation complex to meiosis-specific genes rather than a repressive complex.

Such an activation complex could consist of components which have already been shown to play

a role in meiotic gene activation, such as Myb, Spl, Meisetz, and tTAFs. Relationships between

these proteins and E2Fs have already been suggested but need to be further elucidated.









An explanation for why such a switch in E2F-associated complexes could occur might be

that the activation complex is suddenly able to compete with the repressive complex for binding

at the onset of meiosis. For instance, if the total level of activation complex was upregulated

upon entry into meiosis whereas the repressive complex was downregulated, then this shift in

total protein could be responsible for the switch in E2F6-associated complexes. Further, a

sudden change in cellular localization of activating and repressing components could also

explain the switch in complexes. Interestingly, both a shift in expression patterns and cellular

localization was observed for tTAFs and polycomb proteins at the onset of meiosis (Chen et al.

2005).

An additional function for the upregulation of E2F6 during meiosis could be that E2F6 is

required to repress Gl/S genes from becoming activated during this time. Likewise, E2F 1 may

be downregulated here as it is an activator of genes which promote S phase entry. Therefore,

rather than thinking "narrow-mindedly" about only meiosis-specific gene populations, it is

plausible that there are a multitude of E2Fl1- and E2F6-target genes which need to be regulated

during meiosis. Indeed, experiments in which E2F1 was conditionally overexpressed in mouse

testis resulted in testicular atrophy with seminiferous tubules containing only sertoli cells and

clusters of undifferentiated spermatogonia (Agger et al. 2005). Likewise, E2f6- mice displayed

a lack of mature spermatocytes (Storre et al 2002). These findings suggest that during meiosis,

it is important to keep E2F 1 off and E2F6 on in order to ensure proper completion of meiosis by

preventing the cell from attempting to initiate a cell cycle program in the middle of meiosis. It is

also possible that the role of E2F6, to inhibit Gl/S-regulated genes, may not be as important as

the role of E2F l, pending that E2F 1 is never able to activate these Gl/S regulated genes in the









first place. This would explain why the E2f6-/- mouse is still fertile but not the mice which

overexpress E2Fli.

In summary, E2F6 may play a dual role as both an activator and a repressor during

meiosis. E2F6-containing activation complexes may be bound to meiosis-specific genes while

coexisting repressive E2F6 complexes might be bound to nonmeiotic gene promoters, thereby

preventing the disruption of meiosis. Alternatively, E2F6 may only serve as a repressor, and

through some unknown mechanism, meiosis-specific gene promoters are protected from the

binding of such repressive E2F6 complexes. Future studies using ChlP assays to analyze the

binding of E2F6 to both meiosis-specific genes and Gl/S target genes during meiosis would

clarify this hypothesis.














1.4
1.2 -g E F


.g 0.8 E2F6

'E 0.6
**0.4



pTA TAS TBS PLS LZS JPS APS RS RCB JSC


Figure 5-1. E2fl and E2f6 mRNA transcript levels in the testis. Real-time PCR was used to
measure E2fl (black) and E2f6 (pink) transcript levels in the following purified
spermatogenic cell types: pTA, primitive Type A spermatogonia; TAS, Type A
spermatogonia; TBS, Type B spermatogonia; PLS, Pre-leptotene spermatocytes; LZS,
Leptotene/Zygotene spermatocytes; JPS, Juvenille pachytene spermatocytes; APS,
Adult pachytene spermatocytes; RS, Round Spermatids; RCB, Residual Cytoplasmic
Bodies; JSC, Juvenile (D6) Sertoli Cells. The relative transcript levels are shown with
the transcript level of pTA for E2fl and E2f6 set as 1. All samples were tested in
triplicate and expressed as means + standard deviations.










































Figure 5-2. Immunohistochemical analysis of E2f6 expression in mouse testis. Formalin-fixed,
paraffin-embedded sections of mouse testis from wild type 6-week old mice were
incubated with (A) and B)) IgG antibody as a control or (C) and D)) E2f6 antibody.
Staining was visualized using DAB (brown), and slides were counterstained with
hematoxylin. A) and C) images were taken at a 10X magnification and B) and D)
were at 40X.









CHAPTER 6
CONCLUSIONS AND SIGNIFICANCE

In summary, the research presented here has demonstrated a role for E2F6 in the

repression of meiosis-specific genes in somatic cells. Although many questions remain, we have

successfully achieved the original goal of this research: to study the transcriptional regulation of

gene expression. We have elucidated the relationship between a particular transcription factor

(E2F6) and a specific set of genes (meiosis-specific genes). When transcription factors

controlling gene expression are disrupted, dire consequences may result. Therefore, the

significance of these findings resides with the common observation that many genes are often

aberrantly expressed in cancer cells.

Here we demonstrate that E2F6 depletion results in the expression of a few E2F6-target

genes in inappropriate tissues. Such mischievous expression is a classic example of an event

which can lead to oncogenic transformation (Simpson et al. 2005). Also, the E2F family of

transcription factors is highly involved in the regulation of the cell cycle and one of its most

common binding partners, retinoblastoma, is a pocket protein which has consistently been

demonstrated to play a role in oncogenic transformation (Weinberg 1992). Because E2F6 does

not bind to pocket proteins, it is commonly excluded when review papers discuss the role of

E2Fs and Rb pocket proteins in cancer. Further, this is to be expected given that there was no

indication of tumor formation in the E2f6- mouse (Storre et al. 2002). However, one thing that

may be being overlooked is the potential for E2F6 to serve a negative feedback role in keeping

the expression of genes which are targets of the activating E2Fs in check. Such an important role

for E2F6 may simply be being masked by functional redundancy with E2F4. Although both of

these proteins may be involved in negative feedback, their importance is somewhat diminished

by the fact that they "share" the job. Therefore, we believe that future experiments which









examine the relationship between E2F4, E2F6, and cancer will provide critical insights regarding

the control of E2F-target genes. Taken together, these findings are a small but important

contribution to a broader understanding of the body's finite control of gene expression.









LIST OF REFERENCES


Adams MD 2005 Conserved sequences and the evolution of gene regulatory signals. Current
Opinion in Genetics and Development 15 625-633.

Agger K, Santoni-Rugiu ES, Holmberg C, Karlstrom O & Helin K 2005 Conditional E2F 1
activation in transgenic mice causes testicular atrophy and dysplasia mimicking human CIS.
Oncogene 24 780-789.

Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Walter P 2002 Molecular Biology of the
Cell, 4th edition. New York: Garland Science.

Alsheimer M & Benavente R 1996 Change of karyoskeleton during mammalian
spermatogenesis: expression pattern of nuclear Lamin C2 and its regulation. Experimental Cell
Research 228 181-188.

Arango NA, Pearson EJ, Donahoe PK & Teixeira J 2004 Genomic structure and
expression analysis of the mouse testis-specific ribbon protein (Trib) gene. Gene 343 221-227.

Attwooll C, Oddi S, Cartwright P, Prosperini E, Agger K, Steensgaard P, Wagener C, Sardet
C, Moroni MC & Helin K 2005 A novel repressive E2F6 complex containing the polycomb
group protein, EPC1i, that interacts with EZH2 in a proliferation-specific manner. Journal of
Biological Chemistry 280 1199-1208.

Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL,
Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek
CA, Meade HM, Godke RA, Gavin WG, Overstrom EW & Echelard Y 1999 Production of
goats by somatic cell nuclear transfer. Nature Biotechnology 17 456-461.

Barath P, Polinkova D, Luciakova K & Nelson BD 2004 Identification of NF 1 as a silencer
protein of the human adenine nucleotide translocase-2 gene. European Journal ofBiochemistry
271 1781-1788.

Bartell JG, Davis T, Kremer EJ, Dewey MJ & Kistler WS 1996 Expression of the rat testis-
specific Histone Hit gene in transgenic mice. Journal of Biological Chemistry 271 4046-4054.

Bartusel T, Schubert S & Klempnauer K 2005 Regulation of the cyclin Dl and cyclin Al
promoters by B-Myb is mediated by Spl binding sites. Gene 351 171-180.

Braidotti G & Barlow DP 1997 Identification of a male meiosis-specific gene, Tcte2, which is
differentially spliced in species that form sterile hybrids with laboratory mice and deleted in t
chromosomes showing meiotic drive. DevelopmentalBiology 186 85-99.

Brower JV, Rodic N, Seki T, Jorgensen M, Fliess N, Yachnis AT, McCarrey JR, Oh SP &
Terada N. 2007 Evolutionarily conserved mammalian adenine nucleotide translocase 4 is
essential for spermatogeneis. Journal of Biological Chemistry 282 29658-29666.









Bruin A, Maiti B, Jakoi L, Timmers C, Buerki R & Leone G. 2003 Identification and
characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular
proliferation. Journal of Biological Chemistry 278 42041-42049.

Campbell KHS, Ritchie WA, McWhir J & Wilmut I 1996 Sheep cloned by nuclear transfer from
a cultured cell line. Nature 380 64-66.

Caretti G, Salsi V, Vecchi C, Imbriano C & Mantovani R. 2003 Dynamic recruitment of NF-Y
and histone acetyltransferases on cell-cycle promoters. Journal of Biological Chemistry 278
30435-30440.

Cartwright P, Miiller H, Wagener C, Holm K & Helin K 1998 E2F-6: a novel member of the
E2F family is an inhibitor of E2F-dependent transcription. Oncogene 17 61 1-623.

Chen X, Hiller M, Sancak Y & Fuller MT 2005 Tissue-specific TAFs counteract polycomb to
turn on terminal differentiation. Science 310 869-872.

Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L & Renard JP 2002 Cloned rabbits
produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20 366-369.

Chevrollier A, Loiseau D, Chabi B, Renier G, Douay O, Malthiery Y & Stepien G 2005 ANT2
isoform required for cancer cell glycolysis. Journal ofBioenergetics and'Biomembranes 37 307-
317.

Chung AB, Stepien G, Haraguchi Y, Li K & Wallace D 1992 Transcriptional control of nuclear
genes for the mitochondrial muscle ADP/ATP translocator and the ATP Synthase P Subunit.
Journal of Biological Chemistry 267 21154-21161.

Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce dLn & Robl JM 1998
Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280 1256-1258.

Cloud JE, Rogers C, Reza TL, Ziebold U, Stone JR, Picard MH, Caron AM, Bronson RT &
Lees JA 2002 Mutant mouse models reveal the relative roles of E2F 1 and E2F3 in vivo.
Molecular and' Cellular Biology 22 2663-2672.

Costa Y, Speed R, Ollinger R, Alsheimer M, Semple CA, Gautier P, Maratou K, Novak I,
HS5g C, Benavente R & Cooke H 2005 Two novel proteins recruited by synaptonemal
complex protein 1 (SYCP l) are at the centre of meiosis. Journal of Cell Science 118 2755-2762.

Dahout-Gonzalez C, Nury H, Trezeguet V, Lauquin GJM, Pebay-Paeyroula E & Brandolin G
2006 Molecular, functional, and pathological aspects of the mitochondrial ADP/ATP carrier.
Physiology 21 242-249.

Datta PK, Raychaudhuri P & Bagehi S 1995 Association of pl07 with Spl: genetically separable
regions of pl07 are involved in regulation of E2F- and Spl- dependent transcription. Molecular
and' Cellular Biology 15 5444-5452.










Dejong J 2006 Basic mechanisms for the control of germ cell gene expression. Gene 366 39-50.

DeGregori J & Johnson DG 2006 Distinct and overlapping roles for E2F family members in
transcription, proliferation, and apoptosis. Current Molecular M~edicine 6 739-748.

DiCarlo AD, Travia G & Felici MD 2000 The meiotic specific synaptonemal complex protein
SCP3 is expressed by female and male primordial germ cells of the mouse embryo. International
Journal of Developmental Biology 44 241-244.

Dimova DK & Dyson NJ 2005 The E2F transcriptional network: old acquaintances with new
faces. Oncogene 24 2810-2826.

Dolce V, Scarcia P, lacopetta D & Palmieri F 2005 A fourth ADP/ATP carrier isoform in man:
identification, bacterial expression, functional characterization, and tissue distribution. FEBS
Letters 579 633-637.

Don J & Wolgemuth DJ 1992 Identifieation and characterization of the regulated pattern of
expression of a novel mouse gene, meg1, during the meiotic cell cycle. Cell G; 1,n thr and
Differentiation 3 495-505.

Dorner A, Giessen S, Gaub R, Grosse Siestrup H, Schwimmbeck PL, Hetzer R, Poller W &
Schultheiss HP 2006 An isoform shift in the cardiac adenine nucleotide translocase expression
alters the kinetic properties of the carrier in dilated cardiomyopathy. European Journal ofHeart
Failure 8 8 1-89.

Eberhart CG, Maines JZ & Wasserman SA 1996 Meiotic cell cycle requirement for a fly
homologue of human Deleted in Azoospermia. Nature 381 783-785.

Eddy EM 2002 Male germ cell gene expression. Recent Progress in Hormone Research 57 103-
128.

El-Darwish KS, Parvinen M & Toppari J 2006 Differential expression of members of the E2F
family of transcription factors in rodent testes. Reproductive Biology and Endocrinology 4 63-85.

Ellison JW, Salido EC & Shapiro LJ 1996 Genetic mapping of the adenine nucleotide
translocase-2 gene (Ant2) to the mouse proximal X chromosome. Genomics 36 369-371.

Field SJ, Fong-Ying T, Kuo F, Zubiaga AM, Kaelin WG, Livingston DM, Orkin SH &
Greenberg ME 1996 E2F-1 functions in mice to promote apoptosis and suppress proliferation.
Cell 85 549-561.

Furukawa K, Inagaki H, Naruge T, Tabata S, Tomida T, Yamaguchi A, Yoshikuni M,
Nagahami Y & Hotta Y 1994 cDNA cloning and functional characterization of a meiosis-
specific protein (MNS1) with apparent nuclear association. Chromosome Research 2 99-113.









Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R & Lazzari G 2003
Pregnancy: a cloned horse born to its dam twin. Nature 424 635.

Gaubatz S, Lindeman GJ, Ishida S, Jakoi L, Nevins JR, Livingston DM & Rempel RE 2000
E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Molecular Cell 6
729-735.

Gaubatz S, Wood JG & Livingston DM 1998 Unusual proliferation arrest and transcriptional
control properties of a newly discovered E2F family member, E2F6. Proceedings of the National
Academy ofSciences 95 9190-9195.

Gebara MM & McCarrey JR 1992 Protein-DNA interactions associated with the onset of testis-
specific expression of the mammalian Pgk-2 gene. Molecular and Cellular Biology 12 1422-
1431.

Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K & Daley GQ 2004 Derivation of
embryonic germ cells and male gametes from embryonic stem cells. Nature 427 148-154.

Gentleman JF & Mullin RC 1989 The distribution of the frequency of occurrence of nucleotide
sub sequences, based on their overlap capability. Biometrics 45 35-52.

Giangrande PH, Zhu W, Schlisio S, Sun X, Mori S, Gaubatz S & Nevins JR 2004 A role for
E2F6 in distinguishing Gl/S- and G2/M-specific transcription. Genes and Development 18 2941-
2951.

Graham BH, Waymire KG, Cottrell B, Trounce IA, MacGregor GR & Wallace DC 1997 A
mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the
heart/muscle isoform of the adenine nucleotide translocator. Nature Genetics 16 226-234.

Gurdon JB 1962 Adult frogs derived from the nuclei of single somatic cells. Developmental
Biology 4 256-273.

Habu T, Takuyu T, West A, Nishimune Y & Morita T 1996 The mouse and human homologs of
DMC1i, the yeast meiosis-specific homologous recombination gene, have a common unique form
of exon-skipped transcript in meiosis. Nucleic Acids Research 24 470-477.

Hamer G, Gell K, Kouznetsova A, Novak I, Benavente R & HS5g C 2006 Characterization of a
novel meiosis-specific protein within the central element of the synaptonemal complex. Journal
of Cell Science 119 4025-4032.

Han SY, Xie W, Kim SH, Yue L & DeJong J 2004 A short core promoter drives expression of the
ALF transcription factor in reproductive tissues of male and female mice. Biology of
Reproduction 71 933-941.

Hayashi K, Yoshida K & Matsui Y 2005 A histone H13 methyltransferase controls epigenetic
events required for meiotic prophase. Nature 438 374-378.









Hecht NB, Distel RJ, Yelick PC, Tanhauser SM, Driscoll CE, Goldberg E & Tung KSK 1988
Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in
brain. Molecular and Cellular Biology 8 966-1000.

Hirano M & DiMauro S 2001 ANT1, Twinkle, POLG, and TP: new genes open our eyes to
opthalmoplegia. Neurology 57 2163-2165.

Horvath GC, Kistler WS & Kistler MK 2004 RFX2 is a potential transcriptional regulatory factor
for Histone Hit and other genes expressed during the meiotic phase of spermatogenesis. Biology
ofReproduction 71 1551-1559.

Humbert PO, Rogers C, Ganiastsas S, Landsberg RL, Trimarchi JM, Dandapani S, Brugnara
C, Erdman S, Schrenzel M, Bronson RT & Lees JA 2000 E2F4 is essential for normal
erythrocyte maturation and neonatal viability. Molecular Cell 6 281-291.

Iannello RC, Young J, Sumarsono S, Tymms MJ, Dahl HM, Gould J, Hedger M & Kola I
1997 Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG
methyl ation. Molecular and Cellular Biology 17 612-619.

Jang J & Lee C 2003 Mitochondrial adenine nucleotide translocator 3 is regulated by IL-4 and
IFN-y via STAT-dependent pathways. Cellular Immunology 226 11-19.

Johnson DG, Schwarz JK, Cress WD & Nevins JR 1993 Expression of transcription factor E2F 1
induces quiescent cells to enter S phase. Nature 365 349-35.

Jordens EZ, Palmieri L, Huizing M, van den Heuvel LP, Sengers RC, Dorner A, Ruienbee, W,
Trijbels FJ, Valsson J, Sigfusson G, Palmieri F & Smeitink JA 2002 Adenine nucleotide
translocator 1 deficiency associated with Sengers syndrome. Annals ofNeurology 52 95-99.

Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT, Roskin KM, Schwartz
M, Sugnet CE, Thomas DJ, Weber RJ, Haussler D & Kent WJ 2003 The UCSC Genome
Browser Database. Nucleic Acids Research 31 51-54.

Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H & Tsunoda Y 1998
Eight calves cloned from somatic cells of a single adult. Science 282 2095-2098.

Keeney S, Giroux CN & Kleckner N 1997 Meiosis-specific DNA double-strand breaks are
catalyzed by Spolli, a member of a widely conserved protein family. Cell 88 375-3 84.

Kel AE, Kel-Margoulis OV, Farnham PJ, Bartley SM, Wingender E & Zhang MQ 2001
Computer-assisted identification of cell cycle-related genes: new targets for E2F transcription
factors. Journal of2~olecular Biology 309 99-120.

Kim M, Li D, Cui Y, Mueller K, Chears WC & DeJong J 2006 Regulatory factor interactions
and somatic silencing of the germ cell-specific ALF gene. Journal of Biological Chemistry 281
34288-34298.









Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hwang WS, Hossein MS, Kim JJ, Shin NS, Kang
SK & Lee BC 2007 Endangered wolves cloned from adult somatic cells. Cloning and' Stem Cells
9 130-137

Kim YH, Haidl G, Schaefer M, Egner U, Mandal A & Herr JC 2006 Compartmentalization of a
unique ADP/ATP carrier protein SFEC (Sperm Flagellar Energy Carrier, AAC4) with glycolytic
enzymes in the fibrous sheath of the human sperm flagellar principal piece. Developmental
Biology 302 463-476.

Kong L, Chang JT, Bild AH & Nevins JR 2007 Compensation and specificity of function within
the E2F family. Oncogene 26 321-327.

Lammers JHM, Offenberg HH, Van Aalderen M, Vink ACG, Dietrich AJJ & Heyting C 1994
The gene encoding a maj or component of the lateral elements of synaptonemal complexes of the
rat is related to X-linked lymphocyte-regulated genes. Molecular and' Celullarllllll11~~~~~~~~ Biology 14 1137-
1146.

Latham KE, Litvin J, Orth JM, Patel B, Mettus R & Reddy EP 1996 Temporal patterns of A-
myb and B-myb gene expression during testis development. Oncogene 13 1161-1168.

La Thangue NB & Rigby PW 1987 An adenovirus E1A-like transcription factor regulated during
the differentiation of murine embryonal carcinoma stem cells. Cell 49 507-513.

Lee J, Iwai T, Yokota T, & Yamashita M 2003 Temporally and spatially selective loss of Rec8
protein from meiotic chromosomes during mammalian meiosis. Journal of Cell Science 116
2781-2790.

Lele KM & Wolgemuth DJ 2004 Distinct regions of the mouse Cyclin Al gene, Ccnal, confer
male germ-cell-specific expression and enhancer function. Biology of Reproduction 71 1340-
1347.

Levy SE, Chen YS, Graham BH & Wallace DC 2000 Expression and sequence analysis of the
mouse adenine nucleotide translocase 1 and 2 genes. Gene 254 57-66.

Li FX, Zhu JW, Hogan CJ & DeGregori J 2003 Defective gene expression, S phase progression,
and maturation during hematopoiesis in E2F l/E2F2 mutant mice. Molecular and' Cellular
Biology 23 3607-3622.

Li K, Hodge JA & Wallace DC 1990 OXBOX, a positive transcriptional element of the heart-
skeletal muscle ADP/ATP Translocator Gene. Journal of Biological Chemistry 265 20585-
20588.

Li R, Hodny Z, Luciakova K, Barath P & Nelson BD 1996 Spl activates and inhibits
transcription from separate elements in the proximal promoter of the human adenine nucleotide
translocase 2 (ANT2) gene. Journal of Biological Chemistry 271 18925-18930.









Lin H, Jurk M, Gulick T & Cooper GM 1999 Identifieation of COUP-TF as a transcriptional
repressor of the c-mos proto-oncogene. Journal of Biological Chemistry 274 36796-36800.

Lindeman GJ, Dagnino L, Gaubatz S, Xu Y, Bronson RT, Warren HB & Livingston DM 1998
A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting.
Genes & Development 12 1092-1098.

Looman C, Mark C, Abrink M & L Hellman 2003 MZF6D, a novel KRAB zinc-finger gene
expressed exclusively in meiotic male germ cells. DNA and Cell Biology 22 489-496.

Loots GG & Oveharenko I 2004 rVISTA 2.0: evolutionary analysis of transcription factor binding
sites. Nucleic Acids Research 32 W217-W221.

Lukas J, Petersen BO, Holm K, Bartek J & Helin K 1996 Deregulated expression of E2F family
members induces S-phase entry and overcomes pl6INK4A-mediated growth suppression.
Molecular and Cellular Biology 16 1047-1057.

Lunardi J, Hurko O, Engel WK & Attardi G 1992 The multiple ADP/ATP translocase genes are
differentially expressed during human muscle cell development. Journal of Biological Chemistry
267 15267-15270.

Macaluso M, Cinti C, Russo G, Russo A & Giordano A 2003 pRb2/pl 30-E2F4/5-HDAC 1-
SUV3 9H1-DNMT 1 multimolecular complexes mediate the transcription of estrogen receptor-a
in breast cancer. Oncogene 22 3511-3517.

MacLean II JA & Wilkinson MF 2005 Gene regulation in spermatogenesis. Current Topics in
DevelopmentalBiology 71 13 1-197.

Maiti B, Li J, Bruin A, Gordon F, Timmers C, Opavsky R, Patil K, Tuttle J, Cleghorn W &
Leone G 2005 Cloning and characterization of mouse E2F8, a novel mammalian E2F family
member capable of blocking cellular proliferation. Journal of Biological Chemistry 280 1821 1-
18220.

Marikawa Y, Fujita T & Alarcon V 2005 Heterogeneous DNA methylation status of the
regulatory element of the mouse Oct4 gene in adult somatic cell population. Cloning and Stem
Cells 7 8-16.

Matsui Y & Hayashi K 2007 Epigenetic regulation for the induction of meiosis. Cellular and
Molecular Life Sciences 64 257-262.

Meuwissen RLJ, Offenberg HH, Dietrich AJJ, Riesewijk A, Van Lersel M & Heyting C 1992
A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes
EM~BO Journal 11 5091-5100.









Muller H, Moroni MC, Vigo E, Petersen BO, Bartek J & Helin K 1997 Induction of S-phase
entry by E2F transcription factors depends on their nuclear localization. Molecular and Cellular
Biology 17 5508-5520.

Murga M, Fernandez-Capetillo O, Field S, Moreno B, Borlado LR, Fujiwara Y, Balomenos D,
Vicario A, Carrera AC, Orkin SH, Greenberg ME & Zubiaga AM 2001 Mutation of E2F2 in
mice causes enhanced T lymphocyte proliferation leading to the development of autoimmunity.
Immunity 15 959-970.

Oberley MJ, Inman DR, & Farnham PJ 2003 E2F6 negatively regulates BRCAl in human
cancer cells without methylation of histone H13 on lysine 9. Journal of Biological Chemistry 278
42466-42476.

Offenberg HH, Schalk JAC, Meuwissen RLJ, Aalderen MV, Kester HA, Dietrich AJJ &
Heyting C 1998 SCP2: a maj or protein component of the axial elements of synaptonemal
complexes of the rat. Nucleic Acids Research 26 2572-2579.

Ogawa H, Ishiguro K, Gaubatz S, Livingston DM, & Nakatani Y 2002 A complex with
chromatin modifiers that occupies E2F- and Myc-responsive genes in Go cells. Science 296
1132-1136.

Ollinger R, Alsheimer M & Benavente R 2005 Mammalian protein SCP1 forms synaptonemal
complex-like structures in the absence of meiotic chromosomes. Molecular Biology of the Cell
16 212-217.

Pennisi E 2001 The human genome. Science 291 1177-1180.

Petukhova GV, Pezza RJ, Vanevski F, Ploquin M, Masson J & Camerini-Otero RD 2005 The
Hop2 and Mnd1 proteins act in concert with Rad5 1 and Dmc1 in meiotic recombination. Nature
Structural & M~olecular Biology 12 449-453.

Pezza RJ, Petukhova GV, Ghirlando R & Camerini-Otero RD 2006 Molecular activities of
meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex. Journal of Biological
Chemistry 281 18426-18434.

Pezzi N, Prieto I, Kremer L, Perez-Jurado LA, Valero C, Del-Mazo J, Martinez C & Barbero
JL 2000 STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and
location of STAG3-related genes flanking the Williams-Beuren syndrome deletion. FASEB
Journal 14 581-592.

Pohlers M, Truss M, Frede U, Scholz A, Strehle M, Kuban R, Hoffmann B, Morkel M,
Birchmeier C & Hagemeier C 2005 A role for E2F6 in the restriction of male-germ-cell-
specific gene expression. Current Biology 15 1051-1057.










Polejaeva IA, Chen SH, Vaught TD, Page RL~, Mullins J, Ball S, Dai Y, Boone J, Walker S,
Ayares DL, Colman A & Campbell KHS 2000 Cloned pigs produced by nuclear transfer from
adult somatic cells. Nature 407 86-90.

Pousette A, Leijonhufvud P, Arver S, Kvist U, Pelttari J & HS5g C 1997 Presence of
synaptonemal complex protein 1 transversal filament-like protein in human primary
spermatocytes. Human Reproduction 12 2414-2417.

Prieto I, Suja JA, Pezzi N, Kremer L, Martinez C, Rufas JS & Barbero JL 2001 Mammalian
STAG3 is a cohesion specific to sister chromatid arms in meiosis 1. Nature Cell Biology 3 761-
766.

Qin XQ, Livingston DM, Kaelin WG & Adams PD 1994 Deregulated transcription factor E2F-1
expression leads to S-phase entry and p53-mediated apoptosis. Proceedings of the National
Academy ofSciences 91 10918-10922.

Raynik SE & Wolgemuth DJ 1999 Regulation of meiosis during mammalian spermatogenesis: the
A-type Cyclins and their associated Cyclin-dependent kinases are differentially expressed in the
germ-cell lineage. DevelopnzentalBiology 207 408-418.

Rehling P, Brander K & Pfanner N 2004 Mitochondrial import and the twin pore translocase.
Nature Reviews M~olecular Cell Biology 5 519-530.

Rempel RE, Saenz-Robles MT, Storms R, Morham S, Ishida S, Engel A, Jakoi L, Melhem
MF, Pipas JM, Smith C & Nevins JR 2000 Loss of E2F4 activity leads to abnormal
development of multiple cellular lineages. Molecular Cell 6 293-306.

Revenkova E, Eupe M, Heyting C, Gross B, & Jessberger R 2001 Novel meiosis-specific
isoform of mammalian SMC1i. Molecular and Cellular Biology 21 6984-6998.

Revenkova E & Jessberger R 2005 Keeping sister chromatids together: cohesins in meiosis.
Reproduction 130 783-790.

Ringrose L 2006 Polycomb, trithorax and the decision to differentiate. BioEssayvs 28 330-334.

Robinson MO, McCarrey JR & Simon MI 1989 Transcriptional regulatory regions of testis-
specific PGK-2 defined in transgenic mice. Proceedings of the National Academy of Sciences 86
8437-8441.

Rodic N, Oka M, Hamazaki T, Murawski MR, Jorgensen M, Maatouk DM, Resnick JL, Li E
& Terada N 2005 DNA methylation is required for silencing ofAnt4, an adenine nucleotide
translocase selectively expressed in mouse embryonic stem cells and germ cells. Stent Cells 23
1314-1323.









Rotheneder H, Geymayer S & Haidweger E 1999 Transcription factors of the Spl family:
interaction with E2F and regulation of the murine thymidine kinase promoter. Journal of
Molecular Biology 293 1005-1015.

Sagata N 1997 What does Mos do in oocytes and somatic cells? Bioessay~~ssssssss~ 19 13-21.

Sage J, Martin L, Meuwissen R, Heyting C, Cuzin F & Rassoulzadegan M 1999 Temporal and
spatial control of the Sycpl gene transcription in the mouse meiosis: regulatory elements active
in the male are not sufficient for expression in the female gonad. Mechanisms of Developnzent 80
29-39.

Schwartz JK, Devoto SH, Smith EJ, Chellappan SP, Jakoi L, & Nevins JR. 1993 Interactions of
the pl07 and Rb proteins with E2F during the cell proliferation response. E 180 Journal 3 1013-
1020.

Shannon M, Richardson L, Christian A, Handel MA & Thelen MP 1999 Differential gene
expression of mammalian SPO11 TOP6A homologs during meiosis. FEBS Letters 462 329-334.

Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L &
Westhusin M 2002 Cell biology: a cat cloned by nuclear transplantation. Nature 415 859.

Simpson AJG, Caballero OL, Jungbluth A, Chen Y & Old LJ 2005 Cancer/testis antigens,
gametogenesis and cancer. Nature Reviews Cancer 5 615-625.

Singh AM, Hamazaki T, Hankowski KE & Terada N 2007 A heterogeneous expression pattern
for Nanog in embryonic stem cells. Stent Cells 25 2534-2542.

Singh P, Wong SH & Hong W 1994 Overexpression of E2F-1 in rat embryo fibroblasts leads to
neoplastic transformation. E IBO Journal 13 3329-3338.

Smale ST 1997 Transcription initiation from TATA-less promoters within eukaryotic protein-
coding genes. Biochinsica et Biophysica Acta 1351 73-88.

Stepien G, Torroni A, Chung AB, Hodge JA & Wallace DC 1992 Differential expression of
adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell
differentiation. Journal of Biological Chentistry 267 14592-14597.

Storre J, Elslsser H, Fuchs M, Ullmann D, Livingston DM &Gaubatz S 2002 Homeotic
transformations of the axial skeleton that accompany a targeted deletion of E2f6. E 180 Reports
3 695-700.

Storre J, Schlfer A, Reichert N, Barbero JL, Hauser S, Eilers M, & Gaubatz S 2005 Silencing
of meiotic genes SM~C1; and STAG3 in Somatic Cells by E2F6. Journal of Biological Chentistry
280 41380-41386.










Sweeney C, Murphy M, Kubelka M, Raynik SE, Hawkins CF, Wolgemuth DJ & Carrington
M 1996 A distinct cyclin A is expressed in germ cells in the mouse. Development 122 53-64.

Thomas K, Wu J, Sung DY, Thompson W, Powell M, McCarrey J, Gibbs R & Walker W
2007 SP1 transcription factors in male germ cell development and differentiation. Molecular and
Cellular Endocrinology 270 1-7.

Toscani A, Mettus RV, Coupland R, Simpkins H, Litvin J, Orth J, Hatton KS & Reddy EP
1997 Arrest of spermatogenesis and defective breast development in mice lacking A-myb.
Nature 386 713-717.

Trimarchi JM, Fairchild B, Wen J & Lees JA 2001 The E2F6 transcription factor is a component
of the mammalian Bmili-containing polycomb complex. Proceedings of the National Acadenzy of
Sciences 98 1519-1524.

Trimarchi JM & Lees JA 2002 Sibling rivalry in the E2F family. Nature Reviews M~olecular Cell
Biology 3 11-20.

Tronche F, Ringeisen F, Blumenfeld M, Yaniv M & Pontoglio M 1997 Analysis of the
distribution of binding sites for a tissue-specific transcription factor in the vertebrate genome.
Journal of2~olecular Biology 266 23 1-245.

Tureci O, Sahin U, Zwick C, Koslowski M, Seitz G & Pfreundschuh M 1998 Identification of a
meiosis-specific protein as a member of the class of cancer/testis antigens. Proceedings of the
National Academy of Sciences 95 5211-5216.

Turner JM 2007 Meiotic sex chromosome inactivation. Development 134 1823-1831.

Van-Heldin J 2003. Regulatory sequence analysis tools. Nucleic Acids Research 31 3593-3596.

Vire E, Brenner C, Deplu R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A,
Bernard D, Vanderwinden J, Bollen M, Esteller M, Di Croce L, Launoit Y & Fuks F 2006
The polycomb group protein EZH2 directly controls DNA methylation. Nature 439 871-874.

Wakayama T, Perry AC, Zuccotti M, Johnson KR & Yanagimachi R 1998 Full-term
development of mice from enucleated oocytes inj ected with cumulus cell nuclei. Nature 394
369-374.

Walker JE 1992 The mitochondrial transporter family. Current Opinion in Structural Biology 2
519-526.

Wang PJ, Page DC & McCarrey JR 2005 Differential expression of sex-linked and autosomal
germ-cell-specific genes during spermatogenesis in the mouse. Human M~olecular Genetics 14
2911-2918.










Wang WS, Seki M, Yamoaka T, Seki T, Tada S, Katada T, Fujimoto H & Enomoto T 1998
Cloning of two isoforms of mouse DNA helicase Q 1/RecQL cDNA; alpha form is expressed
ubiquitously and beta form specifically in the testis. Biochemical and Biophysical Research
Communications 1443 198-202.

Wasserman WW & Fickett JW 1998 Identification of regulatory regions which confer muscle-
specific gene expression. Journal of2~olecular Biology 278 167-181.

Watanabe D, Yamada K, Nishina Y, Tajima Y, Koshimizu U, Nagata A & Nishimune Y 1994
Molecular cloning of a novel Ca2+-binding protein (Calmegin) specifically expressed during
male meiotic germ cell development. Journal of Biological Chemistry 269 7744-7749.

Watson JD & Crick FH 1953 Molecular structure of nucleic acids; a structure for deoxyribose
nucleic acid. Nature 171 737-738.

Weil PA, Luse DS, Segall J & Roeder RG 1979 Selective and accurate initiation of transcription
at the Ad2 maj or late promoter in a soluble system dependent on purified RNA polymerase II
and DNA. Cell 18 469-484.

Weinberg RA 1992 The retinoblastoma gene and gene product. Cancer Surveys 12 43-57.

Wilkerson DC, Wolfe SA & Grimes SR 2002 Hlt/GC-Box and Hlt/TE1 element are essential for
promoter activity of the testis-specific Histone Hit gene. Biology of Reproduction 67 1 157-1 164.

Wilmut I, Schnieke AE, McWhir J, Kind AJ & Campbell KHS 1997 Viable offspring derived
from fetal and adult mammalian cells. Nature 385 810-813.

Witte V, Laffert B, Rosorius O, Lischka P, Blume K, Galler G, Stilper A, Willbold D, D'Aloja
P, Sixt M, Kolanus J, Ott M, Kolanus W, Schuler G & Baur AS 2004 HIV-1 Nef mimics an
integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane.
Molecular Cell 13 179-190.

Wu L, Timmers C, Maiti B, Saavedra HI, Sang L, Chong GT, Nuckolls F, Giagrande P,
Wright FA, Field SJ, Greenberg ME, Orkin S, Nevins JR, Robinson ML & Leone G 2001
The E2Fl1-3 transcription factors are essential for cellular proliferation. Nature 414 457-462.

Xu EY, Moore FL & Reijo-Pera RA 2001 A gene family required for human germ cell
development evolved from an ancient meiotic gene conserved in metazoans. Proceedings of the
National Academy of Sciences 98 74 14-74 19.

Xu W & Cooper GM 1995 Identification of a candidate c-mos repressor that restricts transcription
of germ cell-specific genes. Molecular and Cellular Biology 15 5369-5375.

Yamasaki L, Jacks T, Bronson R, Goillot E, Harlow E & Dyson N 1996 Tumor induction and
tissue atrophy in mice lacking E2F-1. Cell 85 537-548.










Yee AS, Reichel R, Kovesdi I & Nevins JR 1987 Promoter interaction of the E1A-inducible factor
E2F and its potential role in the formation of a multi-component complex. EM~BO Journal 6
2061-2068.

Yuan L, Brundell E & Hoog C 1996 Expression of the meiosis-specific Synaptonemal Complex
Protein 1 in a heterologous system results in the formation of large protein structures.
Experimental Cell Research 229 272-275.









BIOGRAPHICAL SKETCH

Sarah Michelle Kehoe was born in Washington D.C. in 1980, to Robert and Patricia

Williams. She was raised in Fairfax, Virginia and graduated from W.T. Woodson High School

in June 1998. Sarah then attended Virginia Polytechnic Institute and State University, where she

graduated both "in honors" and summa cum laude with her Bachelor of Science degree in animal

and poultry sciences in May 2002. During college, in the laboratory of Dr. Paul Siegel, Sarah

conducted poultry genetics research that culminated in the publication of her first-author paper in

the August 2002 issue of Poultry Science. Sarah was also awarded two summer internships at

the National Institutes of Health where she worked at the National Human Genome Research

Institute in the laboratory of Dr. Lawrence Brody. In 2003, Sarah enrolled in the

Interdisciplinary Program in Biomedical Sciences (IDP) at the University of Florida, College of

Medicine and received a Grinter Fellowship upon admission. She began her doctoral study

under the guidance of Dr. Naohiro Terada, in the Department of Molecular and Cell Biology.

Upon completion of her Ph.D. in December 2007, Sarah plans to pursue a career dedicated to the

study of cancer.





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1 E2F6 IS A COMMON REPRESSOR OF MEIOSIS-SPECIFIC GENES By SARAH M. KEHOE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Sarah M. Kehoe

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3 To my dad, who instilled in me his lifelong passion for learning

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4 ACKNOWLEDGMENTS I would first like to thank m y mentor, Dr. Na ohiro Terada, for always being optimistic, no matter how confusing science may seem at times His positive attitude, encouragement, and perseverance will not be forgotten. Dr. Teradas office door being always open for scientific discussions and his willingness to meet regularly with his gra duate students despite his busy schedule, exemplifies his dedication. I would also like to thank a ll of the members of the Terada lab, both past and present, for their support. In particular, Id like to thank lab manager, Amy Meacham, for her frequent assistance, her reliable smile, and her friendship. I would especially like to thank former graduate st udent, Amar Singh, for serving as my mentor at the lab bench; he was a wonderful teacher who answered my many que stions and helped me to get my feet off the ground. I am also grateful for my committee members, Dr. Jrg Bungert, Dr. Hideko Kasahara, Dr. Paul Oh, and Dr. Jim Resnick, for their helpful suggestions, feedback, and expertise. I would like to thank our collaborator, Dr. Stefan Gaubatz at the University of Wurzburg for providing invaluable materials and sound advice for my proj ect. Without him, I would not have had the resources necessary to come to the conclusions that I did. Further, I want to thank my parents, Robert and Patricia and sister, Karen, for their unconditional love and support. Likewise, Id like to thank my husband, Joe, for always being there for me at the end of every day. His hum or and encouragement have been important to surviving graduate school. Lastly, I'd like to thank my horse, Sundance, for keeping my life balanced and my stress levels at bay. Without being able to escap e to the barn and ride my horse on days when the complexity of science seemed too great to ever make headway, I would never have made it this far.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................10 CHAP TER 1 INTRODUCTION..................................................................................................................12 Transcriptional Control of Gene Expression .......................................................................... 12 The General Transcription Machinery............................................................................13 Gene Regulatory Proteins................................................................................................15 The E2F Family of Transcription Factors.............................................................................. 16 The Activating E2Fs........................................................................................................18 Pocket Protein-Depende nt Repressive E2Fs ...................................................................20 Pocket Protein-Independent Repressive E2Fs.................................................................22 Summation..............................................................................................................................25 2 MATERIALS AND METHODS........................................................................................... 30 Cell Culture and Creation of Stable Cell Lines ...................................................................... 30 Gel Mobility Shift Assay........................................................................................................30 Chromatin Immunoprecipitation............................................................................................ 31 Real-Time Quantitative PCR..................................................................................................32 Plasmid Construction and S ite-Directed Mutagenesis ........................................................... 33 Transient Transfection and Reporter Assays .......................................................................... 34 Immunoblotting......................................................................................................................35 Reverse Transcription-PCR....................................................................................................35 Computational Analysis......................................................................................................... .36 Bisulfite Sequencing and Combined Bisu lfite Restriction Analysis (COBRA) ..................... 37 Immunostaining......................................................................................................................38 3 REPRESSION OF ANT4 GENE EXPRESSION BY E2F6 .................................................. 41 Motivation...............................................................................................................................41 Background.............................................................................................................................41 Results.....................................................................................................................................46 Discussion...............................................................................................................................51

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6 4 REPRESSION OF ADDITIONAL MEIOSIS-SPECIFIC GENES BY E 2F6....................... 74 Motivation...............................................................................................................................74 Background.............................................................................................................................74 Results.....................................................................................................................................77 Discussion...............................................................................................................................79 5 THE ROLE OF E2F6 DURING MALE ME IOSIS AND SPERMATOGENESIS ............... 91 Motivation...............................................................................................................................91 Background.............................................................................................................................91 Results.....................................................................................................................................96 Discussion...............................................................................................................................97 6 CONCLUSIONS AND SIGNIFICANCE ............................................................................102 LIST OF REFERENCES.............................................................................................................104 BIOGRAPHICAL SKETCH.......................................................................................................117

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7 LIST OF TABLES Table page 2-1 Primers used for analyzing ChIP e xperim ents with semi-quantitative PCR..................... 39 2-2 Primers used for se m i-quantitative RT-PCR..................................................................... 40 4-1 Locations of E2F6 TFBS (TCCCGC) with in upstream promoter regions of meiosisspecific genes relative to their transcription initiation sites............................................... 86

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8 LIST OF FIGURES Figure page 1-1 Five ways in which regulatory pr oteins can inhibit transcription. ..................................... 27 1-2 The E2F family proteins and their core functional dom ains.............................................. 28 1-3 Changes in the composition and location of E2F com plexes as cells enter the cell cycle.......................................................................................................................... .........29 3-1 Ant4 encodes a novel isofor m of adenine nucleotide translocase...................................... 59 3-2 The conservation of Sp1 binding sites in the Ant4 prom oter............................................. 60 3-3 The proximal promoter region of the Ant4 gene has a conserved E2F6 binding site. ....... 61 3-4 Gel mobility shift assay of E2F6 binding to th e Ant4 promoter........................................ 62 3-5 The proximal promoter region of Ant4 is bound by endogenous E2f6 in undifferentiated W T R1 ES cells....................................................................................... 63 3-6 E2F6-mediated repression of Ant4 requires an intact transcrip ti on factor binding site....64 3-7 E2F6-mediated repression of Ant4 requires an intact C-term inal and DNA binding domain......................................................................................................................... .......65 3-8 Ant4 transcription is repressed by stable E2F6 overexpression in ES cells. ...................... 66 3-9 Ant4 transcription is derepressed in E2f6-/ME Fs. Semi-quantitative RT-PCR analysis is shown for E2f6 Ant4, and -actin expression in WT MEFs and E2f6-/MEFs..................................................................................................................................67 3-10 The polycomb proteins Eed and Ezh2 bind to the Ant4 prom oter in D6 R1 EBs. Chromatin immunoprecipitation (ChIP) was performed using antiE2f6, Eed, Ezh2, Dnmt3, Suz12, and acetylated hi stone H3 (AH3) antibodies............................................ 68 3-11 Ant4 is derepressed in NIH-3T3s after tr eatm ent with 5-AZA-DC but not TSA. 5 M or 10 M of AZA-DC were added either alon e or in combination with 200nm TSA for 65 hours in NIH-3T3s.................................................................................................. 69 3-12 CpG methylation at the Ant4 prom oter is partially reduced in E2f6-/MEFs. A) COBRA analysis of the methylation status of Ant4 in various tissue types...................... 70 3-13 Tuba3 m ethylation decreases in E2f6-/mouse tail DNA.................................................71 3-14 E2F6 overexpression does not in crease CpG m ethylation at the Ant4 promoter..............72 3-15 The All OR None model of E2F4 compensation........................................................ 73

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9 4-1 Frequency of appearance of E2F6 TFBS within upstream regions of genes relative to their transcription initiation sites.......................................................................................87 4-2 E2f6 binds to the promoters of meiosis-specific genes..................................................... 88 4-3 Many meiosis-specific genes are repressed by E2F6. Se mi-quantitative RT-PCR analysis of meiosis-specific gene expression in R 1, HA-E2F6, and HAC-E2F6 ES cells....................................................................................................................................89 4-4 Limited meiosis-specific genes are derepressed in E2f6-/MEFs. Se mi-quantitative RT-PCR analysis of meiosis-specific gene e xpression in WT testis from 6-week old mice, R1 ES cells, WT MEFs, and E2f6-/MEFs.............................................................90 5-1 E2f1 and E2f6 mRNA tr anscript levels in the testis........................................................100 5-2 Immunohistochemical analysis of E2f6 expression in mouse testis................................ 101

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10Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy E2F6 IS A COMMON REPRESSOR OF MEIOSIS-SPECIFIC GENES By Sarah M. Kehoe December 2007 Chair: Naohiro Terada Major: Medical Sciences --Molecular Cell Biology Genes whose protein products function specifi cally during meiosis ar e strictly repressed in somatic cells. Their aberrant expression may lead to mitotic catastrophe and predispose cells to oncogenic transformation. Although previous st udies have demonstrated various mechanisms delineating repression of individual meiosis-specific genes, the existence and identity of a master regulatory protein which coordinately represses multiple meiosis-specific genes in somatic tissues remains to be elucidated. E2F6, a member of the E2F family of tr anscription factors, was recently shown to play an essential role in the repression of a few meiosis-specific genes including Stag3 and Smc1 Here we report that E2F6 is re quired for the repression of another meiosis-specific gene, Ant4, in somatic cells. This discovery prompted us to investigate whether meiosis-specific genes in general, require E2F6 for their repression. We compiled a list of 24 meiosis-specific genes and observed that 19 of them (79.2%) have the core E2F6 -binding element (TCCCGC) within 200bp upstream of their transcription initiation sites. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%) a nd the mathematical probability of random occurrence (9.3%). Further, using embryonic stem cells, we demonstrate that E2F6 indeed binds to the promoters and represses the transcription of these meiosis-specific genes. These data

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11suggest that E2F6 serves as a common repressor of meiosis-specific genes. Of interest, E2F6 deficiency alone did not derepress most of th ese meiosis-specific genes in somatic cells, suggesting that a functional redund ancy exists whereby other safe guards are in place to ensure the repression of most meiosis-specific genes in somatic cells.

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12 CHAPTER 1 INTRODUCTION Transcriptional Control of Gene Expression With the discovery of the structure of DNA by Watson and Crick in the early 1950s (Watson & Crick 1953) came the understanding th at every cell in the body, although diverse, contains the same genetic information as all other cells. Prior to this breakthrough, it was difficult to fathom that two cell types with extr eme differences in shape and function, such as cardiomyocytes and neurons, could have been c oded from the same genome. In fact, it was speculated that different cell types actually contai ned different sets of ge nes due to a selective loss of unnecessary genes as the cells differentiated (Alberts et al. 2002). Today, we know that the vast diversity of cell type s found in the body is a result of differences in gene expression rather than a consequence of changing the presen ce of nucleotide sequences in a cells genome. Experimental evidence leading to this conclusion involved ta king the nucleus from a fully differentiated frog cell and injec ting it into a frog egg whose nuc leus had been removed (Gurdon 1962). The donor nucleus was able to direct th e recipient egg to produce a normal tadpole that contained a full range of differentiated cell type s, thereby proving that the genome must be preserved in the differentiated donor cell. Si milar experiments done in various species of mammals including: sheep (Campbell et al 1996, Wilmut et al 1997), cattle (Cibelli et al. 1998, Kato et al. 1998), mice (Wakayama et al. 1998), goat (Baguisi et al. 1999), pig (Polejaeva et al. 2000), cat (Shin et al. 2002), rabbit (Chesne et al. 2002), horse (Galli et al. 2003), and endangered wolves (Kim et al. 2007), have further validated that the genome is upheld in every somatic cell. Given that the human genome encodes approximately 35,000 genes (Pennisi 2001), specialized cell types have the di fficult job of selectively expressi ng the genes that they need to

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13 support the unique properties of their cell type. If this were not difficult enough, the ability to express the appropriate genes is further complicat ed by the fact that the eukaryotic genome is packaged into nucleosomes and higher order form s of chromatin which can restrict a genes accessibility. Fortunately, cells are equipped with the molecular machinery needed to overcome such obstacles. This machinery controls gene expression by operating at various levels using different mechanisms: 1) on a transcriptional leve l by controlling how often a gene is transcribed to RNA, 2) on an RNA processing level by regu lating RNA transcript sp licing, 3) on a location basis by selecting which messenger RNA (mRNA) in the nucleus gets transported to the cytoplasm for protein synthesis, 4) on a degradation basis by selectively destabilizing certain mRNA molecules in the cytoplasm, 5) on a tr anslational level by sel ecting which mRNAs are translated into proteins by ribosomes, and 6) on a post-translational leve l by affecting protein activity via activation, inactiv ation, degradation, or comp artmentalization (Alberts et al 2002). For the remainder of this dissertation, I will fo cus on the transcriptional regulation of gene expression. The General Transcription Machinery At the heart of transcription is the en zyme, RNA polymerase. RNA polymerase moves along the DNA, unwinding the DNA helix ahead of it to expose a template strand of DNA so that incoming ribonucleotides can complementary base pair a nd form a growing RNA chain. This RNA chain is then released and depending on the type of RNA, goes on to be processed accordingly. In this study, we focus on mRNA which is transcribed by RNA polymerase II, and after splicing goes on to be translated into protein. In order for the RNA polymerase to transcribe a gene accurately, it must be able to recognize where in the genome to start and end transcription. The starting point of transcription is indicated by a special sequence of nucleotides known as the promoter and when the RNA polym erase comes across this sequence, it binds

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14 tightly to it. The polymerase then begins transcription a nd continues to produce an RNA transcript until it encounters a termination signal. This end signal then enab les the release of the newly made RNA chain and the polymerase from the DNA. The observation that unlike bacterial R NA polymerase, purified eukaryotic RNA polymerase II could not in itiate transcription in vitro led to the discovery and purification of additional required factors (Weil et al. 1979). These factors are referred to as general transcription factors because they assemble on all promoters used by RNA Polymerase II. Their job is to help the RNA polymerase position itself correctly at the promoter, to assist in pulling apart the DNA strands, and to release the RNA Po lymerase from the promoter into elongation mode once transcription has begun. These general transcription fact ors assemble at the promoter region in a specific order. Firs t, the general transc ription factor, TFIID, binds to the TATA box, a short DNA sequence located 25 nucle otides upstream from the transc ription start site that is composed primarily of T and A nucleotides. The subunit of TFIID that recognizes the TATA box is called TBP (for TATA-binding protein) TFIID binding to the TATA box causes a distortion in the DNA which bri ngs DNA sequences on both sides of the distortion together to allow for subsequent protein assembly. Howeve r, many promoters do not contain TATA boxes. In some large groups of genes, such as housekee ping genes, the TATA box is often absent and the corresponding promoters are referred to as TA TA-less promoters. In these promoters, the exact position of the tran scription start site and the binding of the general transcription factors is often controlled by an in itiator (Inr) nucleotide sequence in th e transcription initiation region, or by a downstream promoter element (DPE) which is typically observed 30bp downstream of the transcription start site (Smale 1997). Sequences which have the capacity to function as Inrs or DPEs are less characterized than the TATA-box sequence.

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15 After TFIID binding, other general transcription factors including, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH along with the RNA polymerase then assemble at the promoter. This cohort of factors at the promoter is termed the transcription initiation complex. After the RNA polymerase is correctly positioned at the promoter, it then gain s access to the transcription initiation site with the help of TFIIHs DNA helicase activity. TFIIH then phosphorylates the tail of the RNA polymerase causing a conformational change which releases RNA polymerase from the promoter. RNA polymerase then transcribe s along the template DNA strand causing the formation of an elongating RNA transcript (Alberts et al. 2002). Gene Regulatory Proteins Although the general transcripti onal m achinery for protein-enc oded genes is the same for most eukaryotic promoters, gene regulatory prot eins on the other hand are diverse. These are the proteins which regulate gene expression by binding to short stretches of DNA of a defined sequence and in turn, control the accessibility of the general transcriptional machinery to specific promoters. It is estimated that 5-10% of th e coding capacity of a mammalian genome is devoted to the synthesis of these re gulatory proteins (Alberts et al 2002). This high percentage is not surprising given the vast divers ity of cell types found within the body, the constantly changing environmental cues imparted on a cell, and the fact that each gene is regulated by a set of regulatory proteins which are themselves products of genes that are re gulated by a whole other set of proteins. Further complexity results from th e fact that these regulato ry proteins have been shown to regulate transcri ption at distant sites, often thousands of base pairs away from the promoter. The explanation for this is that DNA can form loops to allow the distantly bound regulatory proteins to come into contact with pr oteins bound at the promoter An additional level of complexity comes from the combinatorial nature of transcripti onal regulation, where a

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16 regulatory proteins functi on as either an activator or a repr essor is often determined not by the protein itself but by the proteins with which it interacts (Alberts et al. 2002). Although the diversity and comple xity of transcriptional re gulatory protein networks appears overwhelming, progress has been made to elucidate some of the mechanisms by which regulatory proteins activate and repress transcription. As illu strated in Figure 1-1, regulatory proteins can inhibit transcrip tion via several mechanisms. Th ese include: 1) competing with activator proteins for binding to the same regulatory DNA sequence, 2) binding to the activation domain of an activator protein thereby preventi ng it from carrying out its activator functions even though it is still cap able of binding to DNA, 3) blocki ng assembly of the transcription initiation complex, 4) recruiting chromatin remodeling complexes or histone modifying enzymes which form more compacted chromatin, and 5) re cruiting deacetylases and other histone tail modifying enzymes to add repressive chromatin marks to histones as well as recruiting DNA methyltransferases to methylate DNA and lock a promoter in an inactive state (Alberts et al. 2002). Similar mechanisms are used by activators to produce the opposite effect of transcriptional activation. The remainder of this chapter will focus on a specific family of regulatory proteins known as E2Fs and will discuss their involvement in regulating transcription. The E2F Family of Transcription Factors E2F transcription factors regulate the expr ession of genes whose protein products are essential for cell cycle progression, cellular proliferation, DNA repair, and differentiation (Dimova et al. 2005, Trimarchi & Lees 2002). E2F was or iginally identified as a cellular DNAbinding protein that could bind to the seque nce, TTTCGCGC, within the adenovirus E2 promoter (Yee et al. 1987). Concurrently, La Thangue and coworkers identified DRTF1, a transcription factor which had the same consensus DNA-binding site as E2F during studies of embryonic stem cell differentiation (La Thangue & Rigby 1987). It is no w clear that E2F and

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17 DRTF1 are the same factor. In order to bind to DNA, E2F requires a binding partner known as dimerization partner (DP). The E2F and DP proteins heterodimerize at which point E2F becomes functional. Additionally, E2F associates with and is regulated by the retinoblastoma (RB) protein. RB was the first tumor suppressor protein ever identified and it is absent or mutated in at least one-third of all human tumors (Weinberg 1992). In particular, it is known for being mutated in hereditary and sporadic retinoblastoma. The importance of the RB-E2F interaction is demonstrated by the finding that all naturally occurring RB mutants isolated from human tumors lack the ability to bi nd and negatively regulate E2F. Currently, there are eight known members of the E2F family (DeGregori & Johnson 2006). E2F1 was the first member to be cloned. It was isolated by three independent groups through E2F1's ability to bind to RB. Subsequently, seven additional members of the E2F family were discovered as a result of their homology to E2 F1 or because of their ability to bind to RB. However, some of these E2Fs are unable to bind to RB. Figure 1-2 illust rates the protein binding domains of the E2F family members and s hows that only E2Fs 1-5 possess an RB binding domain. This domain allows for E2Fs 1-5 to in teract with RB and/or other related pocket proteins. E2Fs 1-5 are also the only E2Fs th at have a transcripti onal activation domain. However, only E2Fs 1-3a serve as transc riptional activators whereas E2Fs 3b-5 are transcriptional repressors despite the presence of an activation domai n. E2Fs 6-8 are also repressors but they are considered to be pocket protein-independent transcriptional repressors. E2Fs 6-8 lack both a transactivation domain and a RB binding domain, but they do contain a highly conserved DNA binding domain. E2Fs 1-6 al so contain a dimerization domain for their interaction with the DP proteins, but this domain is absent in E2F7 and E2F8. Generally, the various types of protein doma ins possessed by individual E2F fa mily members dictate their

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18 specific functions. Below we will describe the attributes of the three main classes of E2Fs: 1) activating E2Fs, 2) pocket protei n-dependent repressive E2Fs, a nd 3) pocket protein-independent repressive E2Fs. The Activating E2Fs The activating E2Fs, E2F1-3a, are potent transcriptional activators of E2F-responsive genes. Their key purpose is to activate genes which are essential for cellular proliferation. Briefly, cellular proliferation is regulated by the ce ll cycle which consists of four phases: G1 (the transition phase to prepare for DNA replication), S (the replication phase), G2 (the transition phase to prepare for cell division ), and M (the division phase). Cells which are not cycling are considered to be in a quiescent G0 phase. E2Fs 1-3a activate target ge nes at the G1/S boundary of the cell cycle, thereby prom oting S-phase entry (Schwartz et al. 1993). Interestingly, overexpression of any one of these E2Fs is sufficien t to induce quiescent cells to re-enter the cell cycle (Johnson et al 1993, Qin et al 1994, Lukas et al 1996). Conversely, reducing the levels of individual activating E2Fs can re sult in cell-cycle arrest (DeGregori et al 2006). When all three activating E2Fs are mutated, cellular proliferation is completely blocked (Wu et al. 2001). Given their strong ability to alter cellular proliferation, it is not surprising that these E2Fs are tightly regulated and that their expression and activity is restricted to specific windows during the cell cycle. The activating E2Fs exhi bit maximum expression levels during late G1 and early S phase (DeGregori et al. 2006). During this time, the activity of the E2F protein is also raised and it coinci des with a wave of transcription of E2F-responsive ge nes. The amount of E2F protein activity is largely determined by the interactions between the E2Fs and RB. RB binding to E2Fs 1-3a can inhibit the activation of E2F-responsive genes in one of two ways. First, due to the fact that th e RB binding domain is located in the transactivation domain of E2F

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19 proteins (see Figure 1-2); RB binding can physically mask the activation domain. This association prevents the E2F-DP heterodimer fr om recruiting the general transcription factor, TFIID, thereby inhibiting E2Fs ability to activate. The other way by which RB blocks E2F activity is through active repr ession as a consequence of recruiting HDAC, SWI/SNF, SUV39H1, and other chromatin modi fying enzymes to the promoters of E2F-responsive genes. RB-mediated repression is relinquis hed as quiescent cells are stimulated to enter the cell cycle due to phosphorylation of RB by cell cycle-dependent kinases. Upon RB phosphorylation, E2F is released. This sudden abundan ce of free E2Fs results in the activation of E2F-responsive genes during late G1 of the cell cycle (Trimarchi et al. 2001). During this time, E2F-responsive promoters are bound by activating E2Fs and th is binding is often associated with transcriptionally permissive modifications in local chromatin. In addition to their roles in promoting cell cycle progression, activating E2Fs are also important during development. Th is is evidenced by the various E2F mutant mouse models. For instance, E2f1-/mice are viable and fertile but have tissue-specific abnormalities which include: an excess of T cells, exocrine gland dysplasia, a nd the development of te sticular atrophy (Field et al. 1996, Yamasaki et al. 1996). Also, E2f1-/mice develop various tu mors between 8 and 18 months of age. Although E2F1 overexpression in tissue culture cells stimu lates cell proliferation and can be oncogenic (Singh et al. 1994), the E2f1-/mouse clearly demonstrates that E2F1 can also function as a tumor suppressor. E2f2 mutant mice display enhanced T lymphocyte proliferation which leads to the development of autoimmunity (Murga et al. 2001). E2f1/E2f2 double-knockout mice exhibit severely impaired hematopoiesis due to defective S phase progression in hematopoietic progenitor populations (Li et al 2003). Combined E2f1 and E2f2 loss also results in polyploidy in the live r, salivary gland, and exocrine pancreas. E2f3-/mice

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20 are shown to die in utero and the few that survive to adulthood die prematurely due to congestive heart failure (Cloud et al 2002). It should be noted here that E2f3-/mice are deficient in both E2f3a and E2f3b isoforms. From the phenotype of E2f1/E2f3 double-mutant mice (Cloud et al. 2002), it is clear that almost al l of the developmental and age -related defects arising in the individual E2f1 or E2f3 mutant mice are exacerbated by the combined mutation. Interestingly however, these mice did not have an increased incidence of tumor formation, revealing that tumor suppression is a specific property of E2F1 a nd not E2F3. Overall, in addition to their roles in the induction of proliferation, the activating E2Fs have crucia l functions during development. Pocket Protein-Depende nt Repressive E2Fs Repressing E2Fs that possess an RB bi nding domain are termed pocket proteindependent repressive E2Fs. This class of E2Fs consists of E2Fs 3b-5. It has been suggested that their primary roles are to repr ess growth promoting E2F-responsive genes during G0/early G1 and to induce cell cycl e exit and terminal differentiation (DeGregori et al. 2006). They diverge considerably in sequence from the activating E2Fs in that they lack most of the sequence that is amino-terminal to the DNA binding domain. Fu rther, they use their RB binding domain to interact with additional RB pocke t protein family members. E2F4 interacts with p107, p130, and RB whereas E2F5 interacts mostly with p130. Thes e repressive E2Fs are further distinguished from the activating E2Fs by their expression patte rns. Unlike the activating E2Fs, repressive E2Fs are constitutively expressed and can be found in nearly equivalent levels in both quiescent and proliferating cells. In fact, E2F4 is expr essed at higher levels th an any other E2F and it accounts for at least half of the E2 F-pocket protein activity observed in vivo (Trimarchi et al. 2002). Interestingly, while the protein levels of these E2Fs do not change rapidly, substantial changes in their subcellular location have been observed during the cell cycle.

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21 Unlike the activating E2Fs which are constitu tively nuclear, E2Fs 4-5 are predominately cytoplasmic. This difference in lo cation is attributed to the fact that activating E2F proteins have a nuclear localization signal wher eas repressive E2Fs have a nu clear export signal. However, when the repressive E2Fs associate with pocket pr oteins, it is sufficient to induce their nuclear localization. Once in the nucleus, repressive E2F-pocket protein complexes can be found abundantly and this occurs during G0/G1 phases of the cell cycle (Figure 1-3). During this time, these repressive E2F complexes are bound to the promoters of many E2F-responsive genes and their binding is often correlated with repressive chromatin modi fications. Then around mid to late G1 in the cell cycle, the repressive E2F co mplexes are released from the promoters. This release is due to the separation of the repressive E2F from its pocket protein. The timing of the release is often correlated with the upregula tion of activating E2Fs which may subsequently replace repressing E2F protein occu pancy at E2F-target promoters dur ing S phase. At this point, the repressive E2Fs have been exported back out to the cytoplasm, rendering them inactive (Muller et al 1997). The role of E2Fs 3b-5 in the repression of E2F-responsive genes was elucidated when these proteins were ablated from cell lines. Mouse embryonic fibroblasts (MEFs) deficient in E2f4 and/or E2f5 have defects in their ability to exit the cell cycle in response to various growtharrest signals including p16 overexpre ssion and contact inhibition (DeGregori et al. 2006). However, these mutant cells are able to respond to growth stimulatory signals and there is no change in their proliferative capacity. Thus, the loss of these repressive E2Fs impairs the repression of E2F-responsive genes and therefor e the ability to exit the cell cycle. In agreement with the phenotype of E2f4-/MEFs, cellular proliferation in E2f4-/mice is also normal. However, E2f4-/mice display defects in terminal differentiation. This is

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22 evidenced by several phenotypic abnormalities in E2f4-/mice: 1) fetal anemia and erythrocyte maturation defects, 2) a lack of mature hemat opoietic cell types but an abundance of immature cells, and 3) a thinning of gut epithelium due to a reduction in the density of villi (Rempel et al 2000, Humbert et al 2000). These observations suggest a cr itical role for E2F4 in controlling the maturation of cells in a number of different tissues. A knockout of th e other pocket proteindependent repressive E2F, E2f5 results in mice which are viable but develop hydrocephalus due to an overproduction of cerebral spinal fluid re sulting from a choroid plexus defect (Lindeman et al 1998). Thus, E2F4 and E2F5 clearly have individual roles during development. However, the neonatal lethal phenotype of E2f4/E2f5 double-knockout mice (Gaubatz et al. 2000) demonstrates that E2F4 and E2F5 must also have some overlapping functions in biological processes that are essential to survival. Moreover, the individual and combined knockout phenotypes of both mice and cell lines show that these pocket protein-dependent repressive E2Fs are dispensable for cellular pr oliferation but are required for cell cycle exit and terminal differentiation. Pocket Protein-Independent Repressive E2Fs The previously described E2F proteins roles as transcr iptional activ ators or repressors depended upon whether or not they were bound to a pocket protein. Conve rsely, pocket proteinindependent repressive E2Fs, E2 Fs 6-8, lack an RB binding domain (Figure 1-2) and therefore repress E2F-responsive genes thr ough mechanisms which do not re quire pocket proteins. These E2Fs are localized to the nucleus and their expr ession fluctuates during the cell cycle. In contrast to pocket protein-dependent repressive E2Fs, E2F6 expression is low in serum-starved, quiescent cells. Instead, E2F6 e xpression is increased during re-entry into the cell cycle with peak levels in late G1/early S phase. From that point, E2F6 remains expressed throughout the cell cycle. Similarly, E2F7 and E2F8 are also expressed in a cell growth-dependent manner,

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23 accumulating as cells enter the cell cycle and w ith peak levels found during S phase. This expression pattern suggests that these E2Fs ma y serve as components of a negative feedback system to counterbalance activating E2Fs during the cell cycle. The finding that E2Fs 6-8 bind to E2F-responsive promoters during S phase agrees with this idea. Additionally, these E2Fs may be involved in defining the distinction between G1/Sand G2/Mregulated transcription. This role was proposed after the obs ervation that E2F6 selectively binds and represses only those E2F-responsive genes that were activated at the G1/S boundary and not the G2/M boundary of the cell cycle (Giagrande et al 2004). Overexpression studies have fu rther elucidated the roles of pocket-protein independent E2Fs. Ectopic expression of either E2F6 or E2 F7 is able to block endogenous E2F-responsive gene transcription as well as block the tran sactivating activity of ectopic E2F1 (Cartwright et al. 1998, Gaubatz et al. 1998, Bruin et al. 2003). Further, E2F6 overexpression leads to the accumulation of cells in S-phase (Cartwright et al. 1998). In agreement with this observation is the prediction that following cellular damage, expr ession of E2F6 may be upregulated to inhibit E2F-dependent transcription in S-phase and therefore, delay the progression of the cells through S phase until either the damage has been repaired or the cell has been targeted for apoptosis. Additionally, E2F6 overexpression in quiescent cells delays re-entry into the cell cycle (Gaubatz et al. 1998). This is consistent with the detec tion of an E2F6 chromatin-modifying complex at cell cycle-regulated promot ers during G0. However, E2f6-/MEFs display no de fects in cellular proliferation (Storre et al. 2002). It is speculated that pro liferation remains normal because many of these cell cycle promoters are also occupied by repressive E2F4 complexes, suggesting that E2F6 is redundant. E2F7 or E2F8 overexpression significantly slows down cellular proliferation in MEFs (Bruin et al. 2003, Maiti et al. 2005). Ectopic expression of E2F8 has also been shown

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24 to ablate DNA replication of cells in S phase which may reflect th e downregulation of E2F-target genes required for DNA synthesis. Taken togeth er, these observations propose that pocket protein-independent repressive E2Fs have overlapping and perhap s synergistic roles during the cell cycle. However, the mechanism by which these pocket protein-inde pendent E2Fs mediate transcriptional repression of E2F-target genes is not known for E2Fs 7-8. Further insights will likely be established with the creation of E2f7 and E2f8 knockout mice. Therefore, the remainder of this discussion will focus on the mechanisms of E2F6-mediated repression as there is a substantial amount of experime ntal evidence on this topic. One mechanism by which E2F6 mediates tr anscriptional repression is through its association with polycomb repressive comp lexes. This association is suggested in vivo by the phenotype of E2f6-/mice; posterior homeotic transforma tions of the axial skeleton which are strikingly similar to those observe d in mice deficient in a variet y of polycomb proteins (Storre et al 2002). There are two main types of polycomb repressive complexes: 1) PRC2, which is thought to be required at the in itiating stage of silencing, and 2) PRC1, which is continuously required for the stable maintenance of repressi on once the correct gene expression patterns have been established. E2F6 has been shown to associate with members of the PRC1 complex (Trimarchi et al. 2001). This E2F6-PRC1 complex in cludes the PRC1 complex proteins, RING1, mel-18, mph1, Bmi1, and YY1 binding protein (RYBP), and it is presumed that these polycomb proteins associate with their target promoters thro ugh binding to E2F6. Recently, another E2F6-polycomb complex (E2F6-G0) was described (Ogawa et al. 2002). This complex, containing several novel polycomb pr oteins including Max and HP1 was found to associate with E2F target promoters in G0 but not following re-entry into the cell cycle. This implies a role for E2F6 in the repression of E2F target genes during G0. However, given that E2F6 is

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25 expressed throughout the cell cycle and remains localized to the nuc leus, E2F6 most likely forms alternate complexes in proliferating cells. A dditional studies have revealed that E2F6 is associated with EPC1 and EZH2, a component of the PRC2 complex, only in proliferating cells (Attwooll et al. 2005). Upon overexpression of EPC1, EZH 2, and EED, another component of the PRC2 complex, these three proteins will co-immunoprecipitate. Fu rther, this E2F6 proliferation-specific complex was shown to repr ess reporter activity but a mutation in E2F6 which renders it unable to bind to EPC1 causes a loss of this repression. Interestingly, the PRC2 component EZH2, which is known for its H3K27 me thyltransferase activity, has recently been shown to recruit DNA methyltransferases to the promoters of target genes (Vire et al. 2006). This recruitment could serve to lock in the silent state initiated by the polycombs through DNA methylation. Overall, E2F6 has been shown to associate with components of both the PRC1 and PRC2 complexes suggesting a role for E2F6 in the establishment and the maintenance of E2Ftarget gene repression in both quiescent and pr oliferating cells. Other mechanisms for E2F6-mediated repressi on have been studied but not as thoroughly as the E2F6-polycomb interactions. These incl ude: 1) E2F6 blocking activating E2Fs from binding to the promoters of E2 F-responsive genes (Oberley et al 2003), 2) E2F6 recruiting DNA methyltransferases to induce CpG hypermethylati on and lock target promoters in an inactive state (Pohlers et al 2005), and 3) E2F6 recruiting histone deacetylases and histone methyltransferases which infer repressive chromatin marks on histone tails (Storre et al 2005, Caretti et al 2003, Ogawa et al 2002). For the remainder of this study, we will focus on the E2F family member, E2F6, and E2F6-mediated repression of a select set of target genes. Summation The way by which the hum an body creates so many different cell types from the same genomic sequences is inconceivable. However, we now know that such cellular diversity is at

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26 least made possible through the finite control of gene expr ession. More specifically, the transcriptional regulation of genes through the binding of transcriptional activators and repressors has proven to be especially important. Therefore, it was the goal of this work to study the transcriptional regula tion of gene expression. Considering the complexity of such a task, it is best to simplify the problem into a more manageable solution. Therefore, for the first part of this study, we chose to focus on the tran scriptional regulation of an indi vidual gene and a particular transcription factor. The gene we selected to analyze was adenine nucleotide translocase-4 ( Ant4 ) because it was recently di scovered by our lab (Rodic et al 2005), and prior to this study nothing was known about its transcri ptional regulation. We chose to investigate the E2F family of transcription factors, in particular E2F6, for reasons that will become apparent. The biological significance of Ant4 along with our findings re garding its transcriptiona l regulation, is discussed in Chapter 3. For the second part of this study, we examined the transcriptional regulation of 24 additional genes which were similar in expression to Ant4 because we had reason to believe that they too were regulated by E2F6. More informa tion regarding the transc riptional regulation of these 24 genes is given in Chapter 4. Lastly, in Chapter 5, we present da ta on the expression of E2F1 and E2F6 in the testis. In summation, the overall goal of this research was to analyze the transcriptional regulation of gene expression in the context of a specific gene/set of genes.

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27 Figure 1-1. Five ways in which regulatory proteins can inhibi t transcription. The repressing regulatory proteins can inhi bit transcription by: A) competing with activating regulatory proteins for binding to the same DNA sequence, B) blocking the activity of the activating regulatory protei n even though it may still bind, C) interacting with the general transcription machin ery and blocking it from asse mbling at the promoter, D ) recruiting repressive chromatin remodeli ng complexes, and E) recruiting histone deacetylases and other histone tail an d DNA modifying enzymes (from Alberts et al 2002, Figure 7-49 on page 406). A B C D E

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28 Figure 1-2. The E2F family protei ns and their core functional domains. E2Fs 1-6 share domains which mediate DNA binding (light blue) and DP dimerization (pink). E2Fs 7-8 have two of these DNA binding domains (DBD1 and DBD2), but lack a DP dimerization domain. E2Fs 1-5 also have an Rb bindi ng domain (green) within a transactivation domain that only has transact ivating activity in E2Fs 1-5 (modified from DeGregori and Johnson 2006, Figure 1 on page 740).

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29 Figure 1-3. Changes in the composition and loca tion of E2F complexes as cells enter the cell cycle. A ) During G0, complexes containing E2F6 and pocket proteins bind and repress E2F target genes. The function of activating E2Fs is disabled by association with RB. B) As cells enter the cell cycle, the repressive E2F dissociates from its pocket protein causing release from target promoters and export to the cytoplasm. Also, E2F1 is upregulated and released from RB. It can then replace the repressive E2F complexes by binding to and activating E2F target genes. A B

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30 CHAPTER 2 MATERIALS AND METHODS Cell Culture and Creation of Stable Cell Lines The cell lines used in this study were murine WT R1 ES ce lls (a gift from Dr. A Nagy, Toronto, Canada), HA-E2F6 ES cells, HAC-E2F6 ES cells, and NIH3T3 cells (American Type Culture Collection, Manassa s, VA). Both HA-E2F6 and HAC-E2F6 ES cell lines were established by stable transfection of linearized HA-E2F6 and HAC-E2F6 expression vectors (Gaubatz et al. 1998) into parental R1 ES cells. Stable clones were subsequently confirmed to express E2F6 by western blotting with anti -HA antibody (Cell Signali ng Technology, Danvers, MA), and those with th e highest expression were selected and expanded in the presence of Neomycin (G418) (Calbiochem-EMD Biosciences, San Diego, CA). All ES cells were maintained in an undifferentiated stat e on gelatin-coated dishes in Knock-outTM Dulbeccos Modified Eagle Medium, low glucose (Invitr ogen, Carlsbad, CA) containing 10% KnockoutTM Serum Replacement (Invitrogen), 1% Fetal Bovine Serum (Atlanta Biologicals, Lawrenceville, GA), 1% Penicillin-Streptomycin-Glutamine (100x) liquid (2mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, Invitrogen), 25mM HEPES (Cellgro, Herndon, VA), 300 M monothioglycerol (Sigma-Aldrich, St. Louis, MO), and 1,000 U/ml recombinant mouse LIF (ESGRO) (Millipore, Temecula, CA). NIH3T3 cel ls were maintained in Dulbeccos Modified Eagle Medium (Invitrogen) containing 10% Fetal Bovine Serum, 1% Penicillin-StreptomycinGlutamine (100x) liquid, and 25mM HEPES. Gel Mobility Shift Assay Gel m obility shift assay was performed as described previously (Gaubatz et al. 1998). Briefly, 5 l of reticulocyte lysates with or without in vitro -translated proteins (see below) were incubated with 0.1 picomoles of PAGE purified, 32P-end-labeled, annealed oligonucleotides (5-

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31 TCAGCGCCCGCTTTCCCGC CAGGGTAAAGCT-3) corresponding to the WT E2F6-binding element (underlined) in the murine Ant4 promoter. Competition experiments were performed with wild-type and mutant (5-TCAGCGCCCGCTTTCTTAA CAGGGTAAAGCT-3) unlabeled, double-stranded oligonucleotides corre sponding to the E2F6 site derived from the Ant4 promoter, whereby the amount of unlabeled pr obe relative to the amount of labeled probe was either 20, 50, or 100 fold in excess. For preparation of in vitro -translated proteins, one microgram of HA-E2F6 and Myc-DP2 expression v ectors (constructed as described previously), were co-translated in vitro using a coupled transcription/tran slation reticulocyte lysate system (Promega, Madison, WI) according to ma nufacturers instructions (Gaubatz et al. 1998). Chromatin Immunoprecipitation R1 ES cells were plated on 100-mm plates at a density of 1 x 106 cells per plate and then fixed three days later in 1% formaldehyde for 10 minutes at room temperature. Cross-linking was attenuated by the addition of 125mM glycine. Cells were th en collected by scraping with a cell scraper in cold PBS with the following prot ease inhibitors (PIs): leupeptin, aprotinin, and phenylmethylsulfonyl fluoride. Cells were centrifuge d at 1200rpm for 5 minutes at 4C and then resuspended in cell lysis buffer (5mM PIPES pH 8.0, 85mM KCL, and 0.5% NP-40) plus PIs and incubated for 10 minutes on ice. Nuclei were pelleted at 5000 rpm fo r 5 minutes at 4C and then lysed on ice for 10 minutes in nuclei ly sis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, and 1% SDS) plus PIs. Next, chromatin was shear ed by sonication for 10 re pititions of 10 second bursts with 1 minute rests on ice using a power se tting of on a Fisher Scientific Sonicator Dismembrator Model 100. Lysates were centrif uged for 10 minutes and supernatants were collected, diluted, and a sample was kept as I nput DNA. The supernatant was then pre-cleared with Protein G agarose (Invitrogen) for twenty minutes and collected again following centrifugation. Approximately 2 g of each antibody; control Ig G, (Sigma-Aldrich) and mouse

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32 monoclonal E2f6 (Storre et al 2002), were added to the supern atant and incubated overnight at 4C. The following day, 60 l of Protein G Agarose-50% slurry was added to the reaction and incubated for 1 hour rocking at 4C. The agarose complex was then collected, washed seven times with LiCl wash buffer (0.25M LiCl 0.5% NP-40, 0.5% DOC, 1mM EDTA, and 10mM Tris pH 8.0), and DNA was eluted off with el ution buffer (50mM Tris pH 8.0, 1% SDS, and 10mM EDTA). Crosslinking was reversed by inc ubating the eluted chromatin in 175mM NaCl at 65C overnight followed by 2 hours of Proteinase K treatment at 55C to degrade the proteins. DNA was recovered using phenol/chloroform extrac tion followed by PCR Purification (Qiagen, Valencia, CA). PCR was performed with ei ther semi-quantitative PCR using the Taq DNA polymerase kit (Eppendorf, Westbury, NY) or w ith real-time quantitative PCR (as described below). The primer sequences used for semi-quantitative PCR analysis of Chromatin Immunoprecipitation (ChIP) are desc ribed in Table 2-1. Data from at least three independent experiments were analyzed by semi-quantitative PCR and expressed as means standard deviations (SD). Real-Time Quantitative PCR The prim ers and probes used for real-time PCR were designed with Primer Express software and synthesized at Applied Biosystems. Each real-time PCR reaction was performed in a 25l reaction mixture c ontaining 50ng of cDNA, 10 l of 2X TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), and 1 l of 20X TaqMan Gene Expression Assay Mix (Applied Biosystems) containing the primers and probes. The reaction mixture was incubated for 10 minutes at 95C and repeat ed for 40 cycles (denaturing for 15 seconds at 95C and annealing and extending for 1 minute at 60C) using Applied Biosys tems 7900HT RealTime PCR System. The TaqMan primers and pr obes for monitoring transcription levels of Ant4 E2f1, E2f6, Dmc1, and Nanog were assay-on-demand gene expression products labeled with

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33 FAM reporter dye (Applied Biosys tems). A mixture of mouse -actin primers with VIC-labeled -actin probe was used as endogenous control (A pplied Biosystems, catalog number 4352341E). The primer sequences and probes for quantifyi ng E2f6 enrichment after performing ChIP analysis were custom ordered: -actin : sense primer 5-CGGAGGCTATTCCTGTACATCTG3, antisense primer 5-CGAGATTGAGGAAGA GGATGAAGAG-3, FAM-labeled probe 5CCAGCACCCATCGCC-3; Ant4 : sense primer 5-GCTGTTC TCCCAGCATCCT-3, antisense primer 5-GAGAACTGGAAAACCGCTTC AG-3, FAM-labeled probe 5CTTTCCCGCCAGGGTAA-3; Dmc1 : sense primer 5'-GGCCCCGCCCATCAA-3', antisense primer 5'-CGCCCTGTCGTCGAACA-3', and FA M-labeled probe 5'-CCGCGGCCCTCATT-3' All samples were tested in triplicate and expressed as means standard deviations (SD). Analysis of results was performed using SDS v2.3 software (Applied Biosystems) according to the manufacturers instructions. The comparative CT method ( CT) was used for quantification of gene expression. Plasmid Construction and Site-Directed Mu tagenesis The Ant4 promoter region (from -263bp to +25b p) was PCR-amplified from mouse R1 ES cells genomic DNA with high fidelity LA-TaqTM (Takara, Otsu, Japan) using the following Ant4 primers: sense primer 5-AATCACCG GGTTGGTGTAG-3, antis ense primer 5GCCACACCAACACTCAAGC-3. The 288bp fragme nt was excised with Xho1 and HindIII (New England Biolabs, Ipswich, MA) using a QIAquick gel extraction kit (Qiagen). The Takara DNA ligation kit was then used to ligate the fragment of the Ant4 promoter into a PGL2-basic vector (Promega) containing a luciferase reporter gene. Mutations in the Ant4 -Luciferase reporter vector at the location of the E2F6-b inding element were generated using the QuikChange SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA) in combination with the following PAGE purified primers: Ant4 : sense, 5'-

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34 CCCAGCATCCTCAGCGCCCGCGCCAG GGTAAAGCTGAAGCGG -3', antisense, 5'CCGCTTCAGCTTTACCCTGGC GCGGGCGCTGAGGATGCTGGG -3' (site of mutation is underlined). Reactions consisted of 5 l of 10X reaction buffer, 50ng of vector template, 125ng each primer, 1 l of dNTP mix, and 1 l of pfuTurbo DNA polymerase up to a final volume of 50 l. Reaction conditions consisted of 1 cycle at 95C for 30 seconds, and then 18 cycles of 95C for 30 seconds, 55C for 1 minute, 68C for 9 minutes. Template vector was next degraded by the addition of 1 l of DpnI restriction endonuclease and incubated at 37C for 1 hour. Reactions were transformed into Max Efficiency DH5 chemically competent cells (Invitrogen) analyzed by gel electrop horesis, and sequenced. Transient Transfection and Reporter Assays NIH3T3 cells were plated at a density of 1 x 105 cells per well in 6-well plates. After 24 hours, FuGENE 6 (Roche) was used to transf ect the following according to manufacturers instructions: 0.5 g Ant4 -Luciferase reporter vector (wild-type or mutant), 0.1 g pRL-TKRenilla internal control vector (Promega), and 1.0 g of one of the following vectors: HA-E2F6, HACE2F6, HA-E68-E2F6, HAN-E2F6 (constructed as we prev iously described) or pCMVTag2 empty control vector (Stratagene) (Gaubatz et al. 1998). Twenty-four hours after transfection, the cells were harvested. Firefly and Renilla luciferase activities were measured using a dualluciferase reporter assay system (Promega) according to manufacturers instructions. The firefly luciferase data for each sample was normalized based on transfection efficiency as measured by Renilla luciferase activity. Data from at least three independent experiments were analyzed and expressed as means standard deviations (SD). Statistical analysis was performed by Student's t test and P values of less than 0.05 were considered significant.

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35 Immunoblotting NIH3T3 cells trans iently transfected with one of the following E2F6 expression vectors: HA-E2F6, HAC-E2F6, HA-E68-E2F6, or HAN-E2F6 using Fugene 6 (Roche), were lysed in RIPA buffer (50mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS) plus PIs, and then harvested by scraping with a cell scraper. Ce lls were incubated for 20 minutes on ice and centrifuged. Supernatants were transferred to fresh tubes and total protein was normalized by Lowry assay (Bio-Rad, Hercules, CA). Next, 25 g of total protein was separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking with 4% bovine serum albumin (BSA) solution in TBST buffer (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% Tween 20) for 1 hour at room temperature, the membrane was incubated overnight with anti-HA primary an tibody (1:250 dilution; Cell Signaling Technology). After incubation for one hour with anti-mouse secondary antibody conjugated to horseradish peroxidase (1:5000 dilution; Santa Cruz Biot echnology, Santa Cruz, CA), chemiluminescent detection (GE Healthcare, Piscataway, NJ) of horseradish peroxidase activity was performed. Reverse Transcription-PCR Total RNA was extracted using an RNAqueous kit (Am bion, Austin, TX). The cDNA was synthesized using a SuperScript II firs t-strand synthesis system with oligo(dT) (Invitrogen) according to manufacturers in structions. PCR was performed using Taq DNA polymerase (Eppendorf) with the primer sequences lis ted in Table 2-2. For the samples selected to be quantified using real-time PCR, an al ternative method of reve rse transcription was performed using 1 g of total RNA and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturers instructions. Primer sequences used in realtime PCR are mentioned above. Regardless of which method of PCR was used, the sense

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36 primers were always designed in different exons than the antisense primers in order to ensure that the PCR product represented the specific mRNA species and not ge nomic DNA background. Computational Analysis Evolutionarily conserved regions (ECRs) of the mouse and hum an Ant4 gene and all conserved E2F6 transcription factor binding sites (TFBS) located within the ECRs were identified using the rVista 2.0 software program (Loots & Ovcharenko 2004). Additionally, an interspecies comparison of the Ant4 promoter sequences containi ng the E2F6 TFBS was carried out using rVista. All genomic DNA sequences of meiosis-specific genes were obtained using the University of California at Santa Cruz (UCSC) Ge nome Bioinformatics software (Karolchik et al 2003). A genome-scale DNA pattern matching algorithm was used to identify all core E2F6binding elements (TCCCGC or GCGGGA depending on the direction of binding) within the proximal promoter regions (-1000bp to +1bp) of a ll genes in the entire mouse genome (based on 31,113 genes in the database) using Regulatory Sequence Analysis Tools (RSAT) software (Van-Heldin 2003). The statistical relevance of the rate at which core E2F6 binding elements appear in the genome was then examined. We considered DNA sequences with specified lengths of 100bp, 200bp, 500bp, or 1000bp and the probability that a particular subsequence (the E2F6binding element) appears exactly n times within these sequen ces was computed under the assumption that each of the four nucleotides (A, G, C, or T) appears randomly and with equal probability (Gentleman & Mullin 1989). This inform ation was used to obtain the probability that a particular subsequence appears at least once within a DNA sequence of a given length. Specifically, the individual probabilities correspo nding to each possible outcome of a trial must sum to one as a consequence of the three axio ms upon which probability theory is based. As such, the probability that a subsequence occurs at least once: )1( nP is given by Equation 2-1,

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37 where)0( nP represents the probability that the s ubsequence does not appear at all. The expression for) 0( nP is obtained from Gentleman & Mullin 1989. )0(1)1( nP nP (2-1) The present study examines th e probability that a bi-dir ectional DNA binding element will appear in a given sequence. Therefore, consid eration is required for the case of multiple subsequences, eg. one subsequence corresponding to each binding element. If the subsequences corresponding to each binding element are denoted A and B respectively, the probability that either A or B will occur within a given sequence is denoted as ) ( BAP and is expressed approximately as in Equation 2-2. This expre ssion also results directly from the axioms of probability. The expression shown in Equation 2-2 is actually an upper bound, as the true value must ideally account for cases where A and B occur simultaneously. )()()( BPAPBAP (2-2) Bisulfite Sequencing and Combined Bisu lfite Restriction Analysis (COBRA) Genomic DNA from WT and E2f6-/MEFs was bisulfite converted using EZ DNA Methylation Kit (Zymo Research, Orange, CA). Approximately 80ng of PCR purified bisulfite converted DNA was used as a template for each PCR analysis. Primers used for Ant4 COBRA and bisulfite sequencing were: sense prim er 5'-TTGTTGTGTATTGAT TGAGTATG-3, and antisense primer 5-AAAAAAAACTAAAAAAC-3. COBRA was performed by digesting the PCR products with Hha1. After digestion, th e COBRA reaction was ran on an 8% PAGE Gel and then stained for 5 minutes with ethidium br omide. For bisulfite sequencing, undigested PCR products were cloned into a pCR-2.1 TOPO cloni ng vector (Invitrogen) as we have previously described (Rodic et al 2005). Clones were then screened using blue and white screening, and white colonies were subsequently sequenced.

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38 Immunostaining Testes were harvested from 6-week-old w ild-type male mice. All mice have been maintained under standard specific-pathogenfree (SPF) conditions, and the procedures performed on the mice were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC). The tissu es were then fixed in a mild fixative (10% formalin) overnight with rocking. Following fi xation the tissues were dehydrated using an organic solvent (PBS-Citrasol). The tissues we re then imbedded in paraffin and sectioned. Deparafinized and rehydrated 5 m tissue sections were stained with goat polyclonal antibodies against mouse E2f6, or IgG control. Slides we re blocked for endogenous peroxidase activity and then unmasked in Target Retrieval Solution (Dak o, Carpinteria, CA). Antibody was applied at 1:100 for one hour at room temperature prior to identification usi ng the DAB Envision kit (Dako). Slides were counter stained with hematoxylin.

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39 Table 2-1. Primers used for analyzing Ch IP experiments with semi-quantitative PCR Primer nameOligonucleotide sequence (5' to 3') Ant4 senseACACGTGTTATGGTCACATGC Ant4 antisenseGCCTTCTTTGAAGACTGCTTCT Dmc1 senseCCACCCCCACAACCTGAG Dmc1 antisenseAGAAT TTTCAAGGCGACACC Nanog senseTCACACTGACATGAGTGTGG Nanog antisenseTCTGTGCAGAGCATCTCAGT Oct-4 senseACGCAGAGCCAGCACTTCTC Oct-4 antisenseCCAGTATTTCAGCCCATGTCC Smc1 senseGGAGCAGGGATCCAATTCC Smc1 antisenseGCCACGACTTGAAATTCTCC Spo11 senseAGCTTCATGTCCTCCCTGAA Spo11 antisenseAGGGCGTCGAAGAACGAG Stag3 senseAGGAACGTGGTTAGCACGAG Stag3 antisenseGGCTGAAGAAAGGTCACCAC Sycp1 senseATCCTCCGACAATTTTGAGC Sycp1 antisenseACACCCTCCACACCCTCAC Tuba3 senseTTGTTCCATGCTAAGTTGGATGTC Tuba3 antisenseCCGTTCCTCACCACTGGTCCTCAAC -Actin sense AAATGCTGCACTGTGCGGCG -Actin antisense AGGCAACTTTCGGAACGGCG

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40 Table 2-2. Primers used for semi-quantitative RT-PCR Primer nameOligonucleotide sequence (5' to 3') Ant4 senseTGGAGCAACATCCTTGTGTG Ant4 antisenseAGAAATGGGGTTTCCTTTGG Dmc1 senseAGAAT TTTCAAGGCGACACC Dmc1 antisenseCCACCCCCACAACCTGAG E2F6 senseAAGC TGCAGGCAGAACTCTC E2F6 antisenseTTC ACCCACTCGGGATACTC Nanog senseAGGGTCTGCTACTGAGATGCTCTG Nanog antisenseCAACCACTGGTTTTTCTGCCACCG Oct-4 senseTGGAGACTTTGCAGCCTGAG Oct-4 antisenseTGAATGCATGGGAGAGCCCA Rec8L sense AGGGCGTCGAAGAACGAG Rec8L antisenseAGCTTCATGTCCTCCCTGAA RecQ1 senseACTGGAATCCGTAGCCAGTG RecQ1 antisense AGAGCTGGAAGCATTCAACA RibC2 senseCCATGGAGGTAGCGATGTCT RibC2 antisenseGTGTTCCGCATGTCATTGTC Smc1 senseGCCACGACTTGAAATTCTCC Smc1 antisenseGGAGCAGGGATCCAATTCC Spo11 senseGTTGGCCATGGTGAAGAGAG Spo11 antisenseCTCAAGTTGCCAGCAATCAA Stag3 senseGGCTGAAGAAAGGTCACCAC Stag3 antisenseAGGAACGTGGTTAGCACGAG Sycp1 senseACACCCTCCACACCCTCAC Sycp1 antisenseATCCTCCGACAATTTTGAGC Tuba3 senseGGACCGGATCCGAAAACTGG Tuba3 antisenseGTCTGGAATTCTGTTAAGTCC -Actin sense TTCCTTCTTGGGTATGGAAT -Actin antisense GAGCAATGATCTTGATCTTC

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41 CHAPTER 3 REPRESSION OF ANT4 GENE EXPRESSION BY E2F6 Motivation Eukaryotic cells require energy for their survival. The majority of this energy is produced through oxidative phosphorylation, a pro cess whereby ATP is synthesized from ADP and inorganic phosphate. This occurs within the mitochondrial matrix and therefore requires that ADP and ATP be shuttled across both the inner and outer mitochondrial membranes. Although nonspecific porins can mediate this transport across the outer membrane, the inner membrane is highly selective and requires speci fic carrier proteins for any solu tes to get across. The adenine nucleotide translocases (Ants) are a family of solute carriers that facilitate the exchange of ATP for ADP across the inner membrane of the mitochondria (Dahout-Gonzalez et al. 2006). In this chapter, the Ant isoforms and their critical roles in cellu lar respiration, as witnessed by defects resulting from their mutations, are discussed. In particular, the regulatory mechanisms governing Ant gene expression are emphasized and new results on the transc riptional repression of the Ant4 isoform are presented. Background Adenine nucleotide translocases are the mo st abundant proteins of the mitochondrial inner membrane and are comprised of approxima tely 300-320 amino acid residues which form six transmembrane helices. The Ants transpor t ADP/ATP according to an exchange-diffusion mechanism with a one-to-one stoichiometry, thereby maintaining the adenine nucleotide pool at a constant level within the m itochondrial matrix (Dahout-Gonzalez et al. 2006). The Ants are encoded by nuclear genes whereby the ANT proteins are then tran slocated into the mitochondrial inner membrane (Rehling et al 2004).

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42 Ant genes have been cloned in numerous eukaryotic species including yeast and plants. Mammalian Ant genes have been cloned in human, rat, bovine, and most recently mouse. All of these species have multiple Ant isoforms of varying tissue specif icity. Until recently, it was thought that humans only had thre e members of the ANT family: ANT1 (SLC25A4), which is specific to heart and skeletal muscle, ANT2 (SLC25A5) which is expressed in rapidly growing cells and is inducible, and ANT3 (SLC25A6) which is ubiquitously expressed (Stepien et al. 1992, Lunardi et al. 1992). In contrast, mice have only two isoforms: Ant1 which is expressed in heart, brain, and skeletal muscle and is located on chromosome 8, and Ant2 which is expressed ubiquitously with the exception of skeletal musc le and is located on the X-chromosome (Levy et al 2000). Mouse Ant2 is the ortholog of human ANT2 and is believed to have the combined functions of both human ANT2 and ANT3 (Ellison et al. 1996). Recently, we and others identi fied a novel member of the Ant family, Ant4 in both mouse and humans (Dolce et al. 2005, Rodic et al. 2005, Kim et al. 2007). In humans, the Ant4 gene is located on chromosome 4 whereas in mice it is located on chromosome 3. In contrast to all other known Ants which only have four exons, Ant4 contains six exons in both mouse and human. The Ant4 gene is predicted to encode a 320 amino acid protein and a comparison between mouse and human indicates that the protein is mostly conserved, sharing an 88% overall amino acid identity (Figure 3-1A) (Rodic et al 2005). Ant4 also has high amino acid sequence homology with other previously identified mouse Ant proteins (70.1% and 69.1% overall identity to Ant1 and Ant2, respectively) (Figure 3-1B). Closer examination of Ant4s amino acid sequence reveals the presence of a signature sequence common to me mbers of the mitochondrial carrier family, a PX-[D/E]-X-X-[K/R] motif found three times at ~100 residues apart (Walker 1992). It is noteworthy that Ant4 has a unique extension of amino acids at both the Nterminus and the C-

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43 terminus. From the sequence analysis, it appe ars that Ant4 shares sufficient homology to be classified as a mitochondrial ADP/ATP transpor ter but its regions of unique sequence suggest that it may differ in function or tissue-specificity from the rest of the family. Upon further analysis, we did in fact find that Ant4s expression pattern is di fferent from all other Ants in that its transcripts are detectable only in testis (Brower et al. 2007). Defects resulting from disruption of Ant gene expression have been reported. In humans, there is a clinical manifestat ion associated with ANT1 known as autosomal dominant progressive external opthalmoplegia (adPEO ) (Hirano & DiMauro 2001). Th is adult-onset disorder is characterized by the appearance of external opthalmoplegia, ptosis, and progressive skeletal muscle weakness. Molecularly, adPEO is evidenced by the accumulation of numerous mitochondrial point mutations including many which are found in ANT1: A114P, L98P, A89D, D104G, and V289M substitutions. De ficiency of this ADP/ATP carrier has also been reported in striated muscle of patients affected with myopathy and in patients suffering from Sengers syndrome (Jordens et al. 2002). However, in Sengers syndrome, the ANT1 gene itself was not altered, indicating that the pa thology could result from either impairment of the carrier transcription or from impeded import into the mitochondrial membrane. Studies have also shown an impairment of ADP/ATP carrier function in heart despite the existence of high levels of total ca rrier proteins. Rather than a def ect in total protein levels, this impairment results from a shift in isoform expre ssion. It was shown that an increased amount of ANT1 isoform correlates with a decrease in ANT 2 isoform in dilated cardiomyopathy (Dorner et al. 2006). Phenotypes similar to those found in humans were observed in Ant1-/mice, where the mice were viable but developed mitochondria l myopathy and severe exercise intolerance along with hypertrophic cardiom yopathy as young adults (Graham et al 1997).

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44 Genetic disruption of Ant2 in mice presumably results in peri-neonatal lethality but this observation has not yet been publ ished in a scientific paper (www.patentdebate.com/PATAPP/20050091704). ANT2 has been found to be overexpressed in cancer cells (Chevrollier et al 2005). It is established that cancer cells display a glycolytic phenotype resulting from the downregulation of mitochondria-encoded genes and impairment of mitochondrial function. The ANT2 promoter contains a regulatory element which represses gene expression in response to oxygen. It is hypothesized that ANT2 imports glycolytic ATP into mitochondria in exchange for exporting ADP, a pr ocess which would sust ain basal mitochondrial activity under the predominant glyc olytic status of tumor cell prol iferation. There have been no reports regarding ANT2 or ANT3 mutations in human. Defects resulting from disruption of Ant4 gene expression are evidenced by the Ant4 knockout mice generated by our lab (Brower et al. 2007). We observed that males were sterile and lacked meiotic and post-meiotic cells, thereb y establishing that this ADP/ATP mitochondrial translocase is essential for successful progression of male germ cell meiosis. Furthermore, Ant4 deficient male mice exhibited increased levels of a poptotic cells with in the testis and the majority of these cells were identified as early spermatocytes. Given that Ant2 is encoded on the X chromosome and the X chromosome is inactivat ed via meiotic-sex chromosome inactivation (MSCI) (Turner 2007) at the onset of meiosis, we speculate that Ant4 may have evolved to compensate for the loss of Ant2 during MSCI. Thus, it is likely th at Ant4 functions as the sole mitochondrial ADP/ATP carrier during spermatogenesis. Although the Ants have been stud ied fairly extensively on a prot ein level, very little is known about the regulatory mechanisms governing Ant gene expression. The ANT1 gene is known to contain classic TATA and CCAAT elements within its promoter. Also found within

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45 the promoter is a positive regulatory element known as OXBOX which is bound by proteins present only in muscle cells (Li et al. 1990, Chung et al. 1992). Muscle-specific binding of transcription factors to OXBOX is t hought to account for the induction of ANT1 expression in muscle. The OXBOX element in the ANT1 promoter is overlapped by REBOX, a potentially negative regulatory element. Gel shift assays have demonstrated that proteins binding to REBOX are not muscle-specific, suggesting a potential mechanism for mediating ANT1 repression in non-muscle tissue. However, the mouse Ant1 gene lacks the OXBOX and REBOX elements identified in the human promoter. Similar to ANT1 a classic TATA box is present in the ANT2 promoter. ANT2 also contains three putative Sp1 contro l elements near its transcription start site. Two of the Sp1 elements, known as the AB box, are located 5 of the TATA box and work synergistically to activate ANT2 transcription (Li et al. 1996). However, activation via the AB box is modulated by three separate repressor regions. One of th ese is another Sp1 binding element, termed the C box, and is juxtaposed to the transcrip tion start. When the C box is occupied, ANT2 transcription is decreased. The second repressor region can be f ound in the distal promoter and is the site for NF1 binding (Barath et al. 2004). The third repressor re gion is located between the AB activation box and the distal repressor region and is also bound by NF1. Similar to ANT1 and ANT2 the ANT3 gene also has a classical TATA elem ent within its promoter, as well as 16 identified Sp1 elements. ANT3 has also been shown to be regulated by IL-4 and IFNvia STAT-dependent pathways in T cells (Jang & Lee 2003). In contrast to other ANTs a TATA box is absent in the ANT4 promoter. We have previously shown that CpG methylation is required for Ant4 repression (Rodic et al. 2005). We have also shown a correlati on between methylatio n and expression whereby in testis, Ant4 is

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46 highly expressed and hypomethylated while in all of the somatic tissues examined, Ant4 is repressed and hypermethylated. In particular, we showed Ant4 derepression in D10 embryoid bodies (EBs) upon addition of the DNA demeth ylating agent, 5-aza-dC. Likewise, Ant4 was derepressed in EBs lacking de novo DNA methyltransferases (Dnmts). In this study, we have investigated the transcriptional regulation of Ant4 with the aim of determining how Ant4 is repressed in all somatic cells. We believe that understanding Ant4 gene regulation is important given its unique meiosis-specific expression pattern and its essential role in spermatogenesis. Results After our recent identif ication of the novel Ant4 isoform and further characterization of the Ant4 -/mouse, we next examined the promoter region of Ant4 in search of potential transcription factor binding s ites (TFBS). Given that other Ant isoforms contain multiple Sp1 TFBS in their promoters which have been previous ly shown to play a role in their regulation (Li et al. 1996), we searched the mouse and rat Ant4 promoters for Sp1 TFBS. We compared the sequences of mouse and rat Ant4 ( Slc25a31) genes and surrounding non-coding regions using rVISTA sequence analysis software (Loots & Ovcharenko 2004). The rVISTA program identified several evolutionary conserved regi ons (ECRs) of sequence si milarity and located conserved Sp1 binding sites within the proximal pr omoter region (Figure 3-2A). However, when we compared the mouse Ant4 sequence to human Ant4 no conserved Sp1 binding sites were found (Figure 3-2B). We then ran a search fo r all conserved TFBS between mouse and human Ant4 and found that there was only a handful of conserved TFBS in the proximal promoter region (data not shown). One of these was an E2F binding site with the core E2F6 sequence, TCCCGC (Figure 3-3A). This potential E2F TFBS was located 36bp upstream of the transcription initiation site and an interspecies sequence comparison indicated that the E2F6 TFBS and surrounding flanking regions were mostly conserved (Figure 3-3B). We chose to

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47 investigate this E2F6 site further due to previous literature implicating E2F6 in the repression of meiosis-specific genes (Storre et al 2005, Pohlers et al 2005). Next, we investigated the functional significance of the E2F6 TFBS in the murine Ant4 promoter by analyzing the ability of E2F6 to interact with this site. To address this question, we initially performed a gel retardation assa y to determine whether E2F6 would bind in vitro to a radiolabeled oligonucle otide encompassing the E2F6 TFBS in the Ant4 promoter (-54bp to 24bp). In order to prepare E2F6 protein and its binding co-factor DP2, we used a rabbit reticulocyte lysate in vitro translation (IVT) system to co-express HA-tagged E2F6 (HA-E2F6) and Myc-tagged DP2 (Myc-DP2). As shown in Figure 3-4, addition of the E2F6/DP2 protein complex to radio-labeled Ant4 probe generated an additional re tarded band. The specificity of this interaction was verified by competition w ith wild type (wt) and mutant (mut) cold competitors in 20, 50, or 100 fold excess amounts over radiolabeled Ant4 probe, whereby only the wt competitor was able to efficiently compete for binding to the E2F6 TFBS (filled arrow, Figure 3-4). It should be noted that the reticulocyte lysate us ed for IVT contains an endogenous protein which interacts with the Ant4 probe. This interaction is evidenced by the presence of a retarded band when the probe was incubated with control rabbit reticulocyte lysate in the absence of IVT (open arrow, Figure 3-4). However, ad dition of specific anti bodies against HA and Myc into the protein/probe mixture selectively redu ced the intensity of the bands indicated by the filled arrow as we previously demonstrated (Gaubatz et al. 1998) (data not shown). Subsequently, we analyzed the occupancy of this E2F6 TFBS in vivo using Chromatin immunoprecipitation (ChIP). Here we show th at endogenous E2f6 binds to the proximal promoter of Ant4 but not to -actin in undifferentiated R1 ES cells (Figure 3-5). Both semiquantitative PCR (Figure 3-5A) and quantitative real-time PCR (Figure 3-5B) analysis of ChIP

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48 resulted in the same conclusion. Coll ectively, these observations show that Ant4 contains a conserved E2F6 TFBS that binds E2F6 both in vitro and in vivo To clarify the role that E2F6 plays in the regulation of Ant4 transcription, we performed luciferase assays using an Ant4 reporter vector in combination with various E2F6 expression vectors. The Ant4 -Luciferase reporter contains a 288bp region of the Ant4 promoter (-263bp to +25bp) which includes the E2F6 TFBS. For transi ent transfections, we used NIH3T3 cells due to their high transfection efficiency and thei r apparent absence of endogenous E2f6 expression (data not shown). The activity of the wt Ant4 -Luciferase reporter si gnificantly decreased upon co-transfection with an HA-E2F6 expression vect or (Figure 3-6). However, when the E2F6 TFBS in the Ant4 -Luciferase reporter was mutated usi ng site-directed mutagenesis, cotransfection with HA-E2F6 failed to repress Ant4 transcription (Figure 3-6). Similarly, when the wt Ant4 reporter vector was co-transfected with HAE2F6 expression vectors in which either the DNA binding domain was mutated (HA-E68-E2F6) or the repression domain was mutated (HAC-E2F6), reporter activity was not repressed (Figure 3-7A). E2F6 proteins containing a mutation in the N-terminal domain (HAN-E2F6), which have been shown to retain their repressor activity, had similar repressive effects to that of WT HA-E2F6 (Gaubatz et al. 1998). Western blotting confirmed that th e E2F6 proteins were expressed at comparable levels when equivalent amounts of DNA were transfected (Figure 3-7B). These reporter assays indicate that the E2F6 protein can repress Ant4 providing its DNA binding and repressor domains are intact, and that this repression depends on sequence-specific DNA binding. To further elucidate the potential role of E2F6 in the repression of the Ant4 promoter, we next created stable ES cell lines overexpressing either HA-E2F6 or HAC-E2F6 expression vectors. In contrast to somatic cells many germ-cell-specific genes including Ant4 are

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49 considered to be transcriptionally permissive in ES cells and therefore have detectable mRNA transcripts (Rodic et al 2005, Geijsen et al 2004). The CpG island of the Ant4 gene promoter is hypomethylated in ES cells and a low level of Ant4 transcription is detectable. Using both semiquantitative RT-PCR (Figure 3-8A) and quantitative real-time PCR (F igure 3-8B), we show that overexpression of HA-E2F6 but not mutant HAC-E2F6 is able to reduce Ant4 transcription relative to parental R1 ES cells. This indica tes that increasing the amount of E2F6 protein present in a cell is sufficient to trigger E2F6-m ediated repression. Based on this result, it is reasonable to believe that the opposite scenario, in which E2F6 protein has been depleted from a cell, could potentially resu lt in the derepression of Ant4 transcription. To this end, we examined the expression of Ant4 in mouse embryonic fibroblast (MEF) cell lines derived from both wild type and E2f6 null mice. Using semi-quantitat ive PCR, we demonstrate that Ant4 is in fact aberrantly expressed in E2f6-/MEFs (Figure 3-9). Taken toge ther, these findings indicate that E2F6 is an essential repressor for the Ant4 gene in somatic cells. Given that E2F6 has been shown to be associated with polycomb pr oteins (discussed in Chapter 1), we analyzed the bind ing of the polycomb proteins, Eed and EZH2, to R1 ES cells at D0 and D6 of differentiation (Figure 3-10). Interestingly, we obs erved an increase in binding of Eed and EZH2 to the Ant4 promoter at D6 but not D0 of ES cell differentiation. The polycomb binding at D6 coincides with Ant4 repression by D6. However, it is a bit concerning that we still detected acetylated histone 3 (AH3), a marker of active chromatin, on D6 when Ant4 expression should be silenced. Because the ChIP primers were designed around the E2 F6 binding site in the Ant4 promoter, and both Eed and EZH2 cannot themse lves bind to DNA, we speculate that these proteins maybe using E2F6 as a binding platform. Upon binding to E2F6, they would then be able to exert their re pressive effects on the Ant4 promoter.

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50 An alternative mechanism of E2F6-mediated repression of Ant4 may be through the recruitment of DNA methyltran sferases. It was previous ly shown by our lab that DNA methylation is required for Ant4 repression during embryonic stem cell differentiation (Rodic et al 2005). We first wanted to confirm that Ant4 still required DNA methylation to remain repressed in somatic cells and not just initially during ES cell differentia tion. Therefore, we treated NIH-3T3 cells with the DNA demethylating agent, 5-Aza-Deoxycytidine (5-AZA-DC) (Figure 3-11). We found that DNA methylation was indeed required for the repression of Ant4 in somatic cells indicating the conti nued importance of DNA methylation in Ant4 repression. Additionally, we examined the effects of th e HDAC inihibitor, Trichostatin-A (TSA), on Ant4 expression. We found that in contra st to 5-AZA-DC, TSA had no affect on Ant4 expression indicating that histone acetylation was most likely not the mechanism of E2F6-mediated repression. Perhaps Ant4 repression not being effected by histone acetylation may help to explain how Ant4 can be repressed at D6 of ES cell diffe rentiation despite th e detection of AH3 (Figure 3-10). We next compared the methylation status of Ant4 in WT and E2f6-/MEFs using both COBRA and Bisulfite Sequencing (Figure 3-12). Interestingl y, we observed a reduction in DNA methylation at the Ant4 promoter in E2f6-/MEFs. However, this decrease, from 96% methylation in WT MEFs to 80% methylation in E2f6-/MEFs was minor. This is in contrast to the high percentage of demethylation found in the promoter region of the germ-cell-specific gene, Tuba3 (Figure 3-13). Findings regarding Tuba3 demethylation in E2f6-/MEFs were previously published (Pohlers et al. 2005), and the Tuba3 data presented in Figure 3-13 was generated in collaboration with Emily Smith and Dr. Resnick. Next, we checked for the binding of Dnmt3-containing complexes to the Ant4 promoter using ChIP assa y but the results appeared negative (Figure 3-10A). To further explor e the potential involvement of E2F6 in Ant4

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51 methylation, we compared our HAE2F6 ES cell line to WT R1 ES cells at D0 and D6 of ES cell differentiation. If E2F6 was repressing Ant4 by recruiting DNA methyltransferases, then we would expect to observe an increased rate of DNA methylation in thos e cells overexpressing E2F6. As shown in Figure 3-14, there was no su ch increase in DNA methylation leading us to believe that DNA methylation is not the primary mechanism of E2F6-mediated repression. Discussion In this study, we first demonstrat ed that the meiosis-specific gene, Ant4 contains an evolutionarily conserved E2F6 bindi ng site in its proximal promoter region. This indicates that E2F6-mediated regulation of Ant4 is likely important both within and between species. The importance of conserving such a binding site is highlighted by the fact that two-thirds of the sequence conserved among mammals is not prot ein-coding (Adams 2005). Therefore, the noncoding regulatory regions upon whic h transcription factors bind mu st play a large part in controlling the expression of protein-coding genes. From this knowledge, it is tempting to speculate that an in vivo disruption of the E2F6 bi nding site located at the Ant4 promoter could result in: 1) defects in fertility similar to those observed in the Ant4 knockout mouse, or 2) aberrant expression of Ant4 in somatic tissues. The data pres ented here are in support of the latter effect, although both possi bilities could be true. Addi tional studies whereby mice are created from mutations knocked-in to the E2F6 bi nding site will elucidate if such a binding site is required for Ant4 expression during meiosis and consequen tly fertility. For the remainder of this discussion, we will focus on the mechanisms of Ant4 repression in somatic tissues. From these experiments, it is clear that there are multiple players involved in the repression of Ant4 gene expression. The three key players that we found to be involved are E2F6, polycomb proteins, and DNA me thylation. Examination of these factors on an individual basis is what led us to the conclu sion that they were important to Ant4 repression. For E2F6,

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52 sufficient evidence came from the combined findings that E2F6 binds to the Ant4 promoter (Figures 3-4 and 3-5), that overexpression of E2F6 induces Ant4 repression (Figures 3-6, 3-7, and 3-8), and that Ant4 derepression occurs in E2f6-/MEFs (Figure 3-9). For polycomb proteins, proof came from the binding of Eed and Ezh2 to the Ant4 promoter in D6 EBs (Figure 3-10). Based on previous studi es in our lab, we already knew that DNA methylation was essential to Ant4 repression (Rodic et al. 2005). These experiments showed that Ant4 was derepressed in both ES cells follow ing treatment with 5-AZA-DC and in Dnmt3a Dnmt3b double null ES cells. Additionally, we demonstr ated the importance of DNA methylation in Ant4 repression by verifying that Ant4 was derepressed in somatic cells (NIH-3T3s) after treatment with 5-AZA-DC (Figure 3-11). Thes e findings support a role for E2F6, polycomb proteins, and DNA methylati on in the repression of Ant4 but fail to explain how these key components coordinate with one another to regulate Ant4 gene expression. A relationship between E2F6 and polycomb proteins has been observed in multiple studies (Ogawa et al 2002, Trimarchi et al 2001, Attwooll et al 2005). As discussed in Chapter 1, E2F6 associates with components of both the PRC2 and the PRC1 polycomb repressive complexes. Given that these polycomb protei ns do not bind to DNA directly, E2F6 has been suggested to serve as a platfo rm on which polycombs can bind. Binding to E2F6 would then enable these polycomb proteins to exert their repressive effects on DNA. In agreement with this concept is our finding that the PRC2 components, Ee d and Ezh2, appear to be associated with the Ant4 promoter in the same region that E2F6 is bound (Figure 3-10). Further, we demonstrate that only the endogenous E2F6 platform binds at D0 when Ant4 is expressed, whereas the Eed and Ezh2 polycomb proteins do not bind and assemble until just after Ant4 is repressed at around D6 of differentiation. This s uggests that although a docking plat form for the polycomb proteins

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53 may bind to polycomb-target promoters prior to polycomb complex assembly, gene repression will not occur until the full polycomb repressive complex has arrived and assembled on such a platform. Further studies will need to be performed to confirm these findings and to establish when and where the protein-protein interactions involved in such a repressive complex are occurring. An association between polycom b proteins and DNA methylati on has also been reported in literature. Recently, EZH2, a component of the PRC2 complex th at is known for its ability to methylate histone H3K27, was shown to physically interact with DNA methyltransferases (Vire et al 2006). This 2006 report was the first time that any evidence suggesting a cross-talk between these two silencing pathways had ever been presented. The study showed that knockdown of Dnmt1, 3a, or 3b led to derepression of EZH2-target genes. These target genes were also found to bind both EZH2 and the DNA methyltransferases Additionally, EZH2 overexpression increased the CpG methylation at EZH2 target promoters, whereas EZH2 knockdown decreased CpG methylation. Moreover, EZH2 was needed to bring Dnmts to the promoters of EZH2-target genes. Depletion of EZH2 disturbed this recruitment, and enabled the aberrant expression of target genes. Out of all the possible interactions between the key players involved in Ant4 repression, we focused our efforts on elucidating the c onnection between DNA met hylation and E2F6. Interestingly, a recent study found th at the germ cell-specific gene, Tuba3, was derepressed in E2f6-/MEFs (Pohlers et al. 2005). This derepression was co rrelated with a major reduction in DNA methylation at the Tuba3 promoter, suggesting that E2F6-m ediated repression involves the recruitment of Dnmts to target promoters which then locks them in an inactive state. Given that Ant4 is also a germ cell-specific gene, and that DNA methylation and E2F6 are both essential for

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54 Ant4 repression, we predicted that these key players w ould behave similarly in mediating the repression of Ant4 Surprisingly, Ant4 methylation in E2f6-/MEFs was only slightly reduced (~16%) (Figure 3-12) in comparison to the massive reduction reported for Tuba3 methylation (~69%) (Pohlers et al. 2005). We have proposed the following theories in an effort to explain why the Ant4 promoter remains mostly methylated in E2f6-/MEFs. The first theory to explain why th ere is only a slig ht reduction in Ant4 methylation is that there is heterogeneity among cultured MEF cells wh ere some cells are more differentiated than others. Recent studies have de monstrated that cell populations such as ES cells, which were once believed to be homogeneous, are in fact rather heterogeneous (Singh et al. 2007). Additionally, the methylation status of the germ line and pluripotent cell marker, Oct4 was shown to be heterogeneous among populations of somatic cells indicating that somatic cells may even exhibit some degree of heterogeneity (Marikawa et al. 2005). It is feasib le that within the mixture of cultured MEF cells, some cells may be more dependent on E2F6 for DNA methylation than others depending on their differentiation status. However, if this were the case, one would predict that the Tuba3 promoter would show a simila r distribution of methylation among these mixed cell types as Ant4 We examined the methylation status of the Tuba3 promoter in E2f6-/MEFs using COBRA (data not shown), and found that Tuba3 was indeed less methylated than Ant4 This finding eliminates the possibility that variations in culture conditions between our experiments and those from the previous report on Tuba3 are responsible for this difference in methylation. Thus, it is unlikely that cellular hete rogeneity can account for the slightly reduced Ant4 methylation in E2f6-/MEFs. An alternative theory for why there is very little demethylation of Ant4 in E2f6-/MEFs is that in these cells, Ant4 expression is leaky. The term l eaky generally refers to genes that

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55 are considered to be specific to one tissue type but then become expresse d in another tissue type at much lower levels. Instead of looking at the total percentage of methyl ation that is occurring on the Ant4 promoter in E2f6-/MEFs, examining the spatial arrangement of DNA methylation may provide more clues regarding whether Ant4 expression is indeed leaky. As shown in Figure 3-12, several of the Ant4 clones analyzed during bisu lfite sequencing are entirely demethylated. Thus, rather than thinking of an 80% overall methylation ra te, let us divide the multiple clones of Ant4 from bisulfite sequencing into tw o populations: one group which has 0% methylation (3 of the 18 clones) and another group with methylati on rates nearly equivalent to that found in WT MEFs (15 out of the 18 clones). Interestingly, we noticed this same All OR None distribution of methylation for Tuba3 clones from a previous publication reporting methylation of the Tuba3 promoter in E2f6-/MEFs (Pohlers et al. 2005). We rea lized that 7 out of 10 of these clones were 0% methylated and 3 out of 10 of the clones had high methylation rates equivalent to WT MEFs. Although the percentage of Tuba3 clones which were fully demethylated in E2f6-/MEFs was much greater than th e percentage demethylated for Ant4 the conserved All OR None pattern implies biologi cal relevance. In collaboration with Dr. Resnicks lab, we further showed that this same All OR None pattern exists at the Tuba3 promoter in E2f6-/mouse tail tissue (Figure 3-13) indicatin g that the heterogeneity of cultured MEF cells is most likely not responsible for these two different populations. Thus, if the difference in methylation between the All and None populations is not a result of varying degrees of differentiation among MEFs, but instead occurs in a rather homogeneous population of cells, then there must be some other existing phenomenon to explain these findings. This All OR None effect may be a result of an incomp lete functional redundancy. Although the body has developed backup mechanisms to ensure that processes which are

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56 essential to genome integrity be protected, of tentimes these backup mechanisms are not as efficient as the original. One such process th at may exemplify this point is DNA methylation. For successful DNA methylation to occur there n eeds to be functional DNA methyltransferases (Dnmts) as well as proteins which can recruit thes e Dnmts to target gene promoters. From the previous publication on Tuba3 expression in E2f6-/MEFs, it was suggested that E2F6-mediated repression occurred through E2F6 recruitment of Dnmts (Pohlers et al. 2005). Previous studies have also shown that E2F family members ofte n compensate for each other when another E2F family member is lost (Kong et al. 2007). More specifically, E2F4 has been shown to compensate for E2F6 by repressing E2F6-targe t genes when E2F6 is absent (Giagrande et al. 2004). Further, E2F4 has been found in a complex with Dnmt1 (Macaluso et al 2003). Therefore, we propose a model whereby under normal conditions, E2F4 and E2F6 may both have roles in recruiting Dnmt s to E2F-target genes (such as Tuba3 and Ant4 ) but that in E2f6-/MEFs, E2F4 must attempt to compensate fo r the loss of E2F6 by becoming the sole recruiter of Dnmts (Figure 3-15). It should be noted here th at many other proteins may be involved in the recruitment of Dn mts to promoters but for the purpos es of this discussion, we are specifically focusing on the recruitm ent of Dnmts to E2F-target gene s. Also, here we imply that E2F4 and E2F6 are the only Dnmt recruiters for E2F-target genes, but additional studies are needed to confirm such a theory. However, if this theory holds true, then E2F4-mediated recruitment of Dnmts is serving as a functionally redundant backup for E2F6 in E2f6-/MEFs. We hypothesize that although E2F4 can mostly compen sate for the loss of E2F6, it is likely that the efficiency of Dnmt recruitment to targ et promoters may be lower than under normal conditions. This reduction in efficiency is due to the fact that E2F4 now has double the workload. If E2F4 is successful in recruiting Dn mts to a specific promoter, then the methylation

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57 pattern at that promoter will be nearly identical to WT (Figure 3-15). However, when E2F4 fails to recruit Dnmt to a specific promoter because it is now overwhelmed with having to recruit Dnmts to so many additional promoters, then this promoter will show 0% methylation (Figure 315). This theory could explain w hy in some cells, methylation of Ant4 and Tuba3 promoters is completely missed. Simply stated, we believe that leaky expression of Ant4 is a direct consequence of incomplete functional redundancy. The extent of leaky expression is determined by the number of times in which E2F4 fails to recruit Dnmts to the Ant4 promoter in the absence of E2F6 (the clones having 0% methylation). A possible explanation for why a higher percentage of Tuba3 promoters are demethylated in E2f6-/MEFs could be that Tuba3 primarily relies more on E2F6 for Dnmt recruitment whereas Ant4 depends more on E2F4. Therefore, we predict that in an E2f4-/cell line we would see the opposite effect where Ant4 promoters are more demethylated than Tuba3 promoters. Additionally, if this functi onal redundancy model holds true, in a double E2f4-/E2f6-/cell line we would expect to see a n early 100% overall deme thylation rate for both Ant4 and Tuba3 promoters. Taken together, we pred ict that future experiments such as these will help to furthe r support the theory that Ant4 expression is leaky in E2f6-/MEFs due to incomplete functional redundancy. Additional experiments will be necessary to validate that E2F4 and E2F6 are indeed recruiters of Dnmts. We did not present the data here in this study, but attempts were made to demonstrate a physical interaction between Dnmt3 and E2F6. We transiently transfected tagged E2F6, Dnmt3a and Dnmt3b expression vectors in NIH-3T3 cells and then performed coimmunoprecipitation but saw no interaction between E2F6 and Dnmt3. Similarly, we tried to coimmunoprecipitate endogenous Dnmt3 with ex ogenous HAE2F6 in HAE2F6 ES cell lines but

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58 to no avail. It should be noted here that those studies were pre liminary and several repetitions as well as optimization studies would be needed be fore considering such results as reliable. Given the possibility that E2F6 and Dnmt3 ma y not physically intera ct with each other but could still be part of the same repressor co mplex, we performed ChIP. As shown in figure 310, we were unable to find Dnmt3 association with the Ant4 promoter region despite the presence of prominent E2F6 binding. However, the possibility that the Dnmt3 antibody quality was insufficient for ChIP assays cannot be ruled out. Additionally, because E2F6 overexpression was unable to en hance DNA methylation at the Ant4 promoter (Figure 3-14) we cannot draw any conclusions to suggest a rela tionship between E2F6 and DNA methylation at this time. However, according to our leaky expression theory in combination with the previous publication on Tuba3 (Pohlers et al. 2005), E2F6 should recruit Dnmts to E2F-target promoters. The validity of this theory is pending further results which demonstrate an interaction, either direct or indirect, between E2F6 and Dnmts. Moreover, these experiments have attempted to elucidate the regulatory mechanisms governing Ant4 repression in somatic cells by identifying E2F6 as an essential repressor. In the next chapter, we will further explore the relationship between E2F6 and other genes which are similar to Ant4

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59 Figure 3-1. Ant4 encodes a novel isoform of adenine nucleotide translocase. A) Deduced amino acid sequence of the mouse Ant4 gene is aligned with previously identified mouse Ant proteins. B) Deduced amino acid sequence of the mouse Ant4 gene is aligned with ANT4 human orthologue (from Rodic et. al 2005, Figure 2 on page 1318 with permission). hANT4 -M HREPA K KKAE KR LFD AS SF G KDLLAGGVAAAVSKT A VAPIERVKLLLQVQASSKQISP mAnt4 MS NESSK K QSSK KA LFD PV SF S KDLLAGGVAAAVSKT T VAPIERVKLLLQVQASSKQISP hANT4 EARYKGM V DCLVRIPREQGF F S F WRGNLANVIRYFPTQALNFAFKDKYK Q LFMSGVNKEK mAnt4 EARYKGM L DCLVRIPREQGF L S Y WRGNLANVIRYFPTQALNFAFKDKYK E LFMSGVNKEK hANT4 QFWRWFLANLASGGAAGATSLCVVYPLDFARTRLGVDIGKGPE E RQF K GLGDCIMKIAKS mAnt4 QFWRWFLANLASGGAAGATSLCVVYPLDFARTRLGVDIGKGPE Q RQF T GLGDCIMKIAKS hANT4 DG IA GLYQGFGVSVQGIIVYRASYFGAYDTVKGLLPKPK K TPFLVSF F IAQ V VTTCSGIL mAnt4 DG LI GLYQGFGVSVQGIIVYRASYFGAYDTVKGLLPKPK E TPFLVSF I IAQ I VTTCSGIL hANT4 SYPFDTVRRRMMMQSGE AK RQYKGT L DCF V KIY Q HEG ISS FFRGAFSN V LRGTGGALVLV mAnt4 SYPFDTVRRRMMMQSGE SD RQYKGT I DCF L KIY R HEG VPA FFRGAFSN I LRGTGGALVLV hANT4 LYDKIKEF FH ID I GGR---mAnt4 LYDKIKEF LN ID V GGSSSGDAnt1 -------------MGDQ AL SF L KD F LAGG I AAA V SKTAVAPIERVKLLLQVQ H A SKQI SA Ant2 -------------MTDA AV SF A KD F LAGG V AAA I SKTAVAPIERVKLLLQVQ H A SKQI TA Ant4 MSNESSKKQSSKKALFD PV SF S KD L LAGG V AAA V SKTAVAPIERVKLLLQVQ A S SKQI SP Ant1 EK Q YKG II DC V VRIP K EQG F LS F WRGNLANVIRYFPTQALNFAFKDKYK QI F LG GV DRHK Ant2 DK Q YKG II DC V VRIP K EQG V LS F WRGNLANVIRYFPTQALNFAFKDKYK QI F LG GV DKRT Ant4 EA R YKG ML DC L VRIP R EQG F LS Y WRGNLANVIRYFPTQALNFAFKDKYK EL F MS GV NKEK Ant1 QFWR Y FA G NLASGGAAGATSLC F VYPLDFARTRL AA D V GK GSS Q R E F N GLGDC LT KIF KS Ant2 QFWR Y FA G NLASGGAAGATSLC F VYPLDFARTRL AA D V GK AGA E R E F K GLGDC LV KIY KS Ant4 QFWR W FL A NLASGGAAGATSLC V VYPLDFARTRL GV D I GK GPE Q R Q F T GLGDC IM KIA KS Ant1 DG LK GLYQGF S VSVQGII I YRA A YFG V YDT A KG M LP D PK NVH IIV S WM IAQ S VT AV A G LV Ant2 DG IK GLYQGF N VSVQGII I YRA A YFG I YDT A KG M LP D PK NTH IFI S WM IAQ S VT AV A G LT Ant4 DG LI GLYQGF G VSVQGII V YRA S YFG A YDT V KG L LP K PK ETP FLV S FI IAQ I VT TC S G IL Ant1 SYPFDTVRRRMMMQSG RKG A D IM Y T GT L DC WR KI A KD EGAN AFF K GA W SN V LRGMGGA F V Ant2 SYPFDTVRRRMMMQSG RKG T D IM Y T GT L DC WR KI A RD EGSK AFF K GA W SN V LRGMGGA F V Ant4 SYPFDTVRRRMMMQSG E-S D RQ Y K GT I DC FL KI Y RH EGVP AFF R GA F SN I LRGTGGA L V Ant1 LVLYD E IK KYV----------Ant2 LVLYD E IK KYT----------Ant4 LVLYD K IK EFLNIDVGGSSSGDA B

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60 A B Figure 3-2. The conservation of Sp1 binding sites in the Ant4 promoter. Evolutionary conserved regions (ECRs) betwen A) rat a nd mouse and B) human and mouse Ant4 ( Slc25a31) genomic regions are indicated as peaks on the graph and are color-coded according to the type of genomic DNA in each ECR. Coding exons are in blue, untranslated regions are in yellow, intronic noncoding ECRs are in pink, and intergenic ECRs are in red. The Y-axis represents the per centage of conservation between the species being compared. Sp1 transcription factor binding sites (TFBS) which are conserved between mouse and rat but not between mouse and human are located within an intergenic ECR and an intron as indicated by vertical tick marks above the graph in A)

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61 Figure 3-3. The proximal promoter region of the Ant4 gene has a conserved E2F6 binding site. A) Evolutionary conserved regions (ECRs) of mouse and human Ant4 ( Slc25a31 ) genomic regions are indicated as peaks on the graph and are color-coded according to the type of genomic DNA in each ECR. Coding exons are in blue, untranslated regions are in yellow, intronic noncoding ECRs are in pink, and intergenic ECRs are in red. A scale is shown to indicate the width of the ECRs in kilobases (kb) as measured along the X-axis. The Y-axis re presents the percentage of conservation between mouse and human. The position of a single conserved E2F transcription factor binding site (TFBS) with the preferred E2F6 binding sequence (TCCCGC) was found within an intergenic ECR and is indi cated by a vertical gray tick mark above the graph. B) The E2F6 TFBS is located at 36 base pairs (bp) upstream of the transcription initiation site. The intergenic ECR containing the site is approximately 149bp in length and is conserved between sp ecies. The E2F6 binding site (dark gray) and conserved flanking regions (light gray) are shown for mouse, rat, dog, and human Ant4 A B

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62 Figure 3-4. Gel mobility shift assay of E2F6 binding to the Ant4 promoter. A labeled oligonucleotide corresponding to the E2F6 TFBS in the mouse Ant4 promoter (lane 1) was incubated with control rabbit reticuloc yte lysate alone (lane 2) or in vitro cotranslated HA-E2F6 and myc-DP2 proteins (lanes 3-9). Unlabeled Ant4 wild type (wt) (lanes 4-6) or mutated (mut) (lanes 7-9) oligonucleotides were added in excess amounts over the amount of labeled probe as in dicated. Filled arrow: position of the specific E2F6/DP2 complex, open arro w: endogenous binding activity in the reticulocyte lysate.

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63 Figure 3-5. The proximal prom oter region of Ant4 is bound by endogenous E2f6 in undifferentiated WT R1 ES cells. Chro matin immunoprecipitation (ChIP) was performed using mouse-anti E2f6 antibody. Primers amplifying the Ant4 proximal promoter region containing the E2F6 TFBS and -actin control primers lacking the E2F6 TFBS were used for both A) semiquantitative PCR and B) quantitative RealTime PCR analysis of ChIP. Samples from each primer set were precipitated with IgG to control for nonspecific enrichment. Enrichment of E2f6 binding is expressed as a percentage of the input chromatin. Input samples represen t 1% of the starting amount of chromatin and were analyzed to confirm that the different chromatin preparations contain equal amounts of DNA. All samples were tested in triplicate and expressed as means st andard deviations. A B

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64 Figure 3-6. E2F6-mediated repression of Ant4 requires an intact tran scription factor binding site. Ant4 promoter (-263 to +25bp) lucifera se reporter plasmids containing wild type (wt) or mutated (mut) E2F6 TFBS were tr ansiently co-transfected with an empty vector (-) or an E2F6 expression vector (+) into NIH-3T3 cells. Luciferase activity was measured relative to the Renilla internal control vector as described in Materials and Methods Chapter 2. Sequences for bot h wt and mut E2F6 TFBS reporters are shown above the bar graph. *P<0.05 and data from at least three independent experiments were analyzed and expressed as means standard deviations.

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65 Figure 3-7. E2F6-mediated repression of Ant4 requires an intact C-terminal and DNA binding domain. A) Luciferase reporte r assay in NIH-3T3 cells tran siently co-transfected with either empty vector (-), WT E2F6, or mutant (E68, N, or C) HA-E2F6 expression vectors and the Ant4 wt reporter vector. A schematic representation of the HA-tagged E2F6 vectors is shown above the graph; WT: intact E2F6, E68: a DNA binding domain point mutation, N: an N-terminus deletion mutant, C: a C-terminus deletion mutant, dark gray boxes: DNA binding domains, light gray boxes: dimerization domains, black boxes: marked boxes, and black box inside dark gray box: site of DNA binding domain mutation. B) Western blot anal ysis using anti-HA antibody, showing that all the E2F6 expres sion vectors transiently transfected for luciferase reporter assays in A) expre ssed their respective E2F6 proteins at comparable levels. For A), *P<0.05 and data from at least three independent experiments were analyzed and expressed as means standard deviations. A B

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66 Figure 3-8. Ant4 transcription is repressed by stable E2 F6 overexpression in ES cells. A) Semiquantitative RT-PCR analysis of Ant4 and -actin expression in parental R1, HAE2F6, and HAC-E2F6 ES cell lines. B) Quantit ative Real-Time PCR analysis of samples shown in A). All samples were tested in triplicate and expressed as means standard deviations. B A

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67 Figure 3-9. Ant4 transcription is derepressed in E2f6-/MEFs. Semi-quantitative RT-PCR analysis is shown for E2f6 Ant4, and -actin expression in WT MEFs and E2f6-/MEFs. Reverse transcriptase (RT) minus sa mples were also amplified to eliminate the possibility of genomic DNA contamination.

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68 Figure 3-10. The polycomb protei ns Eed and Ezh2 bind to the Ant4 promoter in D6 R1 EBs. Chromatin immunoprecipitation (ChIP) was pe rformed using antiE2f6, Eed, Ezh2, Dnmt3, Suz12, and acetylated histone H3 (AH3) antibodies. Primers amplifying the Ant4 proximal promoter region co ntaining the E2F6 TFBS and -actin control primers lacking the E2F6 TFBS were used for semi-quantitative PCR for A) undifferentiated ES cells (D0) and B) di fferentiated EBs (D6). Samples from each primer set were precipitate d with IgG to control for nonspecific enrichment. Input samples represent 1% of the starting amount of chromatin and were analyzed to confirm that the different chromatin prepar ations contain equal amounts of DNA. A B

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69 Figure 3-11. Ant4 is derepressed in NIH-3T3s after tr eatment with 5-AZA-DC but not TSA. 5 M or 10 M of AZA-DC were added either alon e or in combination with 200nm TSA for 65 hours in NIH-3T3s. RNA was harvested and Ant4 and -actin primers were used for semi-quantitative RT-PCR. Ant4 -actin

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70 Figure 3-12. CpG methylation at the Ant4 promoter is partially reduced in E2f6-/MEFs. A) COBRA analysis of the methylation status of Ant4 in various tissue types. Top band is undigested PCR product prior to COBR A digestion with Hh a1. Upon digestion there is three bands, the si ngle band marked as U represents unmethylated DNA; the two bands labeled M represent the methylated DNA. B) Bisulfite Sequencing analysis of the Ant4 promoter in WT MEFs (left) and E2f6-/MEFs (right) showing a decrease from 96% methylation in WT to 80% methylation in E2f6-/. A B

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71 Figure 3-13. Tuba3 methylation decreases in E2f6-/mouse tail DNA. Bisulfite Sequencing analysis of the Tuba3 promoter in WT mouse tail (top) and E2f6-/mouse tail (bottom) showing a decrease from 94% met hylation in WT to 37.5% methylation in E2f6-/(with permission from Emily Smith and Dr. Resnick).

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72 Figure 3-14. E2F6 overexpression does not increase CpG me thylation at the Ant4 promoter. Bisulfite Sequencing analysis compar ing the methylation status of the Ant4 promoter between WT R1 ES cells and HAE2F6 ES ce lls at A) D0 and B) D6 of ES cell differentiation. The Ant4 promoter region is shown by a horizontal line with an arrow indicating the transcription initiation site. Each vertical tick mark represents the location of a CpG site with 16 CpG sites residing within the sequenced region. A B

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73 Figure 3-15. The All OR None model of E2F4 compensation. In WT somatic cell types both E2F4 and E2F6 play a role in the methylation of Tuba3 and Ant4 promoters. Then, in E2f6-/tissues one of two things will happen. In the All model, E2F4 will compensate for the loss of E2F6 and completely methylate the Tuba3 and Ant4 promoters, thereby contributing to transcri ptional repression. In the None model, E2F4 is unable to compensate for the loss of E2F6 and will not methylate the Ant4 and Tuba3 promoters, thereby resulti ng in leaky expression.

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74 CHAPTER 4 REPRESSION OF ADDITIONAL MEIO SIS-SPECIFIC GENES B Y E2F6 Motivation The mechanisms controlling germ-cell-speci fic gene expression are diverse (Dejong 2006 Eddy 2002, MacLean & Wilkinson 2005). Studies have found these genes to be regulated extrinsically by hormones secreted from the endocri ne system, interactivel y by factors released from neighboring supportive cells in the gonads and intrinsically by factors affecting transcription, translation, DNA methylation, and histone modifi cations. Among such germ-cellspecific genes, it is especially important that th ose genes which are highly expressed and critical during the meiotic phase of gametogenesis be ap propriately regulated. Aberrant expression of these genes in somatic cells is presumed to be associated with disrupti ons in the mitotic cell cycle and may lead to dire consequences su ch as oncogenic transformation (Sagata 1997, Simpson et al 2005). Many of these critical genes ar e germ-cell-specific and are therefore repressed in all somatic cells in the body. Th is chapter focuses on the somatic repression of a group of germ-cell-specific genes which are similar to Ant4 in that their protein products function specifically during meiosis. Results suggesting the existence of a common transcriptional repressor of these meiosis-sp ecific genes are presented herein. Background Much of our current understanding regarding the transcriptional regulation of genes involved in meiosis can be credited to the use of transgenic mouse models. Studies have demonstrated that short proximal promoter regions are sufficient to specify somatic silencing and germ cell activation for meiotic genes such as: Sycp1 CyclinA1, Pgk2, HistoneH1t, Alf, and Pdha2 (Bartell et al 1996, Han et al 2004, Iannello et al 1997, Lele & Wolgemuth 2004, Robinson et al 1989, Sage et al 1999). A more detailed analysis of their minimal promoters

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75 revealed DNA regulatory elements harboring transcription factor binding sites. Further, incubation of these sequences with nuclear extrac ts from either somatic or testicular tissue indicated that nuclear proteins were indeed binding to these elements. Variations in binding between somatic and testicular nuclear extracts im ply that these proteins mediate testis-specific expression and somatic cell repression. For instance, Mos, a gene found to contain a negative regulatory element in its proximal promoter, is bound by a protein present only in nuclear extracts from somatic cells a nd not from pachytene spermatocytes (Xu & Cooper 1995). This observation, coupled with the absence of expression of Mos in somatic cells, infers that this protein may be serving as a transcriptional repressor. Further studi es revealed that this protein was COUP-TF (Lin et al 1999). Additional transcription factors which bind to various germcell-specific genes expressed during meiosis are beginning to be uncovered and include Sp1, Sp3, Ctf1, Rfx1, Rfx2, Ctcf, and Bmyb (Bartusel et al 2005, Gebara & McCarrey 1992, Horvath et al 2004, Kim et al 2006, Wilkerson et al 2002). However, the existence and identity of a master regulatory protein or protein family which binds to the proximal promoters and coordinately regulates the expression of multip le meiosis-specific genes uniformly as a group remains to be elucidated. Recently, the E2F6 transcription factor was s hown to be required for the repression of a subset of germ-cell-specific-genes in somatic cells. These six genes, Tuba3, Tuba7, XM196054 Tex12 Stag3 and Smc1 are aberrantly expressed in E2f6 null mouse embryonic fibroblasts (MEFs) and/or somatic organs (Pohlers et al 2005, Storre et al 2005). All of these germ-cellspecific genes contain the core E2F6-binding element, TCCCGC, within their proximal promoter regions (Cartwright et al 1998). Although the expression pattern s of these genes are similar in that they are all germ-cell-specif ic, their expression patterns and functions within germ cells are

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76 diverse. Tuba3 and Tuba7 are -tubulins which heterodimerize with -tubulins to form microtubules that constitute the primary structural components of mitotic and meiotic spindles (Hecht et al 1988). Tuba3 and Tuba7 are encoded by separate gene s but have identical protein products which are exclusively expressed in male gonads. More specifically, Tuba3 and Tuba7 are highly expressed in the metaphase spindles of spermatogenic cells undergoing meiosis and in the manchettes of elongated spermatids. Indeed, the Tuba3 gene initiates significant levels of expression at the onset of meiosis in primary spermatocytes (Wang et al 2005). Little is known about XM_196054 other than that it is a gene encodi ng a novel protein of unknown function with testis-specific expression (Storre et al 2005). Tex12 is a male-meiosis-specific component of the synaptonemal complex which is a group of prot eins involved in the al ignment and pairing of homologous chromosomes during prophase one of meiosis (Hamer et al 2006). Stag3 and Smc1 are also meiosis-specific pr oteins, but are components of the cohesion complex which is a group of proteins that act as a molecular glue to hold sist er chromatids together during prophase one of meiosis (Revenkova & Jessberger 2005). Stag3 is restricted to male meiotic germ cells whereas Smc1 can be found in both male and female meiotic germ cells (Pezzi et al 2000, Remelpva et al 2001). In the last chapter (Chapter 3), we presen ted evidence that E2F6 is required for the repression of another germ-cell-specific gene, Ant4 in somatic cells. Ant4 contains the core E2F6-binding element within its proximal prom oter region and, similar to Tex12, Stag3, and Smc1 its protein product is selectively expressed in meiotic cells (Brower et al 2007). This discovery prompted us to investigate whether additional germ-cell-specific genes require E2F6 for their repression. In particular, we focu sed on a subpopulation of germ-cell-specific genes whose expression is not only high in meiotic cells but is also claimed to be restricted to the

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77 meiotic phase of germ cell development. Here we show that most of the 24 meiosis-specific genes examined in this study have the core E2F6-binding element within 200 basepairs (bp) upstream of their transcription in itiation sites. Moreover, using murine embryonic stem (ES) cell culture, we demonstrate that E2F6 indeed plays a broad role in the transc riptional repression of these meiosis-specific genes by binding to their proximal promoter regions. Results The discovery that another meiosis-specific ge ne is regulated by E2F6 prompted us to investigate whether additional meiosis-specific gene s require E2F6 for their repression. After an extensive literature search and without bias we compiled a list of 24 genes which were previously reported to have meiosis-specific expression (Table 4-1). It should be noted here that we excluded genes from the list which continue to have predom inant levels of expression postmeiotically. For each of these meiosis-specific genes, we screened a genomic region spanning 1kb upstream of their transcripti on initiation sites for the pres ence of E2F6-binding elements (TCCCGC or GCGGGA, depending on the direction of binding) using UCSC Genome Browser (Karolchik et al 2003). As shown in Table 4-1, po tential E2F6-binding elements are accumulated within the proximal promoter (~200bp) regions of these meiosis-specific genes. In total, 19 out of the 24 meiosis-specific genes ( 79.2%) contain at least one E2F6-binding element within 200bp upstream of their transcription in itiation sites (white ba rs, Figure 4-1). The relevance of this finding was examined through the application of a genome-scale DNA pattern matching software tool, known as Regulatory Sequence Analysis Tool (RSAT) (Van-Heldin 2003). The RSAT software was used to identif y the E2F6-binding elements within promoter regions (-1000bp to +1bp) of all genes in the en tire mouse genome (based on 31,113 genes in the Ensembl database) (gray bars, Figure 4-1). A comparison between the meiosis-specific genes

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78 and all genes in the mouse genome indicates th at the proximal promoter regions of these meiosis-specific genes are indeed enri ched with E2F6-binding elements. As an additional measure of enrichment, the probability that an E2F6-binding element would randomly occur within a nucleotide sequen ce of a specified length was calculated. This calculation was made under the assu mption that the four nucleotide base pairs (A, G, C, or T) occur at random and with equal probability at each nucleotide position (Gentleman & Mullin 1989). The resulting value can be considered as the expected rate of random occurrence with which the E2F6-binding element appears thr oughout a population of actual DNA sequences, and is used here as a reference poi nt for statistical significance (black bars, Figure 4-1). A comparison between this expected rate of random occurrence and the actual frequency of E2F6binding element appearance indicates that the E2 F6-binding element is specifically enriched towards the proximal, but not the distal, promoter regions of all genes (gray bars, Figure 4-1). The rate of occurrence is shown to be appreciably elevated for meiosis-specific genes (white bars, Figure 4-1). The selectivity of the E2F6 -binding element to the proximal promoter, a genomic region which is known to harbor binding sites of critical tran scriptional regulators, suggests that this E2F6-binding element possesse s a high degree of functional significance. Next, we examined whether E2F6 binds to the E2F6-binding elements found in the proximal promoter regions of meiosis-specific genes. We performed ChIP analysis using undifferentiated ES cells and observed endogenous E2f6 binding to several meiosis-specific genes (Figure 4-2). Figure 4-2A shows E2f6 bi nding to the promoters of genes which are known to be derepressed in E2f6 -/MEFs. To clarify, Tuba3 is one of these derepressed genes but its expression is not restricted to meiotic cells and was therefore not included in Table 1. Figure 42B suggests that several of the newly identified E2F6-binding elements in the proximal

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79 promoters of meiosis-specific gene s (see Table 4-1) are in fact occupied by E2f6. Figure 4-2C verifies the specificity of E2F6 binding as evidenced by the absence of E2f6 binding in both housekeeping and ES cell-pluripotency genes. Fi gure 4-2D is a quantitative analysis from a sampling of genes in Figu res 4-2A, 4-2B, and 4-2C. Next, we looked to see whether exoge nous E2F6 overexpression was capable of repressing meiosis-specific ge nes. Using HA-E2F6 and HAC-E2F6 ES cells, we demonstrate that meiosis-specific transcripts are specifica lly reduced in HA-E2F6 but not in mutant HACE2F6 ES cells relative to pare ntal R1 ES cells (Figure 4-3). This observation brought about the reverse question of whether the absence of E2F6 would result in derepression of additional meiosis-specific genes beyond that of Stag3, Smc1, Tex12 and Ant4 We examined the expression status of meiosis-specific genes in E2f6-/MEFs for the presence of aberrant gene expression, but found that these additional meiosisspecific genes remain repressed (Figure 4-4). Overall, these observations suggest that E2F6 is not required fo r somatic cell repression of most meiotic genes and supports the possibility th at a functional redundancy exists whereby other safeguards are in place to ensure their repression in the absence of E2F6. Discussion During embryonic development, there is a co ordinated regulation of gene expression whereby genes whose protein products functi on in the same physiological processes are concomitantly expressed. Such coordinate regulation is thought to be contro lled, at least in part, by the presence of binding sites for the same tran scription factors in the promoter regions of these genes. Several studies have revealed co mmon transcription factor binding sites in groups of genes having similar tissue-specific expr ession patterns or phys iological roles (Kel et al 2001, Tronche et al 1997, Wasserman & Fickett 1998). For inst ance, previous studi es have screened DNA sequences from the EMBL and Genbank data banks and found that liver-specific gene

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80 promoters harbor binding sites fo r the HNF-1 transcription factor 2.5 times more frequently than do other genes (Tronche et al 1997). Likewise, NFAT/AP-1 bindi ng sites are presen t at rates of 10 times higher in the promoters of immune re sponse genes than in random sequences pulled from EPD and Genbank databases (Kel et al 2001). Further, the promoter regions of genes whose protein products are known to play a role in cell cycle pr ogression were found to have a high frequency of E2F binding site (the traditional TCGCGC site common to E2Fs 1-5 rather than the E2F6-preferred TCCCGC site) occu rrence in comparison to the promoters of functionally different genes (Kel et al 2001). In the present study, our examination of the promoter regions of a group of 24 meiosis-specific genes reveal ed a common E2F6 transcription factor binding site. Kel et al., defined a set of rules to pred ict whether a transcription factor binding site could contribute to the shared pattern of gene expression observed in sets of functionally related genes (Kel et al 2001). If a binding site met these criteria, it was termed a promoter-defining site. Several features of the common E2F6 binding site i nvestigated in this repo rt suggest that it is indeed a promoter-defining site. First, the site occurs in the meiosis-specific gene promoters at a frequency significantly higher th an in random sequences (Figur e 4-1). Second, the frequency of E2F6 binding site occurrence significantly differs between the prom oters of genes belonging to various functional groups. In this case, the occurrence of E2 F6 binding sites in the promoters of the meiosis-specific genes examined in this study was drastically elevated in comparison to the frequency of binding site occurrence in the promoters of all genes in the mouse genome (Figure 4-1). Third, the E2F6 binding site is located in close proximity to the transcription initiation site (Table 4-1). According to Kel et al., in order to be considered a promoterdefining site, the candidate site should o ccur within approximately 300bp upstream of the

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81 transcription initiation site (Kel et al 2001). The observation that 19 (79.2%) of the 24 meiosisspecific genes examined here had an E2F6 binding site within 200bp upstream of their transcription initiation sites is a convincing indicator that this E2F6 site should be considered promoter-defining. One of the fundamental questions raised by our results pertains to why almost every meiosis-specific gene promoter examined in this study contains an E2F6 binding site which binds E2f6 in vivo yet very few of these genes actually require E2F6 for their repression in somatic cells. This discrepancy does not elimin ate the potential role of E2F6 as a broad repressor of meiosis-specific ge nes, but rather suggests the ex istence of a functional redundancy whereby even in the absence of E2F6, another re pressor can compensate. Previous studies have shown that E2f4 can compensate for the loss of E2f6 by binding to the promoters of G1/Sregulated genes (which under normal circumstan ces are bound by E2f6) in the absence of E2f6 (Giagrande et al 2004). E2f4 -knockdown and E2f6 -null MEFs showed no derepression of these genes individually, but when both E2f4 and E2f6 were simultaneously inhibited using E2f4 knockdownE2f6 -null MEFs, specific derepression of G1/S-regulated genes was observed. Other studies have shown that E2F4 and E2F6 are concurrently bound to the promoters of numerous genes including: Cyclin A2 Cyclin B2 Cdc2 Art-27, Hp1 Rpab48 and Ctip (Caretti et al 2003, Oberley et al 2003). Interestingly, ChIP analysis of the Stag3 promoter shows binding of E2f6 and the activating E2f3 but not E2f4 in WT MEFs (Storre et al 2005). Perhaps this lack of redundancy with E2F4 could explain why Stag3 is aberrantly expressed in E2f6 -/MEFs. Conversely, Tuba3 and Tuba7 germ-cell-specific genes are also aberrantly expressed in E2f6 -/MEFs but do in fact bind E2F4 according to affinity chromatography and gel mobility shift assays (Pohlers et al 2005). This finding implies that the presence or absence of E2F4

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82 binding is not a sole criterion by which to decipher the occurrence of E2F6 functional redundancies. It should be noted that the E2F4 binding status of Tuba3 and Tuba7 promoters was not determined in WT MEFs, leaving open th e possibility that the discrepancies in binding between these promoters and Stag3 could be owed to differences in cell type. It would be nave to infer that the only transcription factors capa ble of compensating for the loss of E2F6 are other E2F family members. Transcriptional regulation is a combinatorial process whereby each gene is under the control of a multiplicity of elements, some of which can be dispersed over several kilobases. One exam ple of a redundancy that most likely does not involve other E2F family members may be Ribc2 Ribc2 is one of the many meiosis-specific genes that remain repressed in E2f6 -/MEFs (Figure 4-4). This observation is noteworthy given that Ribc2 and Smc1 genes overlap on chromosome 15 and are transcribed in opposite orientations with their promoter regi ons embedded within each other (Arango et al 2004). These genes are not only transcribed at similar times during meiosis, but they share an E2F6binding element in their overlapping promoter region which we have shown binds E2f6 in vivo (shown as Smc1, Figure 4-2). There are only 261bp of sequence in between the transcription initiation sites of Ribc2 and Smc1 Within this narrow region, the shared E2F6 site is located at -175bp to -170bp relative to Ribc2 and -47bp to -42bp relative to Smc1 transcription initiation sites An explanation regarding why Smc1 is derepressed in E2f6 -/MEFs but not Ribc2 could be partially due to a distance e ffect whereby the proximity of the E2F6-binding element to the transcription initiation site could determine the essentialness of E2F6 binding. Alternatively, DNA elements existing within intr ons and 3' regions specific to each gene may be accountable for the differential regulation. A lthough, the mechanisms responsible for this distinction in gene

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83 regulation remain unclear, a redundancy of an E2F family member at the location of this shared E2F6 binding site would most likely not account for such a phenomenon. The discovery that only a handful of meiosis-sp ecific genes were aberrantly expressed in somatic cells in the absence of E2F6 is in agreem ent with previous studies. Prior reports have described cDNA microarray experime nts with mRNA from WT and E2f6-/MEFs and shown that very few germ-cell-specific genes were upregulated in E2f6-/MEFs (Pohlers et al 2005, Storre et al 2005). Further, our findings are consis tent with the mild phenotype observed in E2f6-/mice. Although these mice display homeotic tr ansformations of the axial skeleton, they are viable, fertile, and display no signs of tumor formation (Storre et al 2002). A higher incidence of abnormally regulated meiosis-specif ic genes would likely result in a more severe phenotype. Evidence supporting this speculation is seen by the ab errant expression of germline genes in cancers whereby expression reflects the acquisition of the silenced gametogenic program in somatic cells (Simpson et al 2005). This acquisition is thought to be one of the driving forces in tumorigenesis. The proteins that become aberrantly expressed as a result of this phenomenon are termed cancer/testis antigens. Two examples of know n cancer/testis antigens which have meiosis-specific expression in normal tissues are Spo11 and Sycp1 (Simpson et al 2005, Tureci et al 1998). They are both components of the synaptonemal protein complex but have different functions. Spo11 causes double-st randed-breaks which are thought to initiate recombination events during meiosis (Keeney et al 1997). Sycp1 makes up the transverse filaments of the synapt onemal complex (Pousette et al 1997). It is theo rized that aberrant expression of either of these meiosis-specific proteins in mitotic cells leads to abnormal chromosome segregation and aneuploidy, the hallmarks of cancer cells (Simpson et al 2005). The finding that overexpression of Sycp1 in COS cells leads to the formation of synaptonemal

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84 complexes supports this theory (Ollinger et al 2005). In agreement with the absence of tumor formation in the E2f6-/mice; we find here that although E2 F6 binds to the promoters of both Spo11 and Sycp1 (Figure 4-2), they remain repressed in E2f6-/MEFs (Figure 4-4). Even though very few meiosis-specific gene s examined in this study require E2F6 for their repression in somatic cells, all of the genes analyzed here were at least partially repressed by E2F6 overexpression (Figure 43). The obvious question raised by this observation concerns the mechanisms by which E2F6-mediated repression occurs. As discussed in Chapter 1, there are several ways that E2F6 can repress its ta rget genes. Whether E2F6-mediated repression occurs via the same mechanism for all the genes we analyzed or whether each individual gene utilizes a different mode of E2F6 -induced repression remains to be determined. In particular, it will be important to distinguish whethe r those genes which are derepressed in E2f6-/MEFs utilize a different mechanism of E2F6-mediated repression than those which remain repressed. We predict that only those ge nes which are derepressed in E2f6-/MEFs will have repression mechanisms similar to those found for Ant4 (discussed in Chapter 3). Further studies analyzing the methylation status as well as polycomb pr otein occupancy at thes e meiosis-specific gene promoters are needed before such conclusions can be drawn. Another intere sting point is that the repression of Stag3 and Smc1 can be restored in E2f6-/MEFs reconstituted with E2F6 (Storre et al 2005). This observation brings into question, the ro le that E2F6 plays in initiating somatic cell repression versus main taining repression. Overall, the present study has provided eviden ce suggesting that E2 F6 may play a broad role in the repression of meiosis-specific genes. However, this role is most likely masked in vivo by the existence of functional redundancies where other repressive mechanisms are in place to ensure the repression of most me iosis-specific genes in somatic cells. Given the catastrophic

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85 outcome that can result from aberrant expr ession of meiosis-specifi c genes (Sagata 1997, Simpson et al 2005); it is not surprising that organism s have developed safeguards to guarantee their repression.

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86 Table 4-1. Locations of E2F6 TFBS (TCCCGC) within upstream promoter regions of meiosisspecific genes relative to their transcription initiation sites Gene Name Upstream Promoter Regions in Basepairs (bp) References +1 to -100 -100 to -200 -200 to -300 -300 to -400 -400 to -500 Ant4 (Slc25a31) (Brower et al. 2007) Boule* (Eberhart et al. 1996, Xu et al. 2001) Cyclin A1 (Ravnick & Wolgemuth 1999, Sweeney et al. 1996) Dmc1 (Lim15h) (Habu et al. 1996) Lamin C2* (Alsheimer et al. 1996) Meg1 (Calmegin) (Don & Wolgemuth 1992, Watanabe et al. 1994) Mnd1 (Petukhova et al. 2005, Pezza et al. 2006) Mns1 (Furukawa et al. 1994) Mzf6d (Looman et al. 2003) Prdm9 (Meisetz)* (Hayashi et al. 2005) Psmc3ip (Hop2)* (Petukhova et al. 2005, Pezza et al. 2006) Rec8 (Lee et al. 2003) RecQL (isoform beta) (Wang et al. 1998) Ribc2 (Trib) (Arango et al. 2004) Smc1 (Renekova et al. 2001) Spo11 (Shannon et al. 1999) Stag3 (Pezzi et al. 2000, Prieto et al. 2001) Syce1 (Costa et al. 2005) Syce2 (Cesc1) (Costa et al. 2005) Sycp1 (Scp1) (Meuwissen et al. 1992, Yuan et al. 1996) Sycp2 (Offenberg et al. 1998) Sycp3 (Di Carlo et al. 2000, Lammers et al. 1994) Tcte2* (Braidotti et al. 1997) Tex12 (Hamer et al. 2006) *These genes have an E2F6 TFBS within their 5 UTRs Denotes the presence of an E2F6 TFBS within the ups tream region of the corresponding to the column

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87 Figure 4-1. Frequency of appearance of E2F6 TFBS within upstream regions of genes relative to their transcription initiation sites. Bar graph indicates the occurrence rate of E2F6 TFBS for the following categories: Random: the probability that an E2F6 TFBS would randomly appear at least once within a sequence of a given length as described in Methods, RSAT: the actual frequency that the E2F6 TFBS occurs at least once within the proximal promoter regions of genes (31,113 genes) in the mouse genome, Meiosis-specific: the actual frequency that the E2F6 TFBS occurs at least once within a population of meiosis-specific genes (those listed in Table 1). The X-axis indicates the location of the E2F6 TFBS to be within 100bp, 200bp, 500bp, or 1000bp upstream of the transcription initiation site; the Y-axis denotes the percentage of genes from each category (see figure legend) th at have at least one E2F6 TFBS within a given upstream region.

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88 Figure 4-2. E2f6 binds to the promoters of meiosis-specific genes. Chromatin immunoprecipitation (ChIP) of endogenous E2f6 binding activity in WT R1 ES cells. ChIP was performed using mouse-anti-E2f6 antibody and analyzed by semiquantitative PCR. Input and IgG are shown as controls. E2f6 binding activity on: A) the promoters of meiotic genes whic h are known to be derepressed in E2f6-/MEFs, B) the promoters of other meiosis-speci fic genes, and C) the promoters of nonmeiotic ES cell pluripotency and -actin genes. D) Real-time PCR quantification of binding as determined by the ratio of speci fic ChIP/IgG ChIP re lative to input. All samples were tested in triplicate and expressed as means standard deviations. A BC D

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89 Figure 4-3. Many meiosis-specific genes are repressed by E2F6. Semi-quantitative RT-PCR analysis of meiosis-specific gene expression in R1, HA-E2F6, and HAC-E2F6 ES cells. A) Genes derepressed in E2f6-/MEFs. B) Other meiosis-specific genes. C) Non-meiotic ES cell pluripotency and -actin genes. D) Quantitative Real-Time PCR analysis of a representative group of samples from A), B), and C). All samples were tested in triplicate and expressed as means standard deviations. A B C 0 0.2 0.4 0.6 0.8 1 1.2 1.4R1 ESHAE2F6 ESDeltaC ESFold Change Oct4 Ant4 Sycp1 D

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90 Figure 4-4. Limited meiosis-speci fic genes are derepressed in E2f6-/MEFs. Semi-quantitative RT-PCR analysis of meiosis-specific gene e xpression in WT testis from 6-week old mice, R1 ES cells, WT MEFs, and E2f6-/MEFs. A) E2f6 and genes showing derepression in E2f6-/MEFs. B) Other meiosis-specific genes. C) Non-meiotic ES cell pluripotency genes and -actin genes. D) Quantitative Real-Time PCR analysis of a representative group of samples from A), B), and C). All samples were tested in triplicate and expressed as means standard deviations. A B C 0 0.2 0.4 0.6 0.8 1 1.2 R1 ES WT MEF E2F6-/MEFFold Change E2f6 Ant4 Dmc1 Nanog D

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91 CHAPTER 5 THE ROLE OF E2F6 DURING MALE MEIOSIS AND SPERMATOGENESIS Motivation From the data presented in Chapters 3 and 4, we can infer that E2F6 is involved in the repression of several meiosis-specific genes in somatic tissues. One of the remaining questions pertains to whether E2F6 also plays a role in regulating meiosi s-specific gene expression in nonsomatic tissues. During male germ cell development, there is a rapid transition in the expression of meiosis-specific genes from a repressed state in pre-meiotic spermatogonia, to a highly active state in meiotic spermatocytes, to a restored repressive state in post-meiotic round spermatids. Therefore, we have analyzed the expression pattern of E2F6 in mouse testis with the expectation of finding that, in agreement with E2F6s role as a transcriptional repressor, there would be high E2F6 expression in spermatogonia, followed by low expression in spermatocytes, and then high expression again in spermatids. Su rprisingly, the data presented he re contradicts our prediction. Such results have convinced us to explore the possibili ty that during meiosis, E2F6 may instead be serving as a transcriptional ac tivator of meiosis-specific genes. Herein, we discuss what is currently known about meiosis-spec ific gene activation in male ge rm cells, as well as speculate how our new findings on E2F6 expression may fit into these already es tablished paradigms. Background There are several ways by which meiosisspecific genes may become activated at the onset of meiosis. These include: 1) activati on by testis-specific tran scription factors, 2) activation by general transcripti on factors, 3) derepression by re moval of repressing factors, and 4) epigenetic alterations in chromatin structure which then promote transcriptional activation. One example of a testis-specific transcription factor that may activate meiotic genes is A-Myb. The murine A-myb gene arises from alternatively spliced mRNA and is expressed predominantly

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92 in the testis (Latham et al. 1996). In situ hybridization analysis of mouse testis shows that A-myb expression increases at post-natal day 10, when pr imary spermatocytes first appear. In the adult, A-myb mRNA is highly expressed in a sub-popul ation of spermatogonia and in primary spermatocytes, but is not detectable in sper matids. This expressi on pattern suggests that A-myb may play a role in activating meiosis-specific gene expression. Additionally, in A-myb-/male mice, germ cells enter meiotic prophase a nd arrest at pachytene, confirming that A-myb is required for the proper progression of meiosis (Toscani et al. 1997). Another myb family member, B-myb, is not testis-specific but is highly expressed in spermatogonia and early spermatocytes (Latham et al 1996). B-myb expression then d ecreases by the late pachytene spermatocyte stage. Further studies compari ng the differences between protein occupancy at meiosis-specific gene promoters incubated with e ither testis or somatic tissue nuclear extracts may reveal additional testis-specific activator proteins. Meiosis-specific genes have also been shown to be activated by general transcription factors which are ubiquitously expressed. For example, the widely expressed Sp1 protein activates transcription of the meiosis-specific gene, Cyclin A1 (Bartusel et al. 2005). Cyclin A1 contains Sp1 binding sites in its promoter region and it is suggested that Sp1s abil ity to activate Cyclin A1 is a result of Sp1 interactions with B-myb. Therefore, it is speculated that Sp1 and Bmyb are binding together as an activation comp lex at these Sp1 sites. Mutation of the Sp1 binding sites prevents Cyclin A1 transcription. Likewise, mutation of the C-terminal domain of B-myb inhibits Cyclin A1 activation, suggesting that th is domain is required for B-myb interaction with Sp1. Sp1 has also been shown to serve as a tr anscriptional repressor, but depending on the factors with which it associates, or alterna tively, depending upon how it is spliced, Sp1 may

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93 switch between being an activator and being a repressor (Thomas et al. 2007). Additionally, Sp1 has been found to associate with E2F family member s. When these two prot eins associate, they too serve as an activation complex. Such is the case at the murine thymidine kinase gene promoter (Rotheneder et al. 1999). Sp1 also interacts with the p107 pocket protein suggesting that Sp1 may change roles during the cell cycle (Datta et al. 1995). Other studies indicate that Sp1 can interact with compone nts of the general transcri ption machinery including TBPassociated factors (TAFs). The TAFs associate with histone modifying enzymes and chromatin remodeling factors to establish open ch romatin during spermatogenesis (Thomas et al. 2007). An alternative way in which meiosis-specifi c gene transcription may be activated, is through the removal of repressive factors which reside at the prom oters of these meiosis-specific genes. For example, polycomb proteins have b een shown to repress genes which are involved in promoting differentiation. Inte restingly, a recent paper using Drosophila as a model organism showed that these polycomb proteins are remove d from gene promoters at the onset of meiosis (Chen et al. 2005). Five testis-specific TAFs (tTAFs), which associate with the TBP portion of the general transcription factor TFIID and have been shown to be required for meiotic cell cycle progression, are responsible for counteracting th ese polycomb proteins. Chen and colleagues found that these tTAF proteins are concentrated in a particular subcompartment of the nucleolus. They then become expressed at the initiati on of spermatocyte differentiation and persist throughout the remainder of the primary spermatocyte stage, disappearing as cells enter the first meiotic division. This tTAF expression co incides with both the localization of PRC1 components to the nucleolus and th e activation of tTAF-target genes. This is in agreement with the finding that polycomb proteins ar e strongly expressed in all cells of the testis until the onset of meiosis (Ringrose et al. 2006).

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94 Further, mutation of tTAFs results in the inab ility of PRC1 component s to localize to the nucleolus, suggesting that tTAFs may be involved in the recruitm ent of PRC1 components away from target promoters and towards the nucleolus (Chen et al. 2005). ChIP analysis revealed that these tTAFs also bound to target promoters just upstream of their transc ription start sites, reduced polycomb binding, and promoted local accumulation of H3K4me3, a mark of Trithorax action. Trithorax proteins have been shown to activate transcription by opposing the repressive action of polycomb proteins (Ringrose et al. 2006). In tTAF mutant testis, PRC1 components continued to bind to tTAF-dependent target gene s. These findings suggest that tTAFs activate germ-cell-specific gene expre ssion by counteracting repression by polycomb proteins. Such transcriptional derepression by sequestration of polycomb proteins has also been observed during HIV-1 infection when the viral Nef protein recr uits the PRC2 component, Eed, to the plasma membrane (Witte et al. 2004). From these findings, it appear s that polycomb proteins may be blocking the expression of meio sis-specific genes. Upon entry into meiosis, polycomb repressive complexes are then disabled through tTAFs and these meiosis-specific genes become expressed. Whether the interactions between tTAF s and polycomb proteins ar e direct or indirect, remains to be elucidated. Meiosis-specific gene transcription may also be activated during meiosis as a result of reorganization in chromatin structure towards a conformation that is more accessible to RNA polymerase II. This restructuring often involves a switch from somatic isoforms to unique germcell-specific isoforms of various histones and histone modify ing enzymes. For example, during male gametogenesis several of the core histones (H2A, H2B, H3, and H4), along with linker histone H1, are partially or comple tely replaced by testis-specific histone isoforms such as H1t, TH2A, TH2B, and H3t (DeJong et al. 2006). These germ-cell-specific histones appear in

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95 spermatogonia, and later in spermatids, but the ma jority are synthesized and incorporated into chromatin during meiosis. Methylation of so matic histone H1 by the H3K27/H1K26 HMT and polycomb protein, Ezh2, seems to be important fo r transcriptional repression. However, testis H1 variants have glycine or al anine residues in place of lysine which makes them unlikely to be targeted by Ezh2, and suggests th at testis-specific H1 variants may represent a more active chromatin status than th eir somatic counterparts. In addition to unique histone isoforms, there are also differences in histone methyltransferases (HMTs) between somatic cells and germ cells. Many of these HMTs have roles in transcriptional repression, but one in particular may aid in the activation of meiosisspecific genes. This meiosis-specific HMT, know n as Meisetz (Prdm9), trimethylates histone H3K4. H3K4 trimethylation has been shown to be associated with active chromatin. Meisetz is specifically expressed in early meiotic germ ce lls in both testis and ovary (Matsui & Hayashi 2007). Interestingly, there is an E2F6 binding site in the proximal promoter region of this HMT (see Table 4-1). It is suggested that Meisetz may trimethyl ate H3K4 around a set of genes essential for meiosis, thereby activating their expression. A role for Meisetz in activating meiosis-specific genes is observed in Meisetz null mice which have specific abnormalities in meiotic prophase and fail to activate meiosis-specific genes (Hayashi et al. 2005). Also, under normal conditions H3K4 trimethylation is upregulated in pac hytene spermatocytes, further supporting the involvement of Meisetz in transcriptional activation. It should be noted here that although DNA demethylation may be an additional mechanism wh ereby meiosis-specific genes are derepressed at the onset of meiosis, it was not discussed here because to date, no DNA demethylases have been identified.

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96 Results After demonstrating that E2F6 indeed plays a role in the repression of meiosis-specific genes, we were curious to see if the expression pattern of E2F6 in the testis could help to explain our findings. As mentioned in the motivation por tion of this chapter, we postulated that E2F6 expression would be high in spermatogonia and then suddenly dr op off upon entry into meiosis, thereby releasing the repression of meiosis-specific genes. At the completion of meiosis, we predicted that E2F6 expression le vels would peak again in orde r to silence the expression of meiosis-specific genes in post-meiotic cells su ch as round spermatids an d sperm. We also predicted that the activ ating E2F, E2F1, would increase upon entry into meiosis in order to activate meiosis-specific gene expression. Surprisingly, when we used real-time PCR to examine the expression patterns of both E2f1 and E2f6 in purified spermatogenic cells harveste d at various stages of spermatogenesis (cells were a kind gift from JR McCarrey), we found expres sion patterns which were almost completely opposite to what we had predicte d (Figure 5-1). According to these data, E2f6 expression was highest in those cell types which were undergoing meiosis whereas E2f1 expression was drastically reduced at this stage. Additionally, both E2f1 and E2f6 expression was low in the somatic population of cells in the testis (sertoli cells). Further confirmation of this expression pattern came from the analysis of E2f6 protein levels in testis from 6 week old mice using immunohistochemistry (Figure 5-2). Staining revealed that E2f6 expression was highest in meiotic cells. The classification of cells as meiotic was determined by cellular morphology and cellular location within the seminiferous tubules. Staining appeared specific when compared to staining using IgG as a negati ve control. Further immunohistochemistry will be needed to confirm these results.

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97 Discussion There has only been one published study whic h describes the expression pattern of E2F family members in the testis (El-Darwish et al. 2006). In this report it was found that the E2F1 protein is stage-specific and mo st abundant in leptotene to ear ly pachytene spermatocytes of mice while strong staining of E2F1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated sp ermatogonia. These findi ngs are in contrast to the expression pattern that we observed for E2F1. Given the inherent nature of high backgrounds during immunohistochemical staining, we are tempted to trust our data more as we have quantitatively shown mRNA expression rather than subjectively characterizing expression. Further, this study showed discrepancies in E2F1 expression patterns between mouse and rat which seems highly unlikely cons idering the high homology of E2 F1 between the two species. Additionally, this study did not examine E2F6 expre ssion in testis. Therefore, for the remainder of this discussion, we will assume that the expression patterns found from our research are correct. One possible explanation for th is unexpected expression patte rn is that E2F6 has dual roles as both an activator and a repressor during di fferent stages of the cell cycle. These roles may be determined by differences in the complexes by which E2F6 is bound. For instance, if E2F6 represses transcription by recruiting either Dnmts or polyc omb protein complexes to the promoter, then the actual repression activity is coming from the proteins which were recruited by E2F6 rather than from E2F6 itself. Therefore, it is possible that in meiotic cells, E2F6 maybe recruiting an activation complex to meiosis-specif ic genes rather than a repressive complex. Such an activation complex could consist of com ponents which have already been shown to play a role in meiotic gene activation, such as Myb, Sp1, Meisetz, and tTAFs. Relationships between these proteins and E2Fs have already been sugg ested but need to be further elucidated.

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98 An explanation for why such a switch in E2Fassociated complexes could occur might be that the activation complex is suddenly able to compete with the repressive complex for binding at the onset of meiosis. For instance, if the total level of activation complex was upregulated upon entry into meiosis whereas th e repressive complex was downre gulated, then this shift in total protein could be responsible for the switch in E2F6-associated complexes. Further, a sudden change in cellular loca lization of activating and repr essing components could also explain the switch in complexes. Interestingly, both a shift in expressi on patterns and cellular localization was observed for tTAFs and polycom b proteins at the on set of meiosis (Chen et al. 2005). An additional function for the upregulation of E2F6 during meiosis could be that E2F6 is required to repress G1/S genes from becoming activated during th is time. Likewise, E2F1 may be downregulated here as it is an activator of genes which promote S phase entry. Therefore, rather than thinking narrow-mindedly about only meiosis-specific gene populations, it is plausible that there are a multitude of E2F1and E2F6-target genes which need to be regulated during meiosis. Indeed, experiments in which E2F1 was conditionally overexpressed in mouse testis resulted in testicular atrophy with semini ferous tubules containing only sertoli cells and clusters of undifferentiated spermatogonia (Agger et al 2005). Likewise, E2f6-/mice displayed a lack of mature spermatocytes (Storre et al. 2002). These findings sugg est that during meiosis, it is important to keep E2F1 off and E2F6 on in order to ensure proper completion of meiosis by preventing the cell from attempting to initiate a cell cycle program in the middle of meiosis. It is also possible that the role of E2F6, to inhibit G1/S-regulated genes, may not be as important as the role of E2F1, pending that E2F1 is never able to activate these G1/S regulated genes in the

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99 first place. This would explain why the E2f6-/mouse is still fertile but not the mice which overexpress E2F1. In summary, E2F6 may play a dual role as both an activator and a repressor during meiosis. E2F6-containing activation complexes may be bound to meiosis-specific genes while coexisting repressive E2F6 complexes might be bound to nonmeiotic gene promoters, thereby preventing the disruption of meiosi s. Alternatively, E2F6 may only serve as a repressor, and through some unknown mechanism, meiosis-specif ic gene promoters are protected from the binding of such repressive E2F6 complexes. Fu ture studies using ChIP assays to analyze the binding of E2F6 to both meiosis-specific genes and G1/S target genes during meiosis would clarify this hypothesis.

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100 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 pTATASTBSPLSLZSJPSAPSRSRCBJSCfold change E2F1 E2F6 Figure 5-1. E2f1 and E2f6 mRNA transcript levels in the testis. Real-time PCR was used to measure E2f1 (black) and E2f6 (pink) transcript levels in the following purified spermatogenic cell types: pTA, primitive Type A spermatogonia; TAS, Type A spermatogonia; TBS, Type B spermatogonia; PL S, Pre-leptotene spermatocytes; LZS, Leptotene/Zygotene spermatocytes; JPS, J uvenille pachytene spermatocytes; APS, Adult pachytene spermatocytes; RS, Round Spermatids; RCB, Residual Cytoplasmic Bodies; JSC, Juvenile (D6) Sertoli Cells. The relative transcript levels are shown with the transcript level of pTA for E2f1 and E2f6 set as 1. All samples were tested in triplicate and expressed as means standard deviations.

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101 Figure 5-2. Immunohistochemical an alysis of E2f6 expression in mouse testis. Formalin-fixed, paraffin-embedded sections of mouse testis from wild type 6-week old mice were incubated with (A) and B)) IgG antibody as a control or (C) and D)) E2f6 antibody. Staining was visualized using DAB ( brown), and slides were counterstained with hematoxylin. A) and C) images were take n at a 10X magnifica tion and B) and D) were at 40X. A B C D

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102 CHAPTER 6 CONCLUSIONS AND SIGNIFICANCE In summ ary, the research presented here has demonstrated a role for E2F6 in the repression of meiosis-specific genes in somatic cells. Although many questions remain, we have successfully achieved the original goal of this re search: to study the tran scriptional regulation of gene expression. We have elucidated the relati onship between a particular transcription factor (E2F6) and a specific set of genes (meiosis-s pecific genes). When transcription factors controlling gene expression are disrupted, dire consequences may result. Therefore, the significance of these findings re sides with the common observati on that many genes are often aberrantly expressed in cancer cells. Here we demonstrate that E2F6 depletion results in the ex pression of a few E2F6-target genes in inappropriate tissues. Such mischievous expression is a classic example of an event which can lead to oncogenic transformation (Simpson et al. 2005). Also, the E2F family of transcription factors is highly i nvolved in the regulation of the cell cycle and one of its most common binding partners, retinoblastoma, is a po cket protein which ha s consistently been demonstrated to play a role in oncogenic tr ansformation (Weinberg 1992). Because E2F6 does not bind to pocket proteins, it is commonly exclud ed when review papers discuss the role of E2Fs and Rb pocket proteins in cancer. Further, this is to be expected given that there was no indication of tumor formation in the E2f6-/mouse (Storre et al 2002). However, one thing that may be being overlooked is the potential for E2F6 to serve a negative feedback role in keeping the expression of genes which are ta rgets of the activating E2Fs in check. Such an important role for E2F6 may simply be being masked by func tional redundancy with E2F4. Although both of these proteins may be involved in negative feed back, their importance is somewhat diminished by the fact that they share the job. Theref ore, we believe that fu ture experiments which

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103 examine the relationship between E2F4, E2F6, and cancer will provide critical insights regarding the control of E2F-target genes. Taken toge ther, these findings are a small but important contribution to a broader understanding of the body s finite control of gene expression.

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104 LIST OF REFERENCES Adams MD 2005 Conserved sequences and the evolut ion of gene regulatory signals. Current Opinion in Genetics and Development 15 625-633. Agger K, Santoni-Rugiu ES, Ho lmberg C, Karlstrom O & Helin K 2005 Conditional E2F1 activation in tran sgenic mice causes testicular atrophy and dysplasia mimicking human CIS. Oncogene 24 780-789. Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Walter P 2002 Molecular Biology of the Cell, 4th edition. New York: Garland Science. Alsheimer M & Benavente R 1996 Change of karyoskeleton during mammalian spermatogenesis: expressi on pattern of nuclear Lamin C2 and its regulation. Experimental Cell Research 228 181-188. Arango NA, Pearson EJ, Donahoe PK & Teixeira J 2004 Genomic structure and expression analysis of the mouse tes tis-specific ribbon prot ein (Trib) gene. Gene 343 221-227. Attwooll C, Oddi S, Cartwright P, Prosperini E, Agger K, Steensgaard P, Wagener C, Sardet C, Moroni MC & Helin K 2005 A novel repressive E2F6 co mplex containing the polycomb group protein, EPC1, that interacts with EZH2 in a proliferation-specific manner. Journal of Biological Chemistry 280 1199-1208. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW & Echelard Y 1999 Production of goats by somatic cell nuclear transfer. Nature Biotechnology 17 456-461. Barath P, Poliakova D, Luciakova K & Nelson BD 2004 Identification of NF1 as a silencer protein of the human adenine nucleotide translocase-2 gene. European Journal of Biochemistry 271 1781-1788. Bartell JG, Davis T, Kremer EJ, Dewey MJ & Kistler WS 1996 Expression of the rat testisspecific Histone H1t gene in transgenic mice. Journal of Biological Chemistry 271 4046-4054. Bartusel T, Schubert S & Klempnauer K 2005 Regulation of the cyclin D1 and cyclin A1 promoters by B-Myb is mediat ed by Sp1 binding sites. Gene 351 171-180. Braidotti G & Barlow DP 1997 Identification of a ma le meiosis-specific gene, Tcte2 which is differentially spliced in species that form st erile hybrids with laborat ory mice and deleted in t chromosomes showing meiotic drive. Developmental Biology 186 85-99. Brower JV, Rodic N, Seki T, Jorgensen M, Fliess N, Yachnis AT, McCarrey JR, Oh SP & Terada N. 2007 Evolutionarily conserved mammalia n adenine nucleotide translocase 4 is essential for spermatogeneis. Journal of Biological Chemistry 282 29658-29666.

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105 Bruin A, Maiti B, Jakoi L, Timmers C, Buerki R & Leone G. 2003 Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation. Journal of Biological Chemistry 278 42041-42049. Campbell KHS, Ritchie WA, McWhir J & Wilmut I 1996 Sheep cloned by nuclear transfer from a cultured cell line. Nature 380 64-66. Caretti G, Salsi V, Vecchi C, Imbriano C & Mantovani R. 2003 Dynamic recruitment of NF-Y and histone acetyltransferases on cell-cycle promoters. Journal of Biological Chemistry 278 30435-30440. Cartwright P, Mller H, Wagener C, Holm K & Helin K 1998 E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription. Oncogene 17 611-623. Chen X, Hiller M, Sancak Y & Fuller MT 2005 Tissue-specific TAFs counteract polycomb to turn on terminal differentiation. Science 310 869-872. Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L & Renard JP 2002 Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20 366-369. Chevrollier A, Loiseau D, Chabi B, Reni er G, Douay O, Malthiery Y & Stepien G 2005 ANT2 isoform required for cancer cell glycolysis. Journal of Bioenergetics and Biomembranes 37 307317. Chung AB, Stepien G, Haraguchi Y, Li K & Wallace D 1992 Transcriptional control of nuclear genes for the mitochondrial muscle ADP/ATP translocator and the ATP Synthase Subunit. Journal of Biological Chemistry 267 21154-21161. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Je rry J, Blackwell C, Ponce dLn & Robl JM 1998 Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280 1256-1258. Cloud JE, Rogers C, Reza TL, Ziebold U, St one JR, Picard MH, Caron AM, Bronson RT & Lees JA 2002 Mutant mouse models reveal the relative roles of E2F1 and E2F3 in vivo. Molecular and Cellular Biology 22 2663-2672. Costa Y, Speed R, Ollinger R, Alsheimer M, Semple CA, Gautier P, Maratou K, Novak I, Hg C, Benavente R & Cooke H 2005 Two novel proteins r ecruited by synaptonemal complex protein 1 (SYCP1) are at the centre of meiosis. Journal of Cell Science 118 2755-2762. Dahout-Gonzalez C, Nury H, Trezeguet V, Lauquin GJM, Pebay-Paeyroula E & Brandolin G 2006 Molecular, functional, and pathological as pects of the mitochondrial ADP/ATP carrier. Physiology 21 242-249. Datta PK, Raychaudhuri P & Bagchi S 1995 Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2Fand S p1dependent transcription. Molecular and Cellular Biology 15 5444-5452.

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106 Dejong J 2006 Basic mechanisms for the contro l of germ cell gene expression. Gene 366 39-50. DeGregori J & Johnson DG 2006 Distinct and overlapping role s for E2F family members in transcription, prolifer ation, and apoptosis. Current Molecular Medicine 6 739-748. DiCarlo AD, Travia G & Felici MD 2000 The meiotic specific synaptonemal complex protein SCP3 is expressed by female and male prim ordial germ cells of the mouse embryo. International Journal of Developmental Biology 44 241-244. Dimova DK & Dyson NJ 2005 The E2F transcriptional networ k: old acquaintances with new faces. Oncogene 24 2810-2826. Dolce V, Scarcia P, Iacopetta D & Palmieri F 2005 A fourth ADP/ATP ca rrier isoform in man: identification, bacterial expression, functiona l characterization, a nd tissue distribution. FEBS Letters 579 633-637. Don J & Wolgemuth DJ 1992 Identification and characterizati on of the regulated pattern of expression of a novel mouse gene, meg1, during the meiotic cell cycle. Cell Growth and Differentiation 3 495-505. Dorner A, Giessen S, Gaub R, Grosse Siestrup H, Schwimmbeck PL, Hetzer R, Poller W & Schultheiss HP 2006 An isoform shift in the cardiac aden ine nucleotide translocase expression alters the kinetic properties of the carrier in dilated cardiomyopathy. European Journal of Heart Failure 8 81-89. Eberhart CG, Maines JZ & Wasserman SA 1996 Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia Nature 381 783-785. Eddy EM 2002 Male germ cell gene expression. Recent Progress in Hormone Research 57 103128. El-Darwish KS, Parvinen M & Toppari J 2006 Differential expression of members of the E2F family of transcription fa ctors in rodent testes. Reproductive Biology and Endocrinology 4 63-85. Ellison JW, Salido EC & Shapiro LJ 1996 Genetic mapping of the adenine nucleotide translocase-2 gene (Ant2) to the mouse proximal X chromosome. Genomics 36 369-371. Field SJ, Fong-Ying T, Kuo F, Zubiaga AM, Kaelin WG, Livingst on DM, Orkin SH & Greenberg ME 1996 E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85 549-561. Furukawa K, Inagaki H, Naruge T, Tabata S, Tomida T, Yamaguchi A, Yoshikuni M, Nagahami Y & Hotta Y 1994 cDNA cloning and functional ch aracterization of a meiosisspecific protein (MNS1) with a pparent nuclear association. Chromosome Research 2 99-113.

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107 Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R & Lazzari G 2003 Pregnancy: a cloned hors e born to its dam twin. Nature 424 635. Gaubatz S, Lindeman GJ, Ishida S, Jakoi L, Nevins JR, Livingst on DM & Rempel RE 2000 E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Molecular Cell 6 729-735. Gaubatz S, Wood JG & Livingston DM 1998 Unusual proliferation ar rest and transcriptional control properties of a newly disc overed E2F family member, E2F6. Proceedings of the National Academy of Sciences 95 9190-9195. Gebara MM & McCarrey JR 1992 Protein-DNA interactions associ ated with the onset of testisspecific expression of the mammalian Pgk-2 gene. Molecular and Cellular Biology 12 14221431. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K & Daley GQ 2004 Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427 148-154. Gentleman JF & Mullin RC 1989 The distribution of the freque ncy of occurrence of nucleotide subsequences, based on their overlap capability. Biometrics 45 35-52. Giangrande PH, Zhu W, Schlisio S, Sun X, Mori S, Gaubatz S & Nevins JR 2004 A role for E2F6 in distinguishing G1/Sand G2/M-specific transcription. Genes and Development 18 29412951. Graham BH, Waymire KG, Cottrell B, Trounce IA, MacGregor GR & Wallace DC 1997 A mouse model for mitochondrial myopathy and cardio myopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator. Nature Genetics 16 226-234. Gurdon JB 1962 Adult frogs derived from the nuclei of single somatic cells. Developmental Biology 4 256-273. Habu T, Takuyu T, West A, Nishimune Y & Morita T 1996 The mouse and human homologs of DMC1, the yeast meiosis-specific homologous re combination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic Acids Research 24 470-477. Hamer G, Gell K, Kouznetsova A, Novak I, Benavente R & Hg C 2006 Characterization of a novel meiosis-specific protein within the centr al element of the synaptonemal complex. Journal of Cell Science 119 4025-4032. Han SY, Xie W, Kim SH, Yue L & DeJong J 2004 A short core promoter drives expression of the ALF transcription factor in reproductiv e tissues of male and female mice. Biology of Reproduction 71 933-941. Hayashi K, Yoshida K & Matsui Y 2005 A histone H3 methyltransf erase controls epigenetic events required for meiotic prophase. Nature 438 374-378.

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108 Hecht NB, Distel RJ, Yelick PC, Tanhauser SM, Driscoll CE, Goldberg E & Tung KSK 1988 Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain. Molecular and Cellular Biology 8 966-1000. Hirano M & DiMauro S 2001 ANT1, Twinkle, POLG, and TP: new genes open our eyes to opthalmoplegia. Neurology 57 2163-2165. Horvath GC, Kistler WS & Kistler MK 2004 RFX2 is a potential tran scriptional regulatory factor for Histone H1t and other genes expressed during the meiotic phase of spermatogenesis. Biology of Reproduction 71 1551-1559. Humbert PO, Rogers C, Ganiastsas S, Landsb erg RL, Trimarchi JM, Dandapani S, Brugnara C, Erdman S, Schrenzel M, Bronson RT & Lees JA 2000 E2F4 is essential for normal erythrocyte maturation and neonatal viability. Molecular Cell 6 281-291. Iannello RC, Young J, Sumarsono S, Tymms MJ, Dahl HM, Gould J, Hedger M & Kola I 1997 Regulation of Pdha-2 expression is medi ated by proximal promoter sequences and CpG methylation. Molecular and Cellular Biology 17 612-619. Jang J & Lee C 2003 Mitochondrial adenine nucleotide tran slocator 3 is regulated by IL-4 and IFNvia STAT-dependent pathways. Cellular Immunology 226 11-19. Johnson DG, Schwarz JK, Cress WD & Nevins JR 1993 Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365 349-35. Jordens EZ, Palmieri L, Huizing M, van den Heuvel LP, Sengers RC, Dorner A, Ruienbee, W, Trijbels FJ, Valsson J, Sigfusso n G, Palmieri F & Smeitink JA 2002 Adenine nucleotide translocator 1 deficiency associated with Sengers syndrome. Annals of Neurology 52 95-99. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT, Roskin KM, Schwartz M, Sugnet CE, Thomas DJ, Weber RJ, Haussler D & Kent WJ 2003 The UCSC Genome Browser Database. Nucleic Acids Research 31 51-54. Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H & Tsunoda Y 1998 Eight calves cloned from somatic cells of a single adult. Science 282 2095-2098. Keeney S, Giroux CN & Kleckner N 1997 Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88 375-384. Kel AE, Kel-Margoulis OV, Farnham P J, Bartley SM, Wingender E & Zhang MQ 2001 Computer-assisted identification of cell cycle-related genes: new targets fo r E2F transcription factors. Journal of Molecular Biology 309 99-120. Kim M, Li D, Cui Y, Muelle r K, Chears WC & DeJong J 2006 Regulatory factor interactions and somatic silencing of the germ cell-specific ALF gene. Journal of Biological Chemistry 281 34288-34298.

PAGE 109

109 Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hwang WS, Hossein MS, Kim JJ, Shin NS, Kang SK & Lee BC 2007 Endangered wolves cloned from adult somatic cells. Cloning and Stem Cells 9 130-137 Kim YH, Haidl G, Schaefer M, Egner U, Mandal A & Herr JC 2006 Compartmentalization of a unique ADP/ATP carrier protein SFEC (Sperm Flag ellar Energy Carrier, AAC4) with glycolytic enzymes in the fibrous sheath of the human sperm flagellar principal piece. Developmental Biology 302 463-476. Kong L, Chang JT, Bild AH & Nevins JR 2007 Compensation and specif icity of function within the E2F family. Oncogene 26 321-327. Lammers JHM, Offenberg HH, Van Aalderen M, Vink ACG, Dietrich AJJ & Heyting C 1994 The gene encoding a major component of the late ral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes. Molecular and Celullar Biology 14 11371146. Latham KE, Litvin J, Orth JM, Patel B, Mettus R & Reddy EP 1996 Temporal patterns of Amyb and B-myb gene expression during testis development. Oncogene 13 1161-1168. La Thangue NB & Rigby PW 1987 An adenovirus E1A-like transc ription factor regulated during the differentiation of murine embryonal carcinoma stem cells. Cell 49 507-513. Lee J, Iwai T, Yokota T, & Yamashita M 2003 Temporally and spatially selective loss of Rec8 protein from meiotic chromoso mes during mammalian meiosis. Journal of Cell Science 116 2781-2790. Lele KM & Wolgemuth DJ 2004 Distinct regions of the mouse Cyclin A1 gene, Ccna1, confer male germ-cell-specific expr ession and enhancer function. Biology of Reproduction 71 13401347. Levy SE, Chen YS, Graham BH & Wallace DC 2000 Expression and sequence analysis of the mouse adenine nucleotide tr anslocase 1 and 2 genes. Gene 254 57-66. Li FX, Zhu JW, Hogan CJ & DeGregori J 2003 Defective gene expres sion, S phase progression, and maturation during hematopoiesis in E2F1/E2F2 mutant mice. Molecular and Cellular Biology 23 3607-3622. Li K, Hodge JA & Wallace DC 1990 OXBOX, a positive transcrip tional element of the heartskeletal muscle ADP/ATP Translocator Gene. Journal of Biological Chemistry 265 2058520588. Li R, Hodny Z, Luciakova K, Barath P & Nelson BD 1996 Sp1 activates and inhibits transcription from separate elements in the prox imal promoter of the human adenine nucleotide translocase 2 (ANT2) gene. Journal of Biological Chemistry 271 18925-18930.

PAGE 110

110 Lin H, Jurk M, Gulick T & Cooper GM 1999 Identification of COUP-T F as a transcriptional repressor of the c-mos proto-oncogene. Journal of Biological Chemistry 274 36796-36800. Lindeman GJ, Dagnino L, Gaubatz S, Xu Y, Bronson RT, Warren HB & Livingston DM 1998 A specific, nonproliferative role for E2F-5 in chor oid plexus function revealed by gene targeting. Genes & Development 12 1092-1098. Looman C, Mark C, Abrink M & L Hellman 2003 MZF6D, a novel KRAB zinc-finger gene expressed exclusively in meiotic male germ cells. DNA and Cell Biology 22 489-496. Loots GG & Ovcharenko I 2004 rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Research 32 W217-W221. Lukas J, Petersen BO, Holm K, Bartek J & Helin K 1996 Deregulated expression of E2F family members induces S-phase entry and overcom es p16INK4A-mediated growth suppression. Molecular and Cellular Biology 16 1047-1057. Lunardi J, Hurko O, Engel WK & Attardi G 1992 The multiple ADP/ATP translocase genes are differentially expressed during human muscle cell development. Journal of Biological Chemistry 267 15267-15270. Macaluso M, Cinti C, Russo G, Russo A & Giordano A 2003 pRb2/p130-E2F4/5-HDAC1SUV39H1-DNMT1 multimolecular complexes mediat e the transcription of estrogen receptorin breast cancer. Oncogene 22 3511-3517. MacLean II JA & Wilkinson MF 2005 Gene regulation in spermatogenesis. Current Topics in Developmental Biology 71 131-197. Maiti B, Li J, Bruin A, Gordon F, Timmers C, Opavsky R, Patil K, Tuttle J, Cleghorn W & Leone G 2005 Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation. Journal of Biological Chemistry 280 1821118220. Marikawa Y, Fujita T & Alarcon V 2005 Heterogeneous DNA met hylation status of the regulatory element of the mouse Oct4 gene in adult somatic cell population. Cloning and Stem Cells 7 8-16. Matsui Y & Hayashi K 2007 Epigenetic regulation fo r the induction of meiosis. Cellular and Molecular Life Sciences 64 257-262. Meuwissen RLJ, Offenberg HH, Dietrich AJJ, Riesewijk A, Van Lersel M & Heyting C 1992 A coiled-coil related protein specific for syna psed regions of meiotic prophase chromosomes EMBO Journal 11 5091-5100.

PAGE 111

111 Muller H, Moroni MC, Vigo E, Pe tersen BO, Bartek J & Helin K 1997 Induction of S-phase entry by E2F transcription factors de pends on their nuclear localization. Molecular and Cellular Biology 17 5508-5520. Murga M, Fernandez-Capetillo O, Field S, Moren o B, Borlado LR, Fujiwara Y, Balomenos D, Vicario A, Carrera AC, Orkin SH, Greenberg ME & Zubiaga AM 2001 Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation leading to the development of autoimmunity. Immunity 15 959-970. Oberley MJ, Inman DR, & Farnham PJ 2003 E2F6 negatively regulates BRCA1 in human cancer cells without methylation of histone H3 on lysine 9. Journal of Biological Chemistry 278 42466-42476. Offenberg HH, Schalk JAC, Meuwissen RLJ, Aalderen MV, Kester HA, Dietrich AJJ & Heyting C 1998 SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat. Nucleic Acids Research 26 2572-2579. Ogawa H, Ishiguro K, Gaubatz S, Livingston DM, & Nakatani Y 2002 A complex with chromatin modifiers that occupies E2Fand Myc-responsive genes in G0 cells. Science 296 1132-1136. Ollinger R, Alsheimer M & Benavente R 2005 Mammalian protein SCP1 forms synaptonemal complex-like structures in the ab sence of meiotic chromosomes. Molecular Biology of the Cell 16 212-217. Pennisi E 2001 The human genome. Science 291 1177-1180. Petukhova GV, Pezza RJ, Vanevski F, Pl oquin M, Masson J & Camerini-Otero RD 2005 The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination. Nature Structural & Molecular Biology 12 449-453. Pezza RJ, Petukhova GV, Ghirlando R & Camerini-Otero RD 2006 Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex. Journal of Biological Chemistry 281 18426-18434. Pezzi N, Prieto I, Kremer L, Perez-Jurado LA, Valero C, Del-Mazo J, Martinez C & Barbero JL 2000 STAG3, a novel gene encoding a protein involved in meiotic chromosome pairing and location of STAG3 -related genes flanking the Williams-Beuren syndrome deletion. FASEB Journal 14 581-592. Pohlers M, Truss M, Frede U, Scholz A, St rehle M, Kuban R, Hoffmann B, Morkel M, Birchmeier C & Hagemeier C 2005 A role for E2F6 in the re striction of male-germ-cellspecific gene expression. Current Biology 15 1051-1057.

PAGE 112

112 Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A & Campbell KHS 2000 Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407 86-90. Pousette A, Leijonhufvud P, Arver S, Kvist U, Pelttari J & Hg C 1997 Presence of synaptonemal complex protein 1 transversal filament-like protein in human primary spermatocytes. Human Reproduction 12 2414-2417. Prieto I, Suja JA, Pezzi N, Kremer L, Martinez C, Rufas JS & Barbero JL 2001 Mammalian STAG3 is a cohesion specific to sister chromatid arms in meiosis 1. Nature Cell Biology 3 761766. Qin XQ, Livingston DM, Kaelin WG & Adams PD 1994 Deregulated transc ription factor E2F-1 expression leads to S-phase en try and p53-mediated apoptosis. Proceedings of the National Academy of Sciences 91 10918-10922. Ravnik SE & Wolgemuth DJ 1999 Regulation of meiosis during mammalian spermatogenesis: the A-type Cyclins and their associated Cyclin-depe ndent kinases are differen tially expressed in the germ-cell lineage. Developmental Biology 207 408-418. Rehling P, Brander K & Pfanner N 2004 Mitochondrial import and th e twin pore translocase. Nature Reviews Molecular Cell Biology 5 519-530. Rempel RE, Saenz-Robles MT, Storms R, Morh am S, Ishida S, Engel A, Jakoi L, Melhem MF, Pipas JM, Smith C & Nevins JR 2000 Loss of E2F4 activity leads to abnormal development of multiple cellular lineages. Molecular Cell 6 293-306. Revenkova E, Eupe M, Heytin g C, Gross B, & Jessberger R 2001 Novel meiosis-specific isoform of mammalian SMC1. Molecular and Cellular Biology 21 6984-6998. Revenkova E & Jessberger R 2005 Keeping sister chromatids together: cohesins in meiosis. Reproduction 130 783-790. Ringrose L 2006 Polycomb, trithorax and the decision to differentiate. BioEssays 28 330-334. Robinson MO, McCarrey JR & Simon MI 1989 Transcriptional regula tory regions of testisspecific PGK-2 defined in transgenic mice. Proceedings of the National Academy of Sciences 86 8437-8441. Rodic N, Oka M, Hamazaki T, Murawski MR, Jorgensen M, Maatouk DM, Resnick JL, Li E & Terada N 2005 DNA methylation is re quired for silencing of Ant4 an adenine nucleotide translocase selectively expressed in mous e embryonic stem cells and germ cells. Stem Cells 23 1314-1323.

PAGE 113

113 Rotheneder H, Geymayer S & Haidweger E 1999 Transcription factors of the Sp1 family: interaction with E2F and re gulation of the murine thymidine kinase promoter. Journal of Molecular Biology 293 1005-1015. Sagata N 1997 What does Mos do in oocytes and somatic cells? Bioessays 19 13-21. Sage J, Martin L, Meuwissen R, Heyting C, Cuzin F & Rassoulzadegan M 1999 Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad. Mechanisms of Development 80 29-39. Schwartz JK, Devoto SH, Smith EJ, Chellappan SP, Jakoi L, & Nevins JR. 1993 Interactions of the p107 and Rb proteins with E2F during the cell prolif eration response. EMBO Journal 3 10131020. Shannon M, Richardson L, Christian A, Handel MA & Thelen MP 1999 Differential gene expression of mammalian SPO11/TOP6A homologs during meiosis. FEBS Letters 462 329-334. Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L & Westhusin M 2002 Cell biology: a cat cloned by nuclear transplantation. Nature 415 859. Simpson AJG, Caballero OL, Jungbluth A, Chen Y & Old LJ 2005 Cancer/testis antigens, gametogenesis and cancer. Nature Reviews Cancer 5 615-625. Singh AM, Hamazaki T, Hankowski KE & Terada N 2007 A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25 2534-2542. Singh P, Wong SH & Hong W 1994 Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation. EMBO Journal 13 3329-3338. Smale ST 1997 Transcription initiation from TATA-le ss promoters within eukaryotic proteincoding genes. Biochimica et Biophysica Acta 1351 73-88. Stepien G, Torroni A, Chung AB, Hodge JA & Wallace DC 1992 Differential expression of adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell differentiation. Journal of Biological Chemistry 267 14592-14597. Storre J, Elssser H, Fuchs M, Ullmann D, Livingston DM &Gaubatz S 2002 Homeotic transformations of the axial skeleton that accompany a targeted deletion of E2f6 EMBO Reports 3 695-700. Storre J, Schfer A, Reichert N, Barbero JL, Hauser S, Eilers M, & Gaubatz S 2005 Silencing of meiotic genes SMC1 and STAG3 in Somatic Cells by E2F6. Journal of Biological Chemistry 280 41380-41386.

PAGE 114

114 Sweeney C, Murphy M, Kubelka M, Ravnik SE, Hawkins CF, Wolgemuth DJ & Carrington M 1996 A distinct cyclin A is expre ssed in germ cells in the mouse. Development 122 53-64. Thomas K, Wu J, Sung DY, Thompson W, Powell M, McCarrey J, Gibbs R & Walker W 2007 SP1 transcription factors in male germ cell development and differentiation. Molecular and Cellular Endocrinology 270 1-7. Toscani A, Mettus RV, Coupland R, Simpkins H, Litvin J, Orth J, Hatton KS & Reddy EP 1997 Arrest of spermatogenesis and defective breast development in mice lacking A-myb. Nature 386 713-717. Trimarchi JM, Fairchild B, Wen J & Lees JA 2001 The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex. Proceedings of the National Academy of Sciences 98 1519-1524. Trimarchi JM & Lees JA 2002 Sibling rivalry in the E2F family. Nature Reviews Molecular Cell Biology 3 11-20. Tronche F, Ringeisen F, Blumenfeld M, Yaniv M & Pontoglio M 1997 Analysis of the distribution of binding sites for a tissue-specific transcription factor in the vertebrate genome. Journal of Molecular Biology 266 231-245. Tureci O, Sahin U, Zwick C, Koslowski M, Seitz G & Pfreundschuh M 1998 Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens. Proceedings of the National Academy of Sciences 95 5211-5216. Turner JM 2007 Meiotic sex chromosome inactivation. Development 134 1823-1831. Van-Heldin J 2003. Regulatory sequence analysis tools. Nucleic Acids Research 31 3593-3596. Vire E, Brenner C, Deplu R, Blanchon L, Fr aga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden J, Bollen M, Estell er M, Di Croce L, Launoit Y & Fuks F 2006 The polycomb group protein EZH2 dire ctly controls DNA methylation. Nature 439 871-874. Wakayama T, Perry AC, Zuccotti M, Johnson KR & Yanagimachi R 1998 Full-term development of mice from enucleated oocyt es injected with cumulus cell nuclei. Nature 394 369-374. Walker JE 1992 The mitochondrial transporter family. Current Opinion in Structural Biology 2 519-526. Wang PJ, Page DC & McCarrey JR 2005 Differential expression of sex-linked and autosomal germ-cell-specific genes during sp ermatogenesis in the mouse. Human Molecular Genetics 14 2911-2918.

PAGE 115

115 Wang WS, Seki M, Yamoaka T, Seki T, Tada S, Katada T, Fujimoto H & Enomoto T 1998 Cloning of two isoforms of mouse DNA helicas e Q1/RecQL cDNA; alpha form is expressed ubiquitously and beta form specifically in the testis. Biochemical and Biophysical Research Communications 1443 198-202. Wasserman WW & Fickett JW 1998 Identification of regulator y regions which confer musclespecific gene expression. Journal of Molecular Biology 278 167-181. Watanabe D, Yamada K, Nishina Y, Tajima Y, Koshimizu U, Nagata A & Nishimune Y 1994 Molecular cloning of a novel Ca2+-binding protein (Calmegin) specifically expressed during male meiotic germ cell development. Journal of Biological Chemistry 269 7744-7749. Watson JD & Crick FH 1953 Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171 737-738. Weil PA, Luse DS, Segall J & Roeder RG 1979 Selective and accurate initiation of transcription at the Ad2 major late promoter in a soluble system dependent on purified RNA polymerase II and DNA. Cell 18 469-484. Weinberg RA 1992 The retinoblastoma ge ne and gene product. Cancer Surveys 12 43-57. Wilkerson DC, Wolfe SA & Grimes SR 2002 H1t/GC-Box and H1t/TE1 element are essential for promoter activity of the testis-specific Histone H1t gene. Biology of Reproduction 67 1157-1164. Wilmut I, Schnieke AE, McWhi r J, Kind AJ & Campbell KHS 1997 Viable offspring derived from fetal and adult mammalian cells. Nature 385 810-813. Witte V, Laffert B, Rosorius O, Lischka P, Blum e K, Galler G, Stilper A, Willbold D, DAloja P, Sixt M, Kolanus J, Ott M, Kolanus W, Schuler G & Baur AS 2004 HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane. Molecular Cell 13 179-190. Wu L, Timmers C, Maiti B, Saavedra HI, Sang L, Chong GT, Nuckolls F, Giagrande P, Wright FA, Field SJ, Greenberg ME, Orkin S, Nevins JR, Robinson ML & Leone G 2001 The E2F1-3 transcription factors are essential for cellular proliferation. Nature 414 457-462. Xu EY, Moore FL & Reijo-Pera RA 2001 A gene family required for human germ cell development evolved from an ancient me iotic gene conserved in metazoans. Proceedings of the National Academy of Sciences 98 7414-7419. Xu W & Cooper GM 1995 Identification of a candidate c-mos repressor that restricts transcription of germ cell-specific genes. Molecular and Cellular Biology 15 5369-5375. Yamasaki L, Jacks T, Bronson R, Goillot E, Harlow E & Dyson N 1996 Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85 537-548.

PAGE 116

116 Yee AS, Reichel R, Kovesdi I & Nevins JR 1987 Promoter interaction of the E1A-inducible factor E2F and its potential role in the fo rmation of a multi-component complex. EMBO Journal 6 2061-2068. Yuan L, Brundell E & Hoog C 1996 Expression of the meiosi s-specific Synaptonemal Complex Protein 1 in a heterologous sy stem results in the formation of large protein structures. Experimental Cell Research 229 272-275.

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117 BIOGRAPHICAL SKETCH Sarah Michelle Kehoe w as born in Washingt on D.C. in 1980, to Robert and Patricia Williams. She was raised in Fairfax, Virgin ia and graduated from W.T. Woodson High School in June 1998. Sarah then attended Virginia Poly technic Institute and Stat e University, where she graduated both in honors and summa cum laude w ith her Bachelor of Science degree in animal and poultry sciences in May 2002. During college, in the laborat ory of Dr. Paul Siegel, Sarah conducted poultry genetics research that culminated in the publication of he r first-author paper in the August 2002 issue of Poultry Science Sarah was also awarded two summer internships at the National Institutes of Health where she wo rked at the National Human Genome Research Institute in the laboratory of Dr. Lawrence Brody. In 2003, Sarah enrolled in the Interdisciplinary Program in Biomedical Sciences (IDP) at the University of Florida, College of Medicine and received a Grin ter Fellowship upon admission. She began her doctoral study under the guidance of Dr. Naohiro Terada, in the Department of Molecular and Cell Biology. Upon completion of her Ph.D. in December 2007, Sa rah plans to pursue a career dedicated to the study of cancer.