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Molecular Analysis of Two Putative Mediator Subunits in Arabidopsis thaliana

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Molecular Analysis of Two Putative Mediator Subunits in Arabidopsis thaliana
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PAN, WEI ( Author, Primary )
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2008

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Cells ( jstor )
Cotyledons ( jstor )
DNA ( jstor )
Genes ( jstor )
Plant roots ( jstor )
RNA ( jstor )
Seedlings ( jstor )
Signals ( jstor )
Transactivators ( jstor )
Yeasts ( jstor )

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Copyright Wei Pan. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS INTArabidopsis
thaliana











By

WEl PAN
















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE


UNIVERSITY OF FLORIDA

2007





























Copyright 2007

by

Wei Pan



























To my family









ACKNOWLEDGMENTS

I thank Dr. Bill Gurley for his kindness, commitment and great mentoring. He is an

excellent advisor because he always encouraged me to develop independent thinking and gave

me the opportunity to pursue my research interests. His careful reading and editing greatly

improved this thesis. I thank Dr. Robert Ferl for his suggestion on my work and giving me the

pBIl01sGFP vector. I thank Dr. Kevin O'Grady for his continuous help with my experiments

and teaching me many molecular biology techniques. I extend thanks to Dr. Eva

Czarnecka-Verner for her suggestions on my research.

I thank Dr. Zhonglin Mou and Ms. Xudong Zhang for their valuable assistance with some

techniques such as Northern blotting and GUS staining, and generously letting me share some of

their facilities. I thank Ms. Donna Williams for her assistance with the con-focal observation.

I thank Dr. Masaharu Suzuki for helping me get started in research in the Plant Molecular

and Cellular Biology Program (PMCB); Dr. Alice Harmon for the invaluable training I gained in

her lab; and Dr. David Clark for his kindness and support for my study in PMCB.

I also wish to express my gratitude to the entire PMCB faculty who taught me in classes

and journal clubs. I benefited a lot from the wonderful courses.

I am extremely grateful for all my family and friends for their understanding and support

over the years.











TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S .........__.. ..... .__. ...............7....


LI ST OF FIGURE S .............. ...............8.....


LI ST OF AB BREVIAT IONS ........._.___..... ..._. ............... 10...


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


1 INTRODUCTION ................. ...............14.......... ......


Assembly of the Preinitiation Complex ................. ...............14........... ...
Identification of the Mediator Complex in Yeast ................. ...............16........... ..
Identification of the Mediator Complex in Human Cells ................ ......... ................17
TRAP Complex .............. ...............17....
SM CC Complex .............. ...............18....
DRIP Complex .............. ...............18....
ARC Complex .............. ...............18....
CRSP Complex............... ...............18
PC2 Complex............... ... .................1
Mediator Interacts with Transactivators .............. ...............20....
Mediator Interacts with RNA pol II............... ...............21...
Phosphorylation of RNA Pol II CTD ..........._... ...__.....__....._ ..............21
Mediator Interacts with Coactivators ................... .......... ...............22......
Mediator Promotes the Formation of a Stable PIC ................. ...............23..............
Mediator is Required in the Reinitiation Scaffold ....__ ......_____ ...... ....__........2
Mediator Stimulates both Basal and Activated Transcription ....._____ ..... .. ...__...........23
Model for Mediator Function in Activated Transcription ................ .......... ...............24
Hypothesis for a Mediator Complex in Arabidopsis ................ .............. ......... .....24

2 MATERIALS AND METHODS .............. ...............29....


Plant Growth Conditions .............. .. ...............29...
Genotyping of the T-DNA Insertion Lines ................. ...............29...............
RN A Analy si s............... ...............3
M icroscopy .............. ...............30....
Plasmid Construction................ .............3
GU S Staining ................. .... ... ...... .......... ............3
Agrobacterium Transformation Technique .............. ...............3 1....
Chromatin Immunoprecipitation .............. ...... ...............32.
PCR Analysis of Chromatin Immunoprecipitation............... ..........3












Bioinf orm atics .............. ...............3 5....


3 RE SULT S .............. ...............36....


Analysis of Arabidopsis 2ed31 Gene by Multiple Sequence Alignments ............._..__.........36
Phenotype Characterization of med31 Mutants ................. ...............37........... ...
M~ed31 Expression in the med31-2 Plants .................. ........... ........ .. .............. ......3
Subcellular Localization and Tissue Expression Pattern of Med3 1::GFP Fusion Proteins....3 9
Tissue Expression Pattern of2~ed31 Promoter::GUS Fusions ........_............. ...... .........39
Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA..........._._... ...............40
ChlP Analysis for Med31 .............. ...............41....
ChlP Analysis for Med6 ........._._ ...... .... ...............42...
Conclusion ........._.___..... .__ ...............43....


4 DI SCUS SSION ............ ..... ._ ............... 8....


Phenotype Characterization of med31 Mutants ................. ...............58........... ...
Evidence for a Mediator Complex in Arabidopsis .............. ...............59....

LIST OF REFERENCES ................. ...............61................


BIOGRAPHICAL SKETCH .............. ...............73....










LIST OF TABLES


Table page

1-1 Interaction of the transactivators with the Mediator subunits in different organisms ..........26

1-2 Mediator subunits in yeast, Arabidopsis, Drosophila and humans ............ ...................28










LIST OF FIGURES


Figure page

3-1 Multiple sequence alignments of Med3 1 homologs in different species. ................... .45

3-2 Multiple alignments of AtMed3 1 with the deduced amino acid sequences of its
homologs in other plant species .............. ...............46....

3-3 Diagrammatic representation of the insertions of the T-DNA in med31-1 and
med31-2............... ...............47

3-4 Germination rate and root length of WT and med31-1 seedlings (9-day-old). ............47

3-5 Nine-day-old WT and med31-2 seedlings grown under continuous light ................... .47

3-6 Nine-day-old WT and med31-2 seedlings grown under dark ................. ................ .48

3-7 Ten-day-old WT and med31-2 seedlings............... ...............4

3-8 Comparison of adult WT plants and med31-2 plants .............. .....................4

3-9 Northern blot analysis of Med31 expression in WT and med31-2 plants ....................49

3-10 Subcellular localization of Med3 1::GFP fusion proteins in the root tip of a
35-day-old plant .............. ...............49....

3-11 Expression of Med3 1::GFP fusion proteins in lateral roots ................ ................ ...50

3-12 Expression of Med3 1::GFP fusion proteins in a root hair ................. ............... .....50

3-13 Expression of Med3 1::GFP fusion proteins in a leaf ................. ................. ......5 1

3-14 Expression of Med3 1::GFP fusion proteins in a trichome. ........._._... ........_._........52

3-15 Expression of Med3 1::GFP fusion proteins in a petiole. .........._.... ......_._...........53

3-16 M~ed31 promoter directed GUS tissue expression pattern in young plants
(16-day-old) ................. ...............54........... ....

3-17 M~ed31 promoter directed GUS tissue expression pattern in adult plants
(46-day-old)................ .............5

3-18 Multiple sequence alignments of Med6 homologs in different species .....................55

3-19 Med3 1 associates with the promoters of CCA1~, Hspl8. 2 and Adhl, but not with
the inter genetic region ................. ...............56........... ....










3-20 Med6 associates with the promoters of CCA1~, Hspl8. 2 and Adhl, but not with
the inter genetic region ................. ...............56........... ....

3-21 Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the
CCA 1 promoter from WT Arabidopsis ..........._..__......__ ....._._ ...........5









LIST OF ABBREVIATIONS

Adh alcohol dehydrogenase

ARC activator-recruited cofactor

CCAl circadian clock associated 1

ChlP chromatin immunoprecipitation

c-Myc cellular myelocytomatosis oncogene

CRSP cofactor required for Spl activation

CTD carboxy-terminal domain

DRIP vitamin D receptor interacting protein

EST expressed sequence tag

GFP green fluorescent protein

GUS P-glucuronidase

HAT histone acetyltransferase

Hsp l 8.2 heat shock protein 18.2

IgG immunoglobulin G

PC2 positive cofactor 2

PIC preinitiation complex

RNA pol RNA polymerase

Sepl0 Separationl0

SMCC SRB/MED Cofactor Complex

Sohl suppressor of hprl1

SWI/SNF switching/sucrose non-fermenting

TAP tandem affinity purification

TFIIA Transcription Factor II A

TFIIB Transcription Factor II B









TFIID Transcription Factor II D

TFIIE Transcription Factor II E

TFIIF Transcription Factor II F

TFIIH Transcription Factor II H

TRAP thyroid hormone receptor-associated protein

UTR untranslated region

VPl16 herpes simplex virus protein 16

WT wild type









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS INTArabidopsis
thaliana


By

Wei Pan

May 2007

Chair: William B. Gurley
Maj or Department: Plant Molecular and Cellular Biology

Mediator is a conserved coactivator complex that has been identified in yeast, Drosophila

and humans. It plays a critical role in gene transcription mediated by RNA polymerase II (RNA

pol II) by serving as a bridge between activators bound to the promoter and other transcription

machineries, including RNA pol II. Despite evidence suggesting such a vital role of Mediator in

gene expression, the subunit composition and function of Mediator has not been determined in

plants. Based on the conserved transcriptional machineries (RNA pol II, general transcription

factors and some coactivators) in plants, metazoans and yeast, we hypothesized the plant also has

the Mediator coactivator. Identification of the homologs of most of the yeast and metazoan

Mediator subunits in Arabidopsis supported this hypothesis.

This study characterized the function of two putative Mediator subunits, Med6 and Med3 1.

Two T-DNA insertion lines in the M~ed31 promoter or 5' untranslated region were identified. The

med31-1 mutant line had shorter root length and a reduced germination rate. The med31-2 plants

had shorter root length, aberrant patterns of cotyledon development, and smaller size compared

with wild type plants. We found the Med31::GFP (green fluorescent protein) fusion proteins

were localized to the nucleus. The Med31::GFP signal was detected in the roots, leaves,









trichomes and petioles. In addition, we found the M~ed31 promoter::GUS fusions were expressed

in the shoot apexes and lateral roots of the young seedlings (16 days old), and in the young

inflorescences, anthers, stigmas of adult plants (46 days old) and in developing seeds. Both

Med6 and Med3 1 proteins were localized to the promoters of three unrelated genes (CCA1,

Hspl8.2 and Adhl). These results strongly support the conclusion that Med6 and Med31 are

members of the Mediator complex in Arabidopsis.









CHAPTER 1

INTRODUCTION

Assembly of the Preinitiation Complex

Transcription is one of the most significant steps that occur during gene expression. It is

carried out by RNA polymerases and additional factors. There are four kinds of RNA

polymerases in plants, RNA polymerase (RNA pol) I, II, III and IV. RNA pol I is located in the

nucleolus, and it transcribes rRNA genes, except 5S rRNA. RNA pol II is located in the

nucleoplasm and transcribes hnRNA, the precursor of mRNA. RNA pol III is also located in the

nucleoplasm and is responsible for the synthesis of tRNA, 5 S rRNA and other small RNAs

(Thomas and Chiang, 2006). And last, an RNA polymerase unique to plants, RNA polymerase

IV, is involved in the siRNA silencing pathway, RNA-dependent DNA methylation and the

formation of heterochromatin (Onodera et al., 2005).

Transcription by RNA pol II can be broadly categorized as basal transcription

(activator-independent) and activated transcription (activator-dependent) A simplified sequence

of activated transcription initiation for RNA pol II has been postulated as follows. Activators

(transactivators or transcription factors) bind the regulatory motifs of DNA and then recruit a

variety of additional factors that prepare the promoter for the arrival of RNA pol II and the

formation of the preinitiation complex (PIC) (Thomas and Chiang, 2006). One of the first

components to arrive is a kinase which phosphorylates histone H3 (Featherstone, 2002). Then

coactivators that can modify chromatin structures are recruited. For example, HAT (histone

acetyltransferase) arrives at the promoter early in the activation process and its role is to

acetylate specific lysines in histone amino-termini and other transcription factors (Roth et al.,

2001; Naar et al., 2001; Clayton et al., 2006). Another complex that is recruited early in the

process of gene activation is SWI/SNF (switching/sucrose non-fermenting), which remodels the









chromatin structure and facilitates the accessibility of other members of the transcriptional

apparatus to the DNA (Gavin et al., 2001, Havas et al., 2000). After the promoter is made

accessible, the TFIID complex is recruited to the TATA box in the promoter (Pugh, 2000).

Activators also recruit Mediator complex which facilitates the formation of pol II PIC, which

consists of RNA pol II and general transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH)

(Conaway et al., 2005).

Next, TFIIH facilitates promoter melting and phosphorylates the CTD (carboxy-terminal

domain) of the largest subunit of RNAP II (Jiang et al., 1996; Kim et al., 1994). This

phosphorylation event is thought to be required for promoter clearance and the start of

transcriptional elongation (Dvir et al., 1997; Kugel and Goodrich, 1998; Kumar et al., 1998).

After the synthesis of the initial transcript, most members of the PIC (with the exception of

TFIIB and TFIIF) remain at the promoter and form a structure know as the "scaffold" that

facilitates the reentry of RNA pol II, TFIIB and TFIIF for subsequent rounds of synthesis

(Yudkovsky et al., 2000). In the process of reinitiation, the CTD of RNA pol II is

dephosphorylated by a CTD phosphatase that is stimulated by TFIIF (Friedl et al., 2003). This

cycle of phosphorylation and dephosphorylation of the CTD is essential to the entry of RNA pol

II to the PIC (with a hypophosphorylated CTD) and subsequent promoter clearance

(hyperphosphorylated CTD) (Oelgeschlager, 2002).

One of the key regulatory complexes involved in the process of promoter activation is the

Mediator. This large assemblage of proteins (~2MDa) is conserved from yeast to humans and is

composed of 25-29 subunits (Boube et al., 2002). It is becoming increasingly clear that Mediator

plays a critical role in both activated and basal transcription mediated by RNA pol II in yeast and

metazoans (Back et al., 2002, Nair et al., 2005), because it serves as a bridge between activators









bound to the promoter and other general transcription factors, as well as RNA pol II (Kornberg,

2005).

Identification of the Mediator Complex in Yeast

The presence of Mediator was proposed because of the discovery of activator inhibition

in yeast. The activator GAL4-VPl16 was found to repress the activation effect of another

activator (a factor binding to a thymidine-rich DNA element) both in vivo and in vitro. This

phenomenon led to the hypothesis that the two activators competed for a common intermediate

factor. Activator interference was relieved in vitro with the addition of the fraction containing

this intermediate factor, which was named Mediator (Kelleher et al., 1990).

The Mediator fraction from column chromatography was shown to be required for

GAL4-VPl16 and GCN4-dependent gene transcription in an in vitro transcription system

(Flanagan et al., 1991). This was the initial direct evidence that Mediator was involved in gene

transcription. The In vitro transcription system was reconstituted with purified RNA pol II and

general transcription factors from yeast and was widely used for checking the presence of

Mediator, thereafter.

Three experimental approaches were originally used to identify proteins as Mediator

subunits: 1) Identify the suppressors of RNA pol II CTD truncation mutations; 2) Isolate the

fraction (RNA pol II holoenzyme) that can stimulate the activator-dependent transcription.

Separate the proteins by electrophoresis in a gel, and then identify the proteins by peptide

sequencing; and 3) Identify the proteins that co-immunoprecipitate with known Mediator

subunits. The presence in the RNA pol II holoenzyme and support of activator-dependent

transcription were two criteria that were used to confirm the identities of Mediator subunits.

In total, 25 Mediator subunits have been identified in S. cerevisiae. Nine Mediator subunits

(Srb2, Srb4, Srb5, Srb6, Srb7, Srb8, Srb9, Srbl0 and SrbI1) were identified based on the










suppression of S. cerevisiae RNA pol II CTD truncation mutations. All of these subunits were

shown to be present in the holoenzyme (Thompson et al., 1993; Kim et al., 1994; Koleske and

Young, 1994; Liao et al., 1995; Hengartner et al., 1995). Fifteen Mediator subunits (Medl, Med2,

Pgdl (Hrsl), Med4, Med7, Med8, Med11, Gallli, Sin4 Rgrl, Mtr32, Rox3, Nutl, Nut2, and

Cse2) were detected in the RNA pol II holoenzyme and identified by peptide sequencing (Kim et

al., 1994; Gustafsson et al., 1997; Gustafsson et al., 1998; Li et al., 1995; Myers et al., 1998).

The mutant yeast strains for Galll, Sin4 and Rgrl showed similar mutant phenotypes, which

suggested they may function in the same pathway (Fassler et al., 1991, Jiang and Stillman, 1995,

Suzuki et al., 1988; Chen et al., 1993, Sakai et al., 1990). More recently, Med31 was found to be

a Mediator subunit in S. cerevisiae and S. pombe based on co-purification with previously

characterized Mediator subunits (Linder and Gustafsson, 2004).

Identification of the Mediator Complex in Human Cells

Two methods were used to identify the Mediator subunits in human cells: 1) Isolate the

nuclear extract fraction that can stimulate the activator-dependent transcription. Separate the

proteins on the gel, and then identify the proteins by peptide sequencing; and 2) Identify the

proteins that co-immunoprecipitate with the transactivators (or their activation domains) or

known Mediator subunits. Most identified subunits are orthologs of the yeast Mediator subunits.

However, various Mediator complexes with different subunit compositions were isolated in

different labs (Sato et al., 2004). A brief description of human Mediator types follows.

TRAP Complex

A thyroid hormone receptor-associated protein (TRAP) complex was isolated based on

co-precipitation with FLAG epitope-tagged hTRalphal (human thyroid hormone receptor alphal)

(Fondell et al., 1996).









SMCC Complex

The human SRB/MED Cofactor Complex (SMCC) was purified by affinity

chromatography of FLAG epitope-tagged human SRB proteins (Gu et al., 1999).

DRIP Complex

The DRIP (vitamin D receptor interacting protein) complex was isolated from the nuclear

extract of human Namalwa B cells based on its interaction with the VDR LBD (vitamin D3

receptor ligand-binding domain) in the presence of hormone. This complex contains 10 proteins

and it can stimulate transcription by VDR-RXR. It was shown that at least one of its subunits has

histone acetyltransferase activity (Rachez et al., 1998).

ARC Complex

The ARC (activator-recruited cofactor) complex was isolated by its affinity for the

activation domains of SREBP-la, VPl16 and the p65 subunit of NF-kB, respectively, from HeLa

cell nuclear extract (Naar et al., 1999). It can not only stimulate transcription by activators such

as SREBP-la/Spl, NF-k
CRSP Complex

The CRSP (cofactor required for Spl activation) complex was isolated from HeLa cell

nuclear extract and shown to be required for Spl-dependent transcriptional activation (Ryu et al.,

1999). This complex consists of 9 subunits and has a mass of approximately 0.7 MDa.

PC2 Complex

The PC2 (positive cofactor 2) complex was isolated from HeLa cell nuclear extracts based

on its ability to stimulate HNF4 (hepatocyte nuclear factor 4) and GAL4-AH dependent

transcription (Malik et al., 2000). This complex consists of at least 15 subunits and is larger than

0.5MDa. The presence of these subunits within the complex was confirmed by the









co-immunoprecipitation of epitope (FLAG and HA)-tagged MED 10. Both PC2 and CRSP were

found to be subcompexes of ARC, DRIP, or TRAP/SMCC (Malik and Roeder, 2000).

Despite being originally isolated by different approaches, some complexes found in human

cells (ARC, DRIP, and TRAP/SMCC) were shown to be very similar in subunit composition

(Naar et al., 1999; Malik and Roeder, 2000).

The finding that various closely related Mediator complexes have slightly different subunit

composition raised the question of whether some of the proteins identified are true subunits, or

just contaminants associated with a particular isolation strategy. Sato and colleagues (Sato et al.,

2004) addressed this question by co-immunoprecipitation of human Mediator using six

FLAG-tagged subunits to individually purify complexes for analysis of subunit composition by

MudPIT (multidimensional protein identification technology). Proteins present in all six

independent Mediator preparations were considered to be true Mediator subunits. Their results

support the conclusion that all proteins identified previously are bona fide Mediator subunits. In

addition, they identified the MED13L and the CDK8-like cyclin-dependent kinase CDKll as

putative Mediator-associated proteins.

The inconsistency in Mediator subunit composition was thought to be due in part to the

dissociation of Mediator subunits during chromatographic purification and to insensitive protein

detection methods. Another possibility is that the distinct Mediator types from different labs may

have various functions, and therefore, slightly different composition. For example, two distinct

Mediator complexes were isolated using VPl6 and SREBP-1 (sterol-responsive enhancer

binding protein) affinity resins, respectively (Taatj es et al., 2002). The larger one was named as

ARC-L, which is almost identical to the TRAP/DRIP/ARC/SMCC complexes. The smaller

complex was the CRSP complex. ARC-L and CRSP have many subunits in common, except that









CRSP has a CRSP70 subunit not present in ARC-L and does not have the following four

subunits present in the ARC-L: ARC240 /TRAP230O/MED 12, ARC250/ TRAP240/MED 13, cdk8,

and Cyclin C. In yeast, homologs (Srb8, -9, -10 and -1 1) of these four proteins comprise a

distinct complex (Borggrefe et al., 2002), designated as the CDK8 module. The ARC-L complex

is transcriptionally inactive, whereas the CRSP complex is highly active in a reconstituted

Spl/SREBP-dependent transcription system (Ryu et al., 1999).

Mediator Interacts with Transactivators

Many Mediator subunits, such as Medl, Medl2, Medl4, Medl5, Medl6, Medl7, Med23,

Med25, Med29, Cdk8 were found to interact with transactivators in human, yeast, or Drosophila

cells (Table 1-1). Some transactivators, such as the glucocorticoid receptor (Hittelman et al.,

1999) and differentiation-inducing factor (Kim et al., 2004), can interact with multiple Mediator

subunits suggesting a mechanism for more efficiently recruiting the Mediator.

The interaction between transactivators and Mediator subunits is important in

transcriptional regulation. Conditions that result in reduced levels of particular subunits may

have a negative influence on transcription. For example, Medl (TRAP220) was shown to

interact with PPARy, which is a nuclear receptor essential for adipogenesis (Zhu et al., 1997). In

7RAP220 null mouse embryos, the adipogenesis markers and PPARy2 target genes were not

expressed in the embryonic fibroblasts (MEFs), and the MEFs failed to differentiate into

adipocytes via the PPARy pathway (Ge et al., 2002). The authors also showed that activated

transcription by PPARy can be greatly increased by the TRAP complex in a reconstituted

transcription system. In addition, RXRa, another Med1 interacting partner (Zhu et al., 1997), was

shown to be able to enhance the effects of PPARy.









Mediator Interacts with RNA pol II

Many lines of evidence indicate that Mediator interacts directly with the CTD of RNA pol

II. Yeast Mediator, without the CDK8 module, and the human CRSP complex were isolated

through CTD-affinity chromatography (Myers et al., 1998; Naar et al., 2002). RNA pol II

lacking a CTD (Pol II ACTD) functions just as well as WT enzyme in basal transcription in vitro

when Mediator is absent. But contrary to the WT polymerase, this mutant RNA pol II cannot

respond to Mediator in basal transcription and in Gal4-VPl6 or GCN4 activated transcription

(Myers et al., 1998).

Precise structural information has revealed that the three modules of Mediator (head,

middle and tail) wrap around the RNA pol II in the holoenzyme. RNA pol II makes multiple

contacts with the head and middle modules and one with the tail. These interactions are centered

on the RNA pol II Rpb3/Rpb 11 heterodimer, but also involve Rpbl1, Rpb2, Rpb6 and Rpbl12

subunits. These contacts between Mediator and RNA pol II only account for 3 5% of the RNA

pol II surface; however, the remaining part is available for interaction with other PIC factors

(Davis et al., 2002, Chadick and Asturias, 2005).

Phosphorylation of RNA Pol II CTD

The cycle of phosphorylation and dephosphorylation of RNA pol II CTD is significant for

gene transcription. During transcription initiation, the recruitment of RNA pol II requires that the

CTD be hypophosphorylated. The Mediators isolated from Fleischmann's yeast (Kim et al.,

1994), S. pombe (Spahr et al., 2000), S. cerevisiae (Myers et al., 1998) and mouse (Jiang et al.,

1998) all stimulate the phosphorylation of the CTD by the TFIIH after PIC formation

(Hengartner et al., 1998). This phosphorylation of the CTD happens during the transition from

the transcriptional initiation to elongation and is thought to trigger promoter clearance

(Hengartner et al., 1998; Oelgeschlager, 2002). An additional role of the hyperphosphorylated









CTD is to promote interaction of the mRNA capping enzyme with the nascent transcript (Cho et

al., 1997).

The Kin28 protein is a subunit of TFIIH in S. cerevisiae and is the primary kinase involved

in the phosphorylation of RNA pol II CTD. Its kinase activity can be stimulated by Mediator in

vitro (Guidi et al., 2004). It was speculated that the Gall11 subunit of Mediator may regulate the

phosphorylation activity of Kin28 due to the interaction of Gall11 with TFIIH (Sakurai and

Fukasawa, 2000).

The CDK8 module of Mediator in yeast contains Srb8, Srb9, Srbl0, and Srbl1 subunits

and seems to exert a negative effect on transcription (Song et al., 1996; Samuelsen et al., 2003).

A plausible mechanism is provided by the action of Srbl10, which was shown to phosphorylate

the CTD prior to PIC formation and, thus, prevent the entry of RNA pol II (Hengartner et al.,

1998).

Mediator Interacts with Coactivators

Mediator has been shown to interact with other coactivators such as mammalian p300 and

TFIID (Black et al., 2006; Koleske et al., 1992; Thompson et al., 1993; Johnson et al., 2002;

Johnson and Carey, 2003). p300 is a coactivator that contains HAT activity, and in addition to

histones, it can acetylate transcription factors, as well as itself (Roth et al., 2001). The

consequence of its interaction with Mediator is an elevation in histone acetylation (Black et al.,

2006), which makes chromatin more accessible to other factors (Roth et al., 2001).

Autophosphorylation of p300 reduces its association with Mediator. The association of TFIID

with Mediator competes with p300 binding and results in a displacement of p300 from the

promoter. The j oining of Mediator with TFIID contributes to the assembly of the PIC and

activating of the promoter (Black et al., 2006).









Mediator Promotes the Formation of a Stable PIC

In vitro and genetic evidence suggest that Mediator contributes to the formation of a stable

PIC. It has been shown by a template commitment assay that Srb2 (Med20) is essential for the

formation of the PIC (Koleske et al., 1992). In addition, mutations in Srb2 (Med20), Srb4

(Medl7), or Srb5 (Medl8) prevent the formation of the PIC (Ranish et al., 1999), and mutations

in Sin4 (Medl6) and Pgdl (Med3) decrease both the rate and amount of PIC formation in yeast

(Reeves and Hahn, 2003).

Mediator is Required in the Reinitiation Scaffold

The association of Mediator with RNA pol II CTD, Gall11 with TFIIH (Sakurai and

Fukasawa, 2000), and Srb2 with TFIID (Koleske et al., 1992) facilitate the formation of a stable

PIC and maintain the reinitiation scaffold (Nair et al., 2005). Reinitiation and then multiple

rounds of transcription occur after RNA pol II, TFIIB, and TFIIF j oin the scaffold to re-form the

PIC (Nair et al., 2005). Mutation of Pgd1 results in dissociation of Mediator from the scaffold

after initiation and, thus, impairs reinitiation in yeast (Reeves and Hahn, 2003).

Mediator Stimulates both Basal and Activated Transcription

The Mediator fraction from yeast has been shown to stimulate GAL4-VPl6 or

GCN4-dependent transcription in a reconstituted system, and has also been shown to increase

basal transcription by 8-fold (Kim et al., 1994). The ARC (activator-recruited cofactor) complex

not only stimulates transcription by activators such as SREBP-la/Spl, NF-k
Gal4-VPl16/Spl, but also enhances basal transcription in vitro (Naar et al., 1999). Genome-wide

expression analysis showed that only 7% of genes were expressed in the Medl7 mutant of S.

cerevisiae (Holstege et al., 1998). Diminished Mediator leads to the reduction of basal and

activator-dependent transcription in yeast and HeLa cells, which can be restored by addition of

purified Mediator complex in vitro (Back et al., 2002, Nair et al., 2005).









Model for Mediator Function in Activated Transcription

Formation of the PIC starts with the binding of transactivators to the DNA, which is

followed by recruitment of TFIID, TFIIA and TFIIB to the promoter (Ranish et al., 1999, Reeves

and Hahn, 2003; Woychik et al., 2002). The Mediator is recruited by transactivators and possibly

by coactivators, such as p300 and TFIID (Koleske et al., 1992; Thompson et al., 1993; Johnson

et al., 2002; Johnson and Carey, 2003; Black et al., 2006). Mediator and TFIID form a platform

for the entry of the following factors. Mediator recruits the RNA pol II through interaction with

the CTD. TFIIF may be enlisted together with RNA pol II. Then TFIIE and TFIIH enter the

preinitiation complex (Thomas and Chiang, 2006). Next, the DNA helicase activity of TFIIH

causes promoter melting (Jiang et al., 1996; Kim et al., 2000), an essential step before the

synthesis of RNA can begin. Mediator greatly enhances the kinase activity of Kin28 of TFIIH,

which hyperphosphorylates the RNAP II CTD (Guidi et al., 2004). After CTD phosphorylation,

RNA pol II leaves the promoter with TFIIF to start transcriptional elongation (Yan et al., 1999;

Shilatifard et al., 2003). Mediator, TFIIA, TFIID, TFIIH and TFIIE stay on the promoter forming

a platform that supports reinitiation. This scaffold structure, in turn, recruits new TFIIB, TFIIF

and RNA pol II repeatedly to support multiple rounds of transcription (Yudkovsky et al., 2000).

Hypothesis for a Mediator Complex in Arabidopsis

Many of the basic mechanisms of transcription are conserved in plants, metazoans and

yeast (Reviewed in Gurley et al., 2006). The structures of many promoters in these three

kingdoms contain a TATA box, CAAT box, transcription start site and cis-elements for the

binding of general transcription factors and transactivators. In addition, RNA pol II and many

general transcription factors are conserved between plants, fungi and metazoans (Coulson and

Ouzounis, 2003). Arabidopsis also has homologs of the subunits of some coactivators such as

SAGA and other HAT containing complexes (Hsieh and Fischer, 2005). This wide array of









evidence for a high degree of conservation in the basic mechanisms of transcription suggests that

plants may also contain the Mediator coactivator. This view is strongly reinforced by the

presence of many putative Mediator subunits in Arabidopsis based on DNA sequence similarity

(Gurley et al., 2006; Boube et al., 2002). A compilation of Mediator subunits from yeast,

Drosophila and humans is presented in Table 1-2, along with putative subunits from Arabidopsis.

This provides the best estimate for Mediator subunit composition in plants and indicates that

plants may have at least 20 Mediator subunits present in other eukaryotes.

Despite evidence suggesting such a vital role for Mediator in gene expression, the precise

subunit composition and function of Mediator has not been determined in plants. Up to now, two

putative Mediator subunits in Arabidopsis thaliana have been studied. SWP (Strawwelpter) is

the orthologue of Medl4 and is involved in pattern formation at the shoot apical meristem, as

well as defining the duration of cell proliferation (Autran et al., 2002). PFT 1 (phytochrome and

flowering time 1) is the orthologue of Med25. It acts downstream of phyB to regulate the gene

expression and induce flowering under low-light conditions (Cerdan and Chory, 2003). The

important functions of these two putative Mediator subunits hint at the significance of the

Mediator in plants. To unravel the mechanism of gene transcription in plants, it is important to

identify the Mediator complex and characterize its function.










Table 1-1. Interaction of the transactivators with the Mediator subunits in different organisms


Saccharomyces
cerevisiae


Drosophila
melanogaster


Transactivator


ERa and ER 13 estrogen receptor (ER)


GATA family of transcription factors
Breast cancer susceptibility gene 1
(BRCA1i)
Thyroid hormone receptor (TRu, TRBl)

Androgen receptor

Glucocorticoid receptor (GR)
Peroxisome proliferator-activated
Med1
receptors (PPAR aand PPARy)
Retinoic acid receptor (RARu)
Retinoid-X-receptor for 9-cis-retinoic
acid (RXRu)
Vitamin D receptor (VDR)

Hepatocyte nuclear factor 4 (HNF-4)

Farnesoid X receptor (FXR)
Retinoid-related orphan receptor
(RORu)
p53

Aryl hydrocarbon receptor (AHR)


Me3 General control nondepressible factor 4
(GCN4)


Homo sapiens
Zhu et al., 1999;
Burakov et al.
2000: Warnmark
et al., 2001
Crawford et al.
2002

Wada et al., 2004
Yuan et al., 1998,
Zhu et al., 1997
Wang et al., 2002
Hittelman et al.,
1999

Zhu et al., 1997

Zhu et al., 1997

Zhu et al., 1997
Rachez et al.,
1999
Malik et al., 2002

Pineda et al., 2004

Atkmns et al., 1999

Drane et al., 1997
Wang et al., 2004


Park et al., 2000


SRY-box containing gene 9 (Sox9)

Replication and transcription activator
(RTA)


Zhou et al., 2002

Gwack et al., 2003


Hittelman et al.,
1999
Malik et al., 2002

Lau et al., 2003

Toth et al., 2004


Medl2


Glucocorticoid receptor (GR)

Hepatocyte nuclear factor 4 (HNF-4)
Medl4 Signal transducer and activator of
transcription (STAT2)
Sterol regulatory element-binding
protein-l a (SREBP-la)










Table 1-1. Continued.


Saccharomyces
cerevisiae


Drosophila
melanogaster


Transactivator


Homo sapiens


Kato et al., 2002


Small mothers against
decapentaplegic 2/3/4 (SMAD2,
SMAD3, SMAD4)
Medl5 VPl6
General control nondepressible factor
4 (GCN4)
Gal4


Medl6 Differentiation-inducing factor (DIF)

p53
VPl6
Medl7 Signal transducer and activator of
(Srb4) transcription (STAT2)
Differentiation-inducing factor (DIF)
Heat-shock factor (HSF)



Early region 1A (E1A)

ETS-like kinase protein-1 (Elk-1)

Med23 Epithelial-restricted with serine box
(ESX)
CCAAT/enhancer binding protein
(C/EBP)
Differentiation-inducing factor (DIF)
HSF (heat-shock factor)

Differentiation-inducing factor (DIF)
Med25 Heat-shock factor (HSF)
VPl6


Lee et al., 1999-'
Park et al., 2000
Lee et al., 1999;
Park et al., 2000
Park et al., 2000


Kim et al., 2004


Ito et al., 1999
Ito et al., 1999
Lau et al., 2003


Kim et al., 2004
Kim et al., 2004


Boyer et al.,
1999; Wang and
Berk, 2002
Stevens et al.
2002

Asada et al., 2002

Mo et al., 2004


Kim et al., 2004
Kim et al., 2004


Kim et al., 2004
Kim et al., 2004



Garrett-Engele et
al., 2002


Mittler et al., 2003


Med29 Doublesex (dsx )


Eberhardy and
Farnham, 2002


Cdk8


Myc










Table 1-2. Mediator subunits in yeast, Arabidopsis, Drosophila and humans (Gurley et al., 2006;
Boube et al., 2002)
Unified


nomenclature
(Bourbon et al.,
2004)
MED1
MED2
MED3
MED4
MED5
MED6
MED7
MED8
MED9


Arabidopsis
thaliana


Drosophila
melanogaster


Homo sapiens


Saccharomyces
cerevisiae

Med1
Med2
Med3
Med4
Nut1
Med6
Med7
Med8
Cse2/Med9

Nut2/Med 10


Trap220


Trap36

Med6
Med7
Arc32


TRAP220-ARC/DRIP205


TRAP36-ARC/DRIP36

hMed6-ARC/DRIP33
ARC/DRIP34-CRSP33
ARC32


At5g02850

At3g21350
At5g03220


At5g41910/
Atlg26665

At4g00450
Atlg55325
At3g04740
(SWP1)
Atl1g5780

At5g20170
At2g22370

At4g09070/
At2g28230
At4g04780
Atlg07950/
Atl1g6430
Atlg23230

Atlg25540
(PFT1)
At3g48060/
At3g48050
At3g09180


MED10

MED11
MED12
MED13

MED14

MED15
MED16
MED17
MED18
MED19

MED20

MED21

MED22

MED23
MED24

MED25


MED26

MED27
MED28
MED29
MED30
MED31
CDK8
CycC


Nut2


hNut2-hMed 10


Med11
Srb8
Srb9

Rgrl

Gall1
Sin4
Srb4
Srb5
Rox3


Med21
Kto
Skd/Pap/Bli

Trapl170

Arcl05
Trap95
Trap80
P28/CG14802
CG5546


HSPC296
TRAP230 ARC/DRIP240
TRAP240 ARC/DRIP250

TRAPl70-DRIP/CRSPl50

ARC105
TRAP95-DRIP92
TRAP80-ARC/DRIP77
p28b
LCMR1


Srb2

Srb7

Srb6


Trfp


hTrfp


Trap19

Med24

Trapl150/7
Trapl100

Arc92


Arc70

Trap37
Med23
Intersex
Trap25
Trap18
CDK8
CycC


hSrb7

SurfS

hSur2/CRSPl30
TRAP/CRSP/DRIP 100

ARC92


CRSP70-ARC70


TRAP3 7-CRSP3 4
Fksg20
Hintersex
TRAP25
hSoh1
CDK8
CycC


Soh1
Srbl0
Srbl1


At5gl9910
At5g63610
At5g48640









CHAPTER 2

MATERIALS AND METHODS

Plant Growth Conditions

The ecotype of Arabidopsis thalianaiiii~~~~~~iiiiii used in this study was Columbia-0. The plants were

grown in soil with continuous light from 40 W fluorescent bulbs at 27+10C. To examine the

germination and root length, the seeds were grown on vertical agar plates. The seeds were

surface sterilized with 70% ethanol for 3-5 min, and then with 10% bleach for 15-20 min. After

rinsing with sterile water (3 X 5 min), the seeds were plated in petri dishes containing 1/2 MS

(Murashige & Skoog) medium supplemented with 1% sucrose, 0.5g/L MES (2-(N-morpholino)

ethanesulfonic acid) and 0.8% agar. The plates were sealed with parafilm and placed vertically in

a growth chamber with a 16h light / 8h dark cycle provided by 40 W fluorescent bulbs at 220C.

For dark treatment, the plates were wrapped in aluminum foil and placed vertically in a growth

chamber at 220C.

Genotyping of the T-DNA Insertion Lines

The nzed31 T-DNA insertion mutants (nzed31-1 and nzed31-2) were obtained from

Arabidopsis Biological Resource Center (ABRC). For the genotyping of nzed31-1,

M~ed31-specific primer 5'- TGGATGTAAGTAGGATTGGCG -3' was paired with the

T-DNA-specific primer LBb 1 5' -GCGTGGACCGCTTGCTGCAACT-3 to produce a 628 base

pair (bp) fragment by polymerase chain reaction (PCR), or with another M~ed31-specific primer

5'- GAACTTGTCTTGGCAAGTTGG -3' to produce a 975 bp fragment. For the genotyping of

nzed31-2, M~ed31-specific primer 5'- TGATGTACTCTGGTCGCTGC -3' was paired with the

T-DNA-specific primer LBb 1 5' -GCGTGGACCGCTTGCTGCAACT-3 to produce a 714 bp

fragment, or with another Med31 specific primer 5' -TTGCGGGGATTACAACATTAC-3 to









produce a 1008 bp fragment. The T-DNA insertion sites were determined by sequencing the

PCR products.

RNA Analysis

The leaves of 40-day-old plants grown on soil were collected and RNA was isolated with

the Concert Plant RNA Reagent (Invitrogen). RNA blots were prepared as described by Cao et al.

(1994) and probed with full-length M~ed31 cDNA.

Microscopy

A Zeiss Axiocam HRm camera was used to examine the subcellular localization of

Med31-GFP fusion proteins in the root tip. GFP fluorescence was monitored with the Zeiss filter

set 10 (excitation, 450 to 490; dichroic, 510 LP; emission, 515 to 565). DAPI

(4',6-diamidino-2-phenylindole) fluorescence was monitored with Zeiss filter set 02 (excitation,

365; dichroic, 395 LP; emission, 420 LP). A Zeiss LSM 5 Pascal confocal laser scanning

microscope was used to localize the Med31::GFP fusion proteins in the plant tissues with an

Argon 488 nm laser and a Band Pass 505-530 fi1ter. A Helium Neon 543 nm laser with a 560 nm

fi1ter was used to record chlorophyll autofluorescence.

Plasmid Construction

The pBIl01sGFP(S65T) vector was provided by Dr. Robert Ferl (Manak et al., 2002). This

vector was constructed by removing the GUS (P-glucuronidase) gene by digestion with

restriction endonucleases Xbal and SacI, and then inserting the sGFP(S65T) gene between the

two restriction sites (Manak et al., 2002). The M~ed31 gene, including the 1.2 kb upstream

sequence and the entire exon and intron region without the stop codon, was amplified from the

genomic DNA by PCR using the primers 5' -tatTGTCGACTCTAATTAATCAGTCTTGGTC-3 '

and 5'- agaTCTAGATATACCCTTCCTGACATTATATGACT -3'. The fragment was inserted

in-frame to the 5' end of the sGFP(S65T) gene in the pBIl01sGFP(S65T) vector using the Sall









and Xbal sites. The M~ed31 1.2 kb upstream sequence was generated from the genomic DNA by

PCR using the pimers 5' -tatTGTCGACTCTAATTAATCAGTCTTGGTC-3 and

5'-ttataT CTAGAGAAC GAAC GGAAC CTGAAGC -3'. Thi s fragm ent was i n serte d i n-frame to

the 5' end of the GUS gene in the pBI101 vector using the Sall and Xbal sites. All of the PCR

amplified fragments were confirmed by DNA sequencing.

GUS Staining

The tissues were immersed in GUS Staining Solution (1 M sodium phosphate (pH 7.0), 0.5

M EDTA (ethylenediaminetetraacetic acid), 50 mM K' ferricyanide, 50 mM K' ferrocyanide,

10% Triton X-100 and 2 mM X-gluc) and vacuum infiltrated for 20 min. The samples were

incubated at 37 OC until blue color appeared. As a final step, 70% ethanol was used to clear the

tissue.

Agrobacterium Transformation Technique

The binary vector was transformed into Agrobacteriunt tunrefaciens strain GV3101 by

electroporation and the T-DNA transferred to Arabidopsis plants via the standard floral dip

protocol (Clough and Bent, 1998). Agrobacterium starter cultures were grown in 30 ml LB

(Loria broth) liquid culture medium with 25 Cpg/ml gentamicin, 50 Cpg/ml rifampicin and 50

Cpg/ml kanamycin with shaking (250rpm) at 28 OC overnight. A 15 ml aliquot of the starter

culture was added to 150 ml of LB liquid medium containing 25 Cpg/ml gentamicin, 50 Cpg/ml

rifampicin and 50 Cpg/ml kanamycin, and the culture was incubated with shaking (250 rpm) at 28

OC until an OD600 of 0.8 was reached. The cells were collected by centrifugation (5000 g, 30

min) and resuspended in 150 ml of 5% sucrose. After addition of 30 Cl~ of Silwet L-77 detergent,

the 3-week-old Arabidopsis plants were dipped in the Agrobacterium solution for several sec,

with gentle agitation. The plants were covered overnight to keep high humidity. Transformants









were selected by germinating the seeds on plates containing 1/2 MS medium with 50 mg/L

kanamycin.

Chromatin Immunoprecipitation

The putative Mediator subunits were mapped to promoter DNA using chromatin

immunoprecipitation (ChIP) according to Gendrel and colleagues (2005), with minor

modifications. The aerial parts ofArabidopsis plants were harvested (1.5-2.0 g) and rinsed with

water. The sample was then placed in 37 ml of 1% formaldehyde for cross-linking and vacuum

infiltrated for 15 min at room temperature. The reaction was quenched by the addition of 2.5 ml

of 2 M glycine, and the sample was placed under vacuum for an additional 5 min. The tissue was

rinsed thoroughly, frozen in liquid nitrogen and stored at -80 OC until further treatment.

Chromatin was extracted by grinding the frozen samples in 30 ml of Extraction Buffer 1

(0.4 M sucrose, 10 mM Tris-HCI (pH 8.0), 10 mM MgCl2, 5 mM P-mercaptoethanol, 0.1 mM

PMSF (phenylmethylsulphonyl fluoride), 1 X protease inhibitor). (To make 200 X Protease

Inhibitor, dissolve 0.16 g TPCK (tosyl phenylalanyl chloromethyl ketone) and 0.16 g TLCK

(tosyl-L-lysine chloromethyl ketone) in 5 ml of DMSO (dimethyl sulfoxide), then dissolve in 10

ml of 0.2 M PMSF in isopropanol.) Next, the sample solution was filtered with Miracloth

(CalBiochem) and then centrifuged at 3000 X g at 4 OC for 20 min. The pellet was dissolved with

1 ml of Extraction Buffer 2 (0.25 M sucrose, 10 mM Tris-HCI (pH 8.0), 10 mM MgCl2, 1%

Triton X-100, 5 mM P-mercaptoethanol, 0.1 mM PMSF, 1 X protease inhibitor) and centrifuged

at 12,000 X g at 4 OC for 10 min. After that, the pellet was resuspended with 300 Cl1 of Extraction

Buffer 3 (1.7 M sucrose, 10 mM Tris-HCI (pH 8.0), 2 mM MgCl2, 0.15% Triton X-100, 5 mM

P-mercaptoethanol, 0.1 mM PMSF, 1 X protease inhibitor), placed on another 300 Cl1 of

extraction buffer 3, and centrifuged at 14,000 X g at 4 OC for 1 hr. The pellet was resuspended

with 300 Cl1 of Nuclei Lysis Buffer (50 mM Tris-HCI (pH8.0), 10 mM EDTA, 1% SDS (sodium









dodecyl sulfate), 1 X protease inhibitor), and the chromatin was sheared to a size of 150 bp to

750 bp by sonication (10 times for 15 sec each at an amplitude setting of 20 using a Tekmar

Sonicator). The sample was centrifuged at 12,000 X g for 10 min and ChlP Dilution Buffer

(1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCI (pH 8.0), 167 mM NaC1) was added to

the supernatant to make a final volume of 3 ml. The solution was divided into three tubes and 40

Cl1 of protein A-agarose (Santa Cruz) was added to each tube of sample for pre-clearing at 4 OC

for 1 hr with gentle agitation. The protein A-agarose was removed by centrifugation (12,000 X g

at 4 OC for 30 sec), and the supernatant was transferred to fresh tubes. A 60 Cl1 aliquot was saved

at -20 oC as the "Input DNA control."

The immunoprecipitation was set up as follows: 10 Cl1 of IgG (Immunoglobulin G)

Sepharose (Amersham Biosciences) and 10 Cl1 of c-Myc (cellular myelocytomatosis oncogene)

antibody (Santa Cruz) were added, respectively, to two tubes to precipitate the TAP-tagged

Mediator subunits. No antibody was added to the third sample, which served as the "no antibody

control." The tubes were incubated at 4 OC overnight with gentle agitation. In order to purify

Mediator-bound complexes, 50 Cl1 of protein A-agarose beads were added to the tubes with

c-Myc antibody and "no antibody control," respectively, and the three tubes were incubated at 4

OC for 1 hr with gentle agitation. The agarose beads were pelleted by centrifugation at 3800 X g

at 4 OC for 30 sec and washed sequentially with Low Salt Wash Buffer (150 mM NaC1, 0.1%

SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCI (pH 8.0)), High Salt Wash Buffer (500

mM NaC1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCI (pH 8.0)), LiCl wash

buffer (0.25 M LiC1, 1% NP-40, 1% DOC (21-hydroxyprogesterone), 1 mM EDTA, 10 mM

Tris-HCI (pH 8.0)) and TE buffer. In order to extract the immune complex from the beads, 250

Cl1 of elution buffer (1% SDS, 8.4 mg/ml NaHCO3) WAS added to each sample, followed by









incubation at 65 OC for 15 min with gentle agitation. The elution step was repeated once to reach

a final volume of 500 Cl1. Elution buffer (440 Cl) was also added to the 60 Cl1 of the "Input DNA

control." After adding 20 Cl1 of 5 M NaCl to each sample, the cross-linking was reversed by

incubation at 650C overnight. The proteins in the sample were digested by incubation with 10 Cl1

of 0.5 M EDTA, 20 Cl1 of 1 M Tris-HCI (pH6.5), and 2 Cl1 of 10 mg/ml proteinase K at 450C for 1

hr. Then the proteins were removed from the DNA by phenol/chloroform extraction, and the

DNA was precipitated with the addition of ethanol (2.5 volume), sodium acetate (1/10 volume,

pH5.2) and 20 Cpg of glycogen. The DNA was resuspended with 50 Cl~ of 10 mM Tris-HCI

(pH7.5).

PCR Analysis of Chromatin Immunoprecipitation

The immunoprecipitated fraction was analyzed by PCR amplification to determine if the

DNA fragments from various promoters were present. The 25 Cl1 of PCR reaction system

contained 12.5 pmol of each primer, 5 nmol of dNTP, 3 Cl1 of DNA sample, 2.5 Cl1 of 10 X PCR

Buffer I and 1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA). The cycling

conditions were 8 min of thermal activation at 95 OC, followed by 50 cycles of 94 OC (30 sec), 55

OC (30 sec), and 72 OC (3 min). The primers used for PCR were as follows: 5'-

cgtggcctagaatacaaagaag -3' and 5'- tcaaacaataagaaagaccatgaca -3' were used for the

amplification of the CCA1 promoter; 5'-agattgttgacattctcggaaatttagtgccaactgt-3' and

5' -aaatgctcctttttctaaaaccttcgcttggagtct- 3' for the amplifi cati on of Hspl~8. 2 promoter;

5' -acaccacggcgtgaccat-3 and 5' -attatccagtcgacatctgta-3 for the amplification ofAdh1 promoter;

and 5'- TGTTTCTTCCCTTTAAGCAACC-3' and 5'-

AACATTTCTTTAGAACATTGACTTGG-3 were used to amplify the intergenic region

between At2g32950 and AT2G32960.









Bioinformatics

Multiple protein sequence alignments were performed with AlignX, which is a component

of Vector NTI Advance 10.3.0 from Invitrogen. The accession numbers at the NCBI (National

Center for Biotechnology Information) for the Med31 homologs are XP_307924 (Anopheles

ga~nbiae), NP_197491 (Arabidopsis thaliana), BQ583133 (Beta vulgaris), CD834180 (Bra~ssica

napus), NP_492413 (Caenorhabditis elegans), EAK92332 (Candida albicans), DY287612

(Citrus clententina), C X05 1496 (Citrus sinensis), E AU9 1557 (Coprinopsis cinerea), X P_62688 1

(Cryptosporidium parvum), DRO63 080 (Cyca~s runsphii), XP_63 83 30 (Dictyosteliunt

discoideunt), NP_649483 (Drosophilanzelan2oga~ster), CAD25946 (Encephalitozoon cuniculi),

DV154959 (Euphorbia esula), BM892402 (Glycine nzax), DT547393 (Gossypium hirsutunt),

CO126156 (Gossypium raintondii), NP_057144 (Homo sapiens), DWO49205 (Lactuca saligna),

DW126099 (Lactuca sativa), CF39363 5 (Loblolly pine), BQ1471 10 (M~edicago truncatula),

NP_080344 (M~us nauscuhts), DY336178 (Ocinsun ba~silicunz), CA902198 (Pha~seobts coccineus),

CF808645 (Phytophthora sojae), DR501487 (Picea sitchensis), CV015282 (Rhododendron

cataw/biense), NP_011388 (Saccharonzyces cerevisiae), NP_587859 (Schizosaccharonzyces

ponabe) and CAD21541 (Taenia solium). The accession numbers for the Med6 homologs are

XP_319180 (Anopheles ga~nbiae), NP_188772 (Arabidopsis thaliana), NP_504791

(Caenorhabditis elegan2s), EAK97077 (Candida albicans), XP_638621 (Dictyosteliunt

discoideunt), NP_731403 (Drosophilanzelan2oga~ster), XP_965884 (Encephalitozoon cuniculi),

NP_005457 (Homo sapiens), NP_081489 (M~us nauscuhts), NP_001057150 (Oryza sativa),

NP_ 1 1925 (Saccharonzyces cerevisiae) and Q9US45 (Schizosaccharonyces ponabe).









CHAPTER 3

RESULTS

Med31/Sohl is a mediator subunit that has been identified in humans (Gu et al., 1999),

Drosophila (Park et al., 2001), S. pombe and S. cerevisiae (Linder and Gustafsson, 2004). Soh1

(suppressor of hprl) was first identified as a suppressor of the S. cerevisiae hprl A mutant which

is temperature-sensitive for growth and can reduce the hyperrecombination phenotype (Fan and

Klein, 1994). Yeast two hybrid analysis showed Sohl interacts with the Rad~p protein, and a

Soh1 mutation exacerbated the DNA repair defect of a rad5-535 mutant (Fan et al., 1996). The

Soh1 orthologue Sepl0 in S. pombe was identified in screening mutants for both sterility and for

defects in cell separation. Sepl0 mutants are temperature-sensitive At a non-permissive

temperature (36 OC), the mutants formed multiple, ill-organized septa (Grallert et al., 1999).

Med31/Sohl was identified in the Mediator complex in humans (Gu et al., 1999) and Drosophila

(Park et al., 2001), but its function has not been determined, and the exact relationship between

mutations in M~ed31 and phenotype is still not clear.

Analysis of Arabidopsis Med31 Gene by Multiple Sequence Alignments

The Med3 1 homologs are present in the protests (Cryptosporidium parvum, Dictyostelium

discoideum, Encephalitozoon cuniculi), fungi (Candida albicans, Coprinopsis cinerea,

Saccharomyces cerevisiae, Schizosaccharomyces pomb), metazoans (Anopheles gamnbiae,

Caenorhabditis elegan2s, Drosophila melan2oga~ster, Homo sapiens, M~us musculus, Taenia.

solium) and plants (Arabidopsis thaliana, Oryza sativa) (Figure 3-1). Based on sequence

homology, we identified AT5Gl9910 in Arabidopsis as the putative M~ed31 gene (AtMed31),

which contains 6 exons and encodes a protein of 196 amino acids (aa), with a calculated

molecular mass of 22.8 kDa. There is a conserved block of 70 aa (aa R30 to R99 of AtMed3 1)

(Figure 3-1) that shows high similarity to Med31 in other species (D. melan2oga~ster, 58.3%









identity and 76.4% similarity; H. sa-piens, 61.1% identity and 72.2% similarity; S. cerevisiae,

45.2% identity and 56.2% similarity; S. pombe, 59.7% identity and 77.8% similarity), where its

functional identity has been demonstrated. Linder and Gustafsson (2004) showed that this region

is required for its assembly within the Mediator complex in S. cerevisiae, which suggests this

conserved domain is important for interaction with other Mediator subunits.

A search of the NCBI EST database using TBLASTN (Altschul et al., 1997) for AtMed3 1

identified the Med31 homologs in many other plant species (Figure 3-2). There is a conserved

block of 139 aa (aa Ml to V139 of AT5Gl9910) between these plant Med3 1 homologs, which

includes the 70 aa domain conserved between different species (Figure 3-1). The C-termini of

the Med31 homologs are less conserved compared with their N-termini (Figure 3-1 and Figure

3-2), and often contain regions that resemble transcriptional activation domains which have

glutamine-rich or serine/proline-rich blocks. Since these domains function in transactivator

proteins to make contact with target transcription factors, it seems reasonable to assume that the

C-terminal region of Med3 1 containing these activator domain-like blocks may be at the outside

of the complex and provides surface for interaction with transactivators or other transcriptional

machinery.

Phenotype Characterization of med31 Mutants

The Mediator plays a vital role for RNA pol II-mediated transcription; therefore, disruption

of the highly conserved Med3 1 subunit is predicted to have a disruptive effect on the expression

of a large number of genes, some leading to abnormal phenotypes. To test this hypothesis and

study the function of this putative subunit, we searched the SIGnAL (signal.salk.edu) T-DNA

insertion collection for mutants (Alonso et al., 2003). Four T-DNA insertion lines were identified

and ordered from the Arabidopsis Biological Resource Center (ABRC). The T-DNAs of

SalkO3 5522 and Salk05 1025 lines insert into the promoter and 3' UTR (untranslated region) of









M~ed31, respectively, but we did not observe any mutant phenotype for these two lines. The

med31-1 (Salkl45479) mutant line has the T-DNA insertion in the promoter region, and the

T-DNA of med31-2 (Salk 143 815) mutant line is located within the 5' UTR (Figure 3-3). The

insertion sites for all Salk lines were confirmed by DNA sequencing.

In contrast to the previous two mutant lines, both med31-1 and med31-2 plants showed

abnormalities in growth. Under our experimental conditions, the germination rate of med31-1

seeds was 17% compared with 100% for the wild type (WT). In addition, their root length was

41.6% of the WT root length (Figure 3-4). The seeds of med31-2 plants germinated as well as the

WT seeds; however, their root length was 47.7% of the WT root length (Figure 3-5). These

differences in growth were not present under dark conditions, where the med31-2 seedlings grew

as well as the WT seedlings (Figure 3-6). This result suggests that the function of Med3 1 during

seed initial development may be dependent on light.

Some of the med31-2 plants had aberrant patterns of cotyledon development, such as three

cotyledons and three first true leaves, a single cotyledon, or forked cotyledons (Figure 3-7).

However, these mutant phenotypes were only inherited by some of their progeny, and the

med31-2 mutants with normal cotyledons also produced progeny with abnormal cotyledons. The

seedlings with abnormal cotyledons segregated 26% (5/19) progeny with abnormal cotyledons;

whereas the seedlings with normal cotyledons segregated 30% (5/20) progeny with abnormal

cotyledons. In addition, the overall sizes of the med31-2 mutants were reduced, their leaves were

smaller, and they had fewer rosette leaves compared with WT plants (Figure 3-8).

Med31 Expression in the med31-2 Plants

In med31-2 plants, the T-DNA inserts into the 5' UTR of the gene and may, therefore,

influence the expression of2~ed31 gene at either the transcriptional or translational level.

Northern blotting was used to examine the expression of2~ed31 in the WT and med31-2 plants.









In med31-2 mutant plants, the corresponding mRNA was more abundant than that in WT plants

(Figure 3-9). The mutant phenotype of med31-2 plants may be caused by the overexpression of

Med3 1 protein, which possibly sequesters the adj acent Mediator subunits or other components of

the transcriptional apparatus. Alternatively, Med31 translation may be inhibited due to the

missing, or changed nucleotides at the 5' end of the transcript.

Subcellular Localization and Tissue Expression Pattern of Med31::GFP Fusion Proteins

To investigate the subcellular localization of Med3 1, the 1.2 kb upstream sequence and the

entire exon and intron region of2~ed31 (without the stop codon) was amplified from the

genomic DNA by PCR. The resulting DNA was ligated in-frame to the 5' end of the sGFP(S65T)

gene in the pBI101 sGFP(S65T) vector. We observed that the Med31::GFP fusion proteins were

expressed in the tip of primary roots and that the signal was confined to the nucleus (Figure

3-10). In addition to being expressed in the lateral root tips, primordia (Figure 3-11) and root

hairs (Figure 3-12), the Med31::GFP signal was also found to be present in the aerial portions of

the plants including leaves (Figure 3-13), trichomes (Figure 3-14) and petioles (Figure 3-15).

Free GFP has been shown to be present in both the nucleus and cytosol (Li et al., 2001; Ye et al.,

2002; Zhong et al., 2005). To further complicate analysis, GFP has been shown to move to other

cells and tissues via the plasmodesmata (Crawford and Zambryski, 2001). However, in each type

of tissue we observed, signal from the Med3 1::GFP fusion protein was almost exclusively found

in the nucleus. The nuclear localization exhibited by Med31::GFP is consistent with its presence

in the nucleus being a property conferred by the Med3 1 portion of the protein, as contrasted with

the more general subcellular localization previously shown for GFP alone.

Tissue Expression Pattern of Med31 Promoter::GUS Fusions

To investigate the tissue expression pattern of Med3 1 protein, the M~ed31 promoter was

fused to the 5' end of the P-glucuronidase (GUS) gene and transferred to Arabidopsis. The GUS









signal was detected in the shoot apexes (Figure 3-16A) and lateral root primordia (Figure 3-16B)

of young seedlings (16 days old) of transformed plants. It was also detected in the whole young

inflorescences (Figure 3-17A), anthers (Figure 3-17B) and stigmas (Figure 3-17C) of adult plants

(46 days old) and in developing seeds (Figure 3-17D). This pattern differs from where

Med31::GFP signal was detected in that no GUS signal was detected in the primary root tips,

leaves and petioles. Two possible explanations for this apparent inconsistency are that GUS

staining sensitivity may be less than that of GFP. Alternatively, the promoter DNA alone as

present in the Med31::GUS construct (without the exons, introns and untranslated regions) is

insufficient to fully reproduce the expression pattern of the endogenous M~ed31 gene.

Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA

Chromatin immunoprecipitation (ChIP) is a powerful tool to explore in vivo protein-DNA

interactions. The ChlP assays conducted here involves the cross-linking of proteins and DNA in

chromatin, followed by co-immunoprecipitation of DNA fragments associated with the

epitope-tagged Mediator subunits. After the proteins have been removed, the pool of DNA

fragments can be queried by PCR amplification for the presence of specific promoter regions.

Mediator associates with promoter DNA indirectly by binding with transactivators and

RNA pol II. Previous studies in yeast using the ChlP technique (Andrau et al., 2006, Zhu et al.,

2006) showed that Mediator could not only bind to the core promoters and upstream activating

sequences, but also to the coding regions of many genes, as well. It can associate with the

promoters of both active and some inactive genes, but genes with higher transcriptional activity

usually have higher promoter occupancy by Mediator. It is thought that the presence of Mediator

at inactive promoters may be required for quick response to environmental changes.

If Med3 1 is a genuine mediator subunit, it should be found associated with promoter DNA.

We used the ChlP assay to test this hypothesis. In addition, we checked if another Arabidopsis









putative Mediator subunit, Med6 (AT3G21350), was also localized to promoter DNA. As with

Med3 1, the assignment of Arabidopsis Med6 as a putative subunit of Mediator was based strictly

on protein sequence homology (Figure 3-18). There is a conserved block of 129 aa (aa M33 to

S161 of AtMed6) that shows similarity to Med6 in other species (D. melan2oga~ster, 36.4%

identity and 51.2% similarity; H. sa-piens, 42.6% identity and 55.0% similarity; S. pombe, 31.1%

identity and 48. 1% similarity) where its functional identity has been demonstrated. It was

predicted to be localized in the nucleus by two web tools (Hua and Sun, 2001; Nair and Rost,

2002). Our prediction is that both proteins we have tentatively identified as AtMed31 and 6,

respectively, should be associated with the promoter regions of a wide array of genes.

The Med6 and Med31 cDNAs were introduced into the pC-TAPa vectors (Rubio et al.,

2005) and individually transformed into Arabidopsis by Dr. Kevin O'Grady (Gurley laboratory,

University of Florida). Their C-termini were fused with nine repeats of the myc epitope,

followed by six histidine residues, the 3C protease cleavage site and two copies of the protein A

IgG binding domain. The fusions of Med6 or Med3 1 with the epitope tags were confirmed by

Western blots. Immunoglobulin G Sepharose and c-Myc antibody were used to

immunoprecipitate the tagged Med6 or Med31 proteins, respectively, in the ChlP experiment.

ChlP Analysis for Med31

Immunoglobulin G Sepharose was used to immunoprecipitate Med31-DNA complexes

from the T1 generation of2~ed31-pC-TAPa transgenic Arabidopsis plants. Primer pairs for the

promoters of CCA1 (AT2g46830), Hspl8.2 (AT5g59720), Adh1 (ATlg77120) and a fragment in

the intergenetic region (between AT2g32950 and AT2g32960) were used to test if Med3 1 binds

to these sequences. The CCA1 circadiann clock associated 1) gene encodes a MYB-related

transcription factor and its expression oscillates with a circadian rhythm (Wang and Tobin, 1998).

Hspl8.2 (heat shock protein 18.2) is a heat inducible gene. Adh (alcohol dehydrogenase) is also









an inducible gene regulated by environmental stresses, such as low oxygen, dehydration, and low

temperature (Dolferus et al., 1994). Both Hspl8.2 and Adh genes are expressed at low levels

under normal conditions (Volkov et al., 2003; Dolferus et al., 1994). Mining of Arabidopsis EST

database failed to find the transcripts of the intergenetic region (>4kb) between At2g32950 and

AT2G32960, suggesting this region is not transcribed. Therefore, we used this region as a

negative control which Mediator may not bind. The promoters of CCA1, Hspl8.2 and Adh1 were

all co-immunoprecipitated with the epitope tagged Med31 protein by IgG Sepharose (Figure

3 -19), demonstrating the localization of Med3 1 to these promoters. As predicted, the intergenetic

region was not co-immunoprecipitated with the tagged Med31 by IgG Sepharose.

ChlP Analysis for Med6

Immunoglobulin G Sepharose and c-Myc antibody were used individually to

immunoprecipitate Med6- DNA complexes from the T2 generation of2~ed6-pC-TAPa transgenic

plants. The same set of primer pairs were used for the amplification from the DNA pool derived

from co-immunoprecipitation with epitope tagged Med6. The promoters of CCA1, Hspl8. 2, and

Adh1 were all co-immunoprecipitated with Med6 by both IgG Sepharose and c-Myc antibody

(Figure 3-20), demonstrating the localization of Med6 with these promoters. Again, as predicted,

the intergenetic region was not co-immunoprecipitated with Med6. It should be noted that both

Med6 and Med3 1 were independently found to be localized to the promoters of three unrelated

genes, CCA1, Hspl8.2 and Adhl, a finding consistent with both proteins belonging to a Mediator

complex.

Wild type plants were also included in ChlP experiment as a negative control to check if

IgG Sepharose and c-Myc antibody can immunoprecipitate the CCA1 promoter in the absence of

epitope tagged Mediator subunits. No PCR product of this promoter was amplified (Figure 3-21),









validating the conclusion that our ChlP protocol serves as a reliable indicator that Med6 and

Med31 can specifically immunoprecipitate promoter DNA.

Conclusion

Two T-DNA insertion lines in either the M~ed31 promoter or 5' untranslated region were

identified. The germination rate of med31-1 plants was lower, and their root length was much

shorter than that of WT plants. The root length of med31-2 plants was also shorter than that of

WT plants. The med31-2 mutants exhibited a dwarfed phenotype with fewer rosettes leaves than

the WT plants, and some of them had aberrant patterns of cotyledon development, such as three

cotyledons and three first true leaves, a single cotyledon, or forked cotyledons. These mutant

phenotypes imply that M~ed31 plays an important role in many aspects of plant development,

such as germination, root elongation and cotyledon development.

Using Med31::GFP constructs, we found that the Med31 protein was localized in the

nucleus. The Med31::GFP signal was detected in all the tissues that were examined, including

roots, root hairs, leaves, trichomes and petioles. In another experiment, the M~ed31 promoter was

fused to GUS gene to study its tissue expression pattern. The M~ed31 promoter::GUS reporter

was detected in the shoot apexes and lateral roots of young seedlings (16 days old) and in the

young inflorescences, anthers, stigmas of the adult plants (46 days old) and in developing seeds.

The promoters of three unrelated genes (CCA1~, Hspl8. 2 and Adhl) were all

co-immunoprecipitated with Med31 by IgG Sepharose and with Med6 by both IgG Sepharose

and c-Myc antibody. These results demonstrate the localization of Med6 and Med3 1 to these

promoters, which is consistent with the function of Mediator.

Taken together, this study provides evidence that Med6 and Med31 are both Mediator

subunits because 1) Med31 was localized in the nucleus; 2) Med31 was expressed in every type

of tissue that were examined; 3) Disruption of Med3 1 resulted in abnormal plant growth; and 4)









Both Med6 and Med31 proteins were localized to promoters. These data strongly support our

hypothesis that the Mediator complex found in fungi and metazoans is also present in plants.


















(1) 1 3 5 7 9 0 1 2
Arabd~opss~thalpna (1) M S E M D A EPPIT K EG ~RLEEl
Orymautwa (1) MEEnAP ELnn LEElCP E YHL
Drtyostelum~dscod~eum (1) M S S IELG I N N T I E G N E I K D N
Cryptorpor~um~parvum (1) MS G SLIL N I RSEE~CS EYaL
Anophezs~gambpe (1) L~V LFaLNN LF~RYKP F~L
aorophibamelnogaster (1) Mi~cGTIs ~an ~ aEE~CS E Y
Homo~saprzns (1) MEVMTL GRRaEE~CPEYNLa
Mus~murcubs (1) MEVMTL GRRaEE~CPEYNLa
Caenorhabdas~elgans (1) MSEETF VCF(LN NLF ~ R YKE
Taenapsolum (1) M~Ps LPnGsW NLLk RaEEVS
Cand~a~abrans (1) M~ Ta I~a (~~Y NI SE RoILF
Saccharomyces cerevena e (1) MsNNPTs oNr TFV LF(LN ( T
Encephaltozooncunrul (1) MGREEEVL CEYRL RGF SERY
Schaosaccharomyces pombe (1) M T WL K EDSFILF(LN kLF ~ H
Coprnopsscnerea (1) M P Pa P V A T P S NAFLLF(LN Y
Consensus (1) nn EEVCEE YNL Y FIYK LYKEYKIY LME~
(121) 2 3 4 5 6 7 8 9 0 1 2 3 0
Arabd~opss~thalpna (96) PF T M H EKLH (Fro NI~LIIP
Orymautwa (84) PF NMHEKVH(Y~KY~RIHLR
Drtyostelum~dscodeum(120)RRENH(TFH((YFoYI~R SKa
Cryptorpor~um~parvum (84) DRNSEV(I(T aolDKEH L
Anophezs~gambpe (67) EFRIS(CFD(ILoHTRTL AG
aorophibamelnogaster (86) EF R I N (CFD (Iao H T KILE V
Homo~saprzns (82) EFKLN(AFD(II~(YRRRaA
Mus~murcubs (82) EFKLN(AFD(II~(YRRRaA
Caenorhabdas~elgans (75) arLM Y E AF E (VaoFLK HLM P
Taenapsolum (95) onvHsnro(rIuKYR AE\T
Cand~a~abrans (95) EFKINLMSM LVRoSP~DNEE
Saccharomyces cere vena e (88) MSIN LLED LKI(G aNMVRP
Encephaltozooncunrull (70) NNMSE FLG(YlkIHKGK
Schaosaccharomyces pombe (81) PrNIR LSaNEY ~ LK ~ aGADT
Coprnopsscnerea (89) ArELKELL~(( L~o~~D iHN
Conrenur(121) F I (




Figure 3-1. Multiple sequence alignments of Med3 1 homologs in different species. Identical

amino acids are indicated in yellow, conservative amino acids in light blue and


similar amino acids in green.



















(1)1 1 2 3 4 5 6 7 8 9 10 ~ 1 2
ARabd~opss thalna (1) MSEM EPPKTKPG~RLEE CPPYH LC
Brassra~napus (1) lRRFED aPPA LSVVSAPE LE KTKPV aRLEE CNTYHA,
G ocne~max (1) svnsssTssKEESL SP YDDaaFLLFaPNYI La
Medrago~truncatuba (1) ESENILRVVWDVMSTSSRT YDDGaFEEFCPPYH La
Phytophthora~solae (1) AKEESL SP YDDaaFEE FCPNTI La
Phaseobs coceneus (1) IL~r(R(RE LELVH LWCETS SPNYDDGaFLLFaLNTIY~
Cirus clmentna (1) (~s~ ~or~sCKIM KN SASPKYDDGaFLLFaLNTIY~
Cirus~snenss (1) SCKIM K SASP YDDGaFLLFCPNTH La
Rhododendron~catawbrz nse (1) REFP LFTMS aTE SP YDDGRLEE FCPNYI La
Euphorbapesuba (1) RXNRGHPXSE DPSK KPDRRLEE CPPYH LC
Gossvpum~ralmondll (1) IFPCVI FRN F SVVKLLLFkHT( LI ILSFIK PSK KPDRRLE F LNTYYA,
Betauubars (1) VFSLNLLFITNFECFE aKDS S STNYDDGaFLLFaLNTIY~
Lactuca~ulgna (1) GLklRR LRRENPECSASE HSK OPDRRLEE LNTYYA,
Lactuca~satwa (1) G~A~lRLETTVS LaRSLSVN NHSK OPDRRLEE CPPYHLC
Ocimum~basilrum (1) HFCINDM S P S P TYDDGaFLLFaLKTIY~
Cycas rumphil (1) GRGMLLPIKPNPD~RLEE~CPPYH
P rea~srchenss (1) GG EPIKPKPD~RLEE CPPYH LC
Pnus taeda (1) CST~lR DDVL kNSRT(RIEN EPIKPKPD~RLEE CPPYHLC
Consensus (1) A L TSE YLLG( FLLFaPN YILa
(121) 11 ~ 3 10 ~ 5 10 ~ 7 10 ~ 9 20 ~ 1 20 ~ 3 4
Arabd~opss thalna (54) RFDAIYKaaREIF MPCYLL WWIRLHLRLEV aEESTPEST P
Brassra~napus (83) RFDAIYKaYa PYKIYHLF E WWIRLHLRLEV aEVSSPEPT
G ocne~max (70) N YEEFGLYaa P YKI YHLFE~
Medrago~truncatuba (80) RF D AI YKaa REIFM P CYLL ELEETEPV LPPTSVT a
Phytophthora~solae (54) RF D AI YKaa R EIFM P CYLL EEEETSAV LPPTVVAPa
Phaseobs coccneus (93) RF D AIYKara P YKI YHLLE TSVEST
Cirus clmentna (76) NYEEFGL YaaPYKIYHLF E ~ WWI~LHLRLE AEPEEEEPLPT G
Cirus~snenss (61) NYEEFGL YaaPYKIYHLF E ~ WWI~LHLRLE AEPEEEEPPPT G
Rhododendron~catawbrz nse (68) RFDAIYK aaREI FMPCY LL NRAA NEA aa WWI KI EPEEPEV EEESTVAPPP P
Euphorbapesuba (66) NYEEF GL YaaPYKIYHLF E ~ WWI~LHLRLE EESPP~PPVPTGPGA
Gossvpum~ralmondl(118) RFDAIYK~~REIFMPCYLL a A~a WIY~LFL PEVTELESE~SPSIMTP A
Betauubars (98) NY EFG LY aaPYKIYH LFE~NNRAAP SEP~aYWW I~LHLRLEL
Lactuca~ulgna (83) NYEEFG LY aaPYK I Y HLF E~N SR P P P N ET~aYW P
Lactuca~satwa (93) NYEEFG LY aaPYK I Y HLF E~N SR P P P N ET~aYW L
Ocimum~basilrum (59) RFDAIYK aaRE FMPC LLoPFNMHPK LH arFIY~R PP TES ELLEVPVNPAP a
Cycas rumphil (52) Nu Er cLuaaPu~lYHLFE~NNRPPPNEP ~aYWWI~L PEL
Prea~sTchenss (50) R DAIYK aaREI FMPC LLoPFSMHTK LH a FIY~RKIPPP EPE EAAEEPVTVVS T
Pnus taeda(113) NY EFG LY aaPYKIYH LFE~NNRPPPNEP ~ a WWI~LHLRLE ASEETEEPAP~TVVS a(
Consenus(121) NuEEr cLuaaPY~rYHrr~r EE E
(241) 4 5 6 7 8 9 0 1 2 3 4 5 0
Arabdopss thalna( 164) LP OTM SWTNGT RKKG
Brassra~napur(191) SMTL W IVE RKKGEA LaPDYSC

MedK asornaua Zo SMY PS IDXTE
Phytophtoaoau llSPPGPGGAWMNE NRR LNTKIIP oU LoI LF
Phass~Locns ..en .9101


Rhododendronctwrne16 LSMO PHS IIPTG L

Gosq rpeuania~ P LSMY :S IDRT R KKEPISElnPI











edr ago mrunatu( 226) -


Ctmas snens(267O) LFRGTG RR RK NNLFFLIIIFISE YL FR
Rhddnr nucatawense(213) --R S VE R R YI LR T
Euporbaesub(231)A -- G WDR I RR

AlBeostauar(2185) -
Latuass alna (269) --

Ldastruncatutwa(37) -
vOcimumobasilaeu(230) -

Cyru cas rumnzphl5) --~ r CS S Y GCLS SILKLSLE~
Prearsncenss(237) --LLFLKK

Cuponbenag36Z1)



Figure 3-.Mutpl linensofAme3 wt tedeuedain ci eqece f t

homlog inohe latsece. dniclamn aisar ndctd nyelw

cosevtie mnoacd i lgt lu ndsiiaramn aid n ren












I I I I


med31-2


100 bp


Figure 3-3. Diagrammatic representation of the insertions of the T-DNA in nzed31-1 and
nzed31-2. The M~ed31 gene contains six exons, which are represented by red boxes.
UTR regions are indicated by blue boxes. The position of triangle represents the
T-DNA insertion site.


Figure 3-4. Germination rate and root length of WT and nzed31-1 seedlings (9-day-old). A) WT.
B) nzed31-1. The size bars represent 0.5 cm.


Figure 3-5. Nine-day-old WT and nzed31-2 seedlings grown under continuous light. A) WT. B)
nzed31-2. The size bars represent 0.5 cm.

























Figure 3-6. Nine-day-old WT and med31-2 seedlings grown under dark. A) WT. B) med31-2.
The size bars represent 0.5 cm.


U


U


Figure 3-7. Ten-day-old WT and med31-2 seedlings. A) WT. B) med31-2 with three cotyledons
and three first true leaves. C) med31-2 with forked cotyledon. D) med31-2 with a
single cotyledon.


Figure 3-8. Comparison of adult WT plants and med31-2 plants. A) 30-day-old WT and med31-2
plants. B) 55-day-old WT and med31-2 plants. In both panels, the left plant is
med31-2, and the right plant is WT.










WT mdd31-2


Figure 3-9. Northern blot analysis of2~ed31 expression in WT and med31-2 plants.




















Figure 3-10. Subcellular localization of Med3 1::GFP fusion proteins in the root tip of a
35-day-old plant. A) Image of GFP. B) Image of DAPI staining.





























Figure 3-11i. Expression of Med3 1::GFP fusion proteins in lateral roots. A) A lateral root. B) A
lateral root primordium.


:S*;;


V.~~';' ;d
ii r ,~flr*~",~'!
..~..
d"i..* *e ..":~?*Y~1
I~
,r b=

-;~* -.


Figure 3-12. Expression of Med3 1::GFP fusion proteins in a root hair. A) Image of GFP signal.
B) DIC image. C) Overlay of the DIC and GFP images.



















A 11 IIB













Figure 3-13. Expression of Med3 1::GFP fusion proteins in a leaf. A) Image of GFP signal. B)
DIC image. C) Image of autofluorescence. The chloroplasts are red because of
autofluorescence of chlorophyll. D) Overlay of the GFP and autofluorescence images.













~g~,
-
T,


Figure 3-14. Expression of Med3 1::GFP fusion proteins in a trichome. A) Image of GFP signal.
B) DIC image. C) Image of autofluorescence. The chloroplasts are red because of
autofluorescence of chlorophyll. D) Overlay of the GFP and autofluorescence images.





































Figure 3-15. Expression of Med3 1::GFP fusion proteins in a petiole. A) Image of GFP signal. B)
DIC image. C) Image of autofluorescence. The chloroplasts are red because of
autofluorescence of chlorophyll. D) Overlay of the GFP and autofluorescence images.




















A L~


Figure 3-16. M~ed31 promoter directed GUS tissue expression pattern in young plants
(16-day-old). GUS signal was detected in A) A shoot apex. B) Lateral root primordia
and tips.















A B C D

Figure 3-17. M~ed31 promoter directed GUS tissue expression pattern in adult plants (46-day-old).
GUS signal was detected in A) A young inflorescence. B) Anthers. C) A stigma. D)
Developing seeds.
















(1) 1 ,10 ,20 ,30 ,40 5 O 7 0 9 0
Arabidopss~thalina (1) -----------------MDS S LSAATADTFGEU PPCPTM RULIS LDN YAS YT
Oryza~sativa (1) --TLPAPPGDTGICRaWNY LRNTF~F L F~
Dictyostellu m~dsco de um (1) MEDFNNDDPMNFDKDDMINNNN~NDNNNN~NNDNDNNNENNEDSNNNSNEEDTC~W~PWCMPN~T ILCFYSF~
Anopheles_gambae (1) --LCNLISH~~~ TLPNV~F KNF~
Drosophila~mehnogaster (1) --RMNHRSHTIMEL~T~~FRS
Homo~saplens (1) --T~IRNL ST~ WPILSGV~F RNF~
Mus~musculus (1) -
Caenorhabdits~elgans (1) --RGPERDPH~SRPPNIKNI
Candda~albrans (1) --LECKPFCR LNNT LYSSF~
Saccharomyces_cerevsne (1) --~PD LWSEIT~GRET LYASF
Schmosaccharomyces~pombe (1) --GPSDTIWMEWCMGRET LY~
Encephalitozoon~cun cull (1) --RE SF~~R LSPDTT~EFG F~
Consensus (1) LIW~WC LTYYSSF~


(101) 13 ,140 150
Arabidopss~thalina (67) TN EL RSHLLHS~TLYLDTPL
Oryza~sativa (53) TN ELSaHLMHT~T M YLD~~P
Dictyostellum~dscoldeum(100) N N~L ~ RDSL~GE ELKVPF IK~R P
Anopheles_gambae (43) T N EV~~SEL~GEIL~DIYI~
Drosophib~melanogaster (44) C N TRaLPHHILY HVE YIKCRNS
Homo~saplens (44) C N V KC R LHNMrGE LHCE F IaaSP
Mus_musculus (1) CR LHNMrGE LHCE F IaaSP
Caenorhabditis_elegans (51) N Ca I~NV R V ELTP~YLYaP F
Candida~albicans (38) T NaL~r~aa P G S~YaRS M GE
Saccharomyces~cerevislae (40) TS~VKaa~LD NA~aIMTLPDGKNGNLEEEAVPRQL
Sch mosaccharomyces~pombe (46) KN EL~~rADGL~LRT~rIH R
Encephalitozoon~cunicul (39) S N EL~~rG~ISLS~GFEES H
Consensus(101) CNIK R GEVL FIK P


(201) 201 ,210 ,220 ,230 ,240 ,250 ,260 ,270 ,280 ,290 300
Arabidopss~thalina(117) ----KVTPMLTYYI LDGSIYQAPQLCSVFAARVSRTI YNI SKAFTDAASKLETIRQVDTENQNEPAE----SKPASET----VD LKEM KR
Oryza~sativa(103) ----KSNAMLAYYI LDGSIYQAPQLCSVFASRI SRAMHHI SKAFTTACSKLEKIGHVETEPDTAASE----SKTQK Dictyostellum~dscoldeum(147) ----DVLINT LYYVINGNIYQ~APE LHVVFKSRVSQ~SISHLSEAFNSI SSIVNWUDIVNGYS LNLDPSN---QEKSKLAAYS--RK--IEDTKRL
Anopheles_gambae (90) ----EATPMADYYIIAGTVYQAPDLASVFNSRI LSTVHHLQTAFDEASSYSRYHPSKGYSWDFSSNKAIAEKTKQKEPEPSIFRV
Drosophib~melanogaster (91) ----EATPIADYYI IGGTVYKAPDLANVrINSRI LNTVVNILQSAFEEASSYARYHPNKGYTWD FSSNK VFSRSKDKDNSKENGLFQ~KQRV
Homo~saplens (91) -----VIPLADYYIIAGVIYQAPDLGSVINSRVLTAVHGIQSAFDEMYRHSGWHKH--QKRKK-KESSFRR
Mus_musculus (40) -----VIPLADYYIIAGVIYQAPDLGSVINSRVLTAVHGIQSAFDEMYRHSGWHKH--QKKKK-KESSFRR
Caenorhabditis_elegans(101) ----NVSPIAYYYVINGSVHQAPDMYSLVQSRLLGALEPLRNAFETYRNAGWFKPVKEEKDE-LDSNQTT
Candida~albicans(101) ----TVTLQDYYIIGANVYQAPRIYDVLSSRLL ASVLSIKNSTDLNMSHDGSYSHSSKQSAKP-TTTATP
Saccharomyces~cerevislae(139) VGSAKGPEIIPLQ DYYIIGANIYQSPTIFKIVQSRLMSTSYLSELDIFQQGHKPTTAATNNG-GNSSRGA
Schmosaccharomyces_pombe(101) ----EVKPLTVYFVCNENIYMAPNAYTLL ATRMLNATYCFQALTIKPYPEGTPLNNEVHNNPD-------N
Encephalitozoon~cunicul (92) ---AETLGMYYIIHGHVYAAPTNYSIYRCRMGDSMWQLNS FID RMK FN S- --P---GRL S------ED D
Consensus(201) V PLADYYIIAG IYQ)APDL SVINSRVL AVH LQ)SAFDEA SY RY PS GY W KSK SI RV


(0)301 ,310 ,320 ,330 ,340 ,350 ,360
Arab dopss5thalina(19 6) -----------QVDPP GY~CELGKEL~aG S PP V P I IDQGPAKRMKF-
O ryzasativa(18 2) -----------QVDPP GT~S CEASD LA EL P~ P I IDQGPAKRPRFQ-
Drtyostellum~dscod eu m(231) -----------Q------QQ~GGGI TU C PSQP--
Anophees~gamblae(182) -------GDSDHVGADATLIKQEPTEGGVASNNHG
Drosoph la~melanogaster(184) -----P-PI P~N LCPEG~NARA MNEG LDIKTEGVDMKPPPEKKSK--
Homoaplens(178) -----ETTKEVQQTVSAKGPPEKRNRLQ-KEAEPI
Mus_musculus(127) -----ESTKNIQQTVSEPT~T~CEKEAPLTKGPPEKRNRLQ-
Caenorhabdits~elegans(193) --------TEAEREKEVEEE TSTDEPEPTTRTSQ
Candida~albicans(191) ITIPLYG--EGSTLERLGLKGNKDAGLSL--NDVV
Saccharomyces~cerev slae(237) -----------LMVTSIRSTPNYI--RTG GNMG
Sch izosaccharomyces~po mbe(181) ---------------SW AF US HSS-KEAPD~K --
Encephalitozoon~cunicul(160) LFMINFK~
Consensus(301) KP K P KR




Figure 3-18. Multiple sequence alignments of Med6 homologs in different species. Identical

amino acids are indicated in yellow, conservative amino acids are indicated in light

blue, and similar amino acids are indicated in green.










CCAl Equl.2 Adlh Inerge esreg
ML2 4M234M123 Y1Z44M~I 2 34








Figure 3 -19. Med3 1 associates with the promoters of CCA1~, Hspl8. 2 and Adhl, but not with the
intergenetic region. The promoters used are indicated above the gels. Lane M was
loaded with 100 bp DNA Ladder from New England Biolabs. The templates for each
PCR are as follows: Lane 1: Genomic DNA from wild-type plants; Lane 2: Input
DNA control (sonicated genomic DNA from M~ed31-pC-TAPa transgenic plants);
Lane 3: Negative control chromatinn extract without antibody immunoprecipitation
from M~ed31-pC-TAPa transgenic plants); Lane 4: Chromatin immunoprecipitated
with IgG Sepharose from M~ed31-pC-TAPa transgenic plants.




(TAl I~slB. Adh In~eimpEh ~eH@D
M 12394 5 1M l 2 3 4 5 Ml 2 34 5 ~M I2 34









Figure 3-20. Med6 associates with the promoters of CCA1, Hspl8.2 and Adhl, but not with the
intergenetic region. The promoters used are indicated above the gels. Lane M was
loaded with 100 bp DNA Ladder from New England Biolabs. The templates for each
PCR are as follows: Lane 1: genomic DNA from wild-type plants; Lane 2: Input
DNA control (sonicated genomic DNA from M~ed6-pC-TAPa transgenic plants); Lane
3: Negative control chromatinn extract without antibody immunoprecipitation from
M~ed6-pC-TAPa transgenic plants); Lane 4: Chromatin immunoprecipitated with IgG
Sepharose from M~ed6-pC-TAPa transgenic plants. Lane 5: Chromatin
immunoprecipitated with c-Myc antibody from M~ed6-pC-TAPa transgenic plants.










O4C~I
Mu 1 2 4 5









Figure 3-21. Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the
CCA1 promoter from WT Arabidopsis. The templates for each PCR are as follows.
Lane M was loaded with 100 bp DNA Ladder from New England Biolabs. Lane 1:
genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic
DNA from wild-type plants); Lane 3: Negative control chromatinn extract without
antibody immunoprecipitation from wild-type plants); Lane 4: Chromatin
immunoprecipitated with IgG Sepharose from wild-type plants. Lane 5: Chromatin
immunoprecipitated with c-Myc antibody from wild-type plants.









CHAPTER 4

DISCUSSION

Phenotype Characterization of med31 Mutants

The M~ed31 promoter or 5' UTR were disrupted by T-DNA insertion in med31-1 and

med31-2 lines. Both mutant lines had shorter roots than WT plants under our experimental

conditions. In addition, seeds were examined in preliminary studies (data not included) for their

responses to a variety of hormones. The med31-2 seedlings were insensitive to ABA, kinetin,

and 2, 4-D, compared with WT seedlings. A possible cause for the mutant phenotypes of these

two insertion lines is due to the disruption of either transcriptional or translational expression of

M~ed31. The T-DNA in med31-1 breaks a GT-1 cis-element (identified by AthaMap web tools;

www.athamap.de), which has been shown in other promoters to play a role in the gene regulation

by light, pathogens and salt (Villain et al., 1994; Park et al., 2004). Likewise, the T-DNA in

med31-2 breaks the CCAAT BOX1 (identified in the PLACE database;

www. dna. affrc.go.j p/PLACE), which has been reported to be involved in transcriptional

expression by heat stress (Rieping and Schoffl, 1992; Haralampidis et al., 2002). T-DNA

insertions not only disrupt the inserted cis-elements, but also impede the function of the

cis-elements upstream of the insertion sites. The location of the two T-DNA insertions found in

med31-1 and -2 are predicted to strongly interfere with the regulation of2~ed31 gene expression.

Med3 1 is a subunit of Mediator complex, which is important in gene transcription mediated by

RNA pol II. Defective M~ed31 expression has the potential to influence the binding of the

transactivators to Med3 1 subunit, alter the structure of the mediator, hinder the entry of other

subunits into the Mediator, or the entry of general transcription factor or RNA pol II into the PIC,

and thus, cause pleiotropic effects by impeding RNA pol II-dependent transcription. Consistent









with this hypothesis, our preliminary data showed multiple aspects of plant development were

influenced for the med31-2 mutant.

Evidence for a Mediator Complex in Arabidopsis

The transcription apparatus of plants, metazoans and yeast are conserved (Gurley et al.,

2006). Many of the promoters in these three kingdoms contain the TATA motif and CAAT box

for the binding of RNA pol II and general transcription factors. Transactivators generally bind

the upstream cis-elements to regulate gene expression. The RNA pol II in all the three kingdoms

contains 12 conserved subunits. In addition, plants possess the genes coding for all the general

transcription factors (TFIIA, B, D, E, F, and H) that are present in metazoans and fungi (Coulson

and Ouzounis, 2003). Arabidopsis also has the homologs of the subunits of some coactivators

(Hsieh and Fischer, 2005), such as the SAGA (Spt-Ada-Gen5-acetyltransferase) (Stockinger et

al., 2001) and SWI/SNF complexes (Brzeski et al., 1999; Eshed et al., 1999; Ogas et al., 1999).

This high degree in conservation of the transcription machinery suggests that the plants may also

have the Mediator coactivator which has been shown to play an essential role in RNA pol

II-mediated transcription in other eukaryotes. Identification of the homologs of most of the yeast

and metazoan Mediator subunits in Arabidopsis strongly supports this hypothesis (Gurley et al.,

2006; Boube et al., 2002).

The experiments described here explore various aspects of gene expression for two

putative Mediator subunits from Arabidopsis, Med31 and Med6. By all measures tested, these

two proteins behaved as expected for bona fide members of plant Mediator. AtMed3 1 was

localized in the nucleus, and was widely expressed throughout the plants. Both AtMed6 and

AtMed3 1 were localized to the promoters of three unrelated genes: CCA1, Hspl8.2 and Adhl.

Together with the sequence homology between Arabidopsis proteins and known Mediator

subunits from other eukaryotes, these data strongly support the presence of a Mediator complex









in Arabidopsis, and higher plants in general, that shows strong conservation in both form and

function with analogous complexes in fungi and metazoans.










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BIOGRAPHICAL SKETCH

Wei Pan received his Bachelor of Science degree in biology from Northeast Normal

University in Changchun, China, and then a Master of Science degree in biophysics from the

Chinese Academy of Agricultural Sciences in Beijing, China. His current research interests are

centered on genetics, development and molecular biology.




Full Text

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1 MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS IN Arabidopsis thaliana By WEI PAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 Copyright 2007 by Wei Pan

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3 To my family

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4 ACKNOWLEDGMENTS I thank Dr. Bill Gurley for his kindness, commitment and great mentoring. He is an excellent advisor because he always encouraged me to develop indepe ndent thinking and gave me the opportunity to pursue my research inte rests. His careful reading and editing greatly improved this thesis. I thank Dr. Robert Ferl fo r his suggestion on my wo rk and giving me the pBI101sGFP vector. I thank Dr. Kevin OGrady for his continuous help with my experiments and teaching me many molecular biology tec hniques. I extend thanks to Dr. Eva Czarnecka-Verner for her suggestions on my research. I thank Dr. Zhonglin Mou and Ms. Xudong Zhang fo r their valuable assistance with some techniques such as Northern blotting and GUS st aining, and generously letting me share some of their facilities. I thank Ms. Donna Williams fo r her assistance with the con-focal observation. I thank Dr. Masaharu Suzuki for helping me ge t started in research in the Plant Molecular and Cellular Biology Program (PMCB); Dr. Alice Ha rmon for the invaluable training I gained in her lab; and Dr. David Cl ark for his kindness and support for my study in PMCB. I also wish to express my gratitude to the entire PMCB faculty who taught me in classes and journal clubs. I benefited a lo t from the wonderful courses. I am extremely grateful for all my family and friends for their understanding and support over the years.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION..................................................................................................................14 Assembly of the Preinitiation Complex..................................................................................14 Identification of the Mediator Complex in Yeast...................................................................16 Identification of the Mediator Complex in Human Cells.......................................................17 TRAP Complex...............................................................................................................17 SMCC Complex..............................................................................................................18 DRIP Complex................................................................................................................18 ARC Complex.................................................................................................................18 CRSP Complex................................................................................................................18 PC2 Complex...................................................................................................................18 Mediator Interacts w ith Transactivators.................................................................................20 Mediator Interacts with RNA pol II........................................................................................21 Phosphorylation of RNA Pol II CTD.....................................................................................21 Mediator Interacts with Coactivators......................................................................................22 Mediator Promotes the Formation of a Stable PIC.................................................................23 Mediator is Required in the Reinitiation Scaffold..................................................................23 Mediator Stimulates both Basal and Activated Transcription................................................23 Model for Mediator Function in Activated Transcription......................................................24 Hypothesis for a Mediator Complex in Arabidopsis ..............................................................24 2 MATERIALS AND METHODS...........................................................................................29 Plant Growth Conditions........................................................................................................29 Genotyping of the T-DN A Insertion Lines.............................................................................29 RNA Analysis................................................................................................................... ......30 Microscopy..................................................................................................................... ........30 Plasmid Construction........................................................................................................... ...30 GUS Staining................................................................................................................... .......31 Agrobacterium Transformation Technique............................................................................31 Chromatin Immunoprecipitation............................................................................................32 PCR Analysis of Chroma tin Immunoprecipitation.................................................................34

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6 Bioinformatics................................................................................................................. .......35 3 RESULTS........................................................................................................................ .......36 Analysis of Arabidopsis Med31 Gene by Multiple Sequence Alignments............................36 Phenotype Characterization of med31 Mutants......................................................................37 Med31 Expression in the med31-2 Plants...............................................................................38 Subcellular Localization and Tissue Expression Pattern of Med31::GFP Fusion Proteins....39 Tissue Expression Pattern of Med31 Promoter::GUS Fusions...............................................39 Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA....................................40 ChIP Analysis for Med31................................................................................................41 ChIP Analysis for Med6..................................................................................................42 Conclusion..................................................................................................................... .........43 4 DISCUSSION..................................................................................................................... ....58 Phenotype Characterization of med31 Mutants......................................................................58 Evidence for a Mediator Complex in Arabidopsis .................................................................59 LIST OF REFERENCES............................................................................................................. ..61 BIOGRAPHICAL SKETCH.........................................................................................................73

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7 LIST OF TABLES Table page 1-1 Interaction of the transact ivators with the Mediator subu nits in different organisms..........26 1-2 Mediator subunits in yeast, Arabidopsis Drosophila and humans ....................................28

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8 LIST OF FIGURES Figure page 3-1 Multiple sequence alignments of Med31 homologs in different species.....................45 3-2 Multiple alignments of AtMed31 with the deduced amino acid sequences of its homologs in other plant species................................................................................ ...46 3-3 Diagrammatic representati on of the insertions of the T-DNA in med31-1 and med31-2........................................................................................................................47 3-4 Germination rate and root length of WT and med31-1 seedlings (9-day-old).............47 3-5 Nine-day-old WT and med31-2 seedlings grown under continuous light....................47 3-6 Nine-day-old WT and med31-2 seedlings grown under dark.......................................48 3-7 Ten-day-old WT and med31-2 seedlings......................................................................48 3-8 Comparison of adult WT plants and med31-2 plants...................................................48 3-9 Northern blot analysis of Med31 expression in WT and med31-2 plants....................49 3-10 Subcellular locali zation of Med31::GFP fusion prot eins in the root tip of a 35-day-old plant............................................................................................... ............49 3-11 Expression of Med31::GFP fusion proteins in lateral roots.........................................50 3-12 Expression of Med31::GFP fusion proteins in a root hair............................................50 3-13 Expression of Med31::G FP fusion proteins in a leaf...................................................51 3-14 Expression of Med31::GFP fusion proteins in a trichome...........................................52 3-15 Expression of Med31::GFP fusion proteins in a petiole...............................................53 3-16 Med31 promoter directed GUS tissue e xpression pattern in young plants (16-day-old)..................................................................................................................54 3-17 Med31 promoter directed GUS tissue expr ession pattern in adult plants (46-day-old)..................................................................................................................54 3-18 Multiple sequence alignments of Med6 homologs in different species.......................55 3-19 Med31 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region........................................................................................ ..........56

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9 3-20 Med6 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region........................................................................................ ..........56 3-21 Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the CCA1 promoter from WT Arabidopsis ........................................................................57

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10 LIST OF ABBREVIATIONS Adh ARC CCA1 ChIP c-Myc CRSP CTD DRIP EST GFP GUS HAT Hsp18.2 IgG PC2 PIC RNA pol Sep10 SMCC Soh1 SWI/SNF TAP TFIIA TFIIB alcohol dehydrogenase activator-recruited cofactor circadian clock associated 1 chromatin immunoprecipitation cellular myelocytomatosis oncogene cofactor required for Sp1 activation carboxy-terminal domain vitamin D receptor interacting protein expressed sequence tag green fluorescent protein -glucuronidase histone acetyltransferase heat shock protein 18.2 immunoglobulin G positive cofactor 2 preinitiation complex RNA polymerase Separation10 SRB/MED Cofactor Complex suppressor of hpr1 switching/sucrose non-fermenting tandem affinity purification Transcription Factor II A Transcription Factor II B

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11 TFIID TFIIE TFIIF TFIIH TRAP UTR VP16 WT Transcription Factor II D Transcription Factor II E Transcription Factor II F Transcription Factor II H thyroid hormone receptor-associated protein untranslated region herpes simplex virus protein 16 wild type

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12 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS IN Arabidopsis thaliana By Wei Pan May 2007 Chair: William B. Gurley Major Department: Plant Molecular and Cellular Biology Mediator is a conserved coactivator comp lex that has been identified in yeast, Drosophila and humans. It plays a critical role in gene transcription me diated by RNA polymerase II (RNA pol II) by serving as a bridge between activators bound to the prom oter and other transcription machineries, including RNA pol II. Despite eviden ce suggesting such a vital role of Mediator in gene expression, the subunit composition and functi on of Mediator has not been determined in plants. Based on the conserved transcriptional machineries (RNA pol II, general transcription factors and some coactivators) in plants, metazo ans and yeast, we hypothesized the plant also has the Mediator coactivator. Identification of the homologs of most of the yeast and metazoan Mediator subunits in Arabidopsis supported this hypothesis. This study characterized the function of tw o putative Mediator subunits, Med6 and Med31. Two T-DNA insertion lines in the Med31 promoter or 5 untranslate d region were identified. The med31-1 mutant line had shorter root length and a reduced germination rate. The med31-2 plants had shorter root length, aberrant patterns of cotyledon development, and smaller size compared with wild type plants. We found the Med31::GFP (green fluorescent prot ein) fusion proteins were localized to the nucleus. The Med31::GFP signal was detected in the roots, leaves,

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13 trichomes and petioles. In addition, we found the Med31 promoter::GUS fusions were expressed in the shoot apexes and latera l roots of the young seedlings (1 6 days old), and in the young inflorescences, anthers, stigmas of adult plants (46 days old) and in developing seeds. Both Med6 and Med31 proteins were localized to the promoters of three unrelated genes ( CCA1, Hsp18.2 and Adh1 ). These results strongly support th e conclusion that Med6 and Med31 are members of the Mediator complex in Arabidopsis

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14 CHAPTER 1 INTRODUCTION Assembly of the Preinitiation Complex Transcription is one of the most significant st eps that occur during ge ne expression. It is carried out by RNA polymerases and additiona l factors. There are four kinds of RNA polymerases in plants, RNA polymerase (RNA pol) I, II, III and IV. RNA pol I is located in the nucleolus, and it transcribes rRNA genes, excep t 5S rRNA. RNA pol II is located in the nucleoplasm and transcribes hnRNA, the precursor of mRNA. RNA pol III is also located in the nucleoplasm and is responsible for the synthe sis of tRNA, 5S rRNA and other small RNAs (Thomas and Chiang, 2006). And last, an RNA pol ymerase unique to plants, RNA polymerase IV, is involved in the siRNA silencing pa thway, RNA-dependent DNA methylation and the formation of heterochroma tin (Onodera et al., 2005). Transcription by RNA pol II can be broadl y categorized as basal transcription (activator-independent) and activ ated transcription (activator-dependent). A simplified sequence of activated transcription initiation for RNA pol II has been postulated as follows. Activators (transactivators or transcripti on factors) bind the re gulatory motifs of DNA and then recruit a variety of additional factors that prepare the promoter for the arrival of RNA pol II and the formation of the preinitiation complex (PIC ) (Thomas and Chiang, 2006). One of the first components to arrive is a kinase which phosphor ylates histone H3 (Featherstone, 2002). Then coactivators that can modify chromatin structur es are recruited. For example, HAT (histone acetyltransferase) arrives at the promoter early in the activation proce ss and its role is to acetylate specific lysines in histone amino-termin i and other transcriptio n factors (Roth et al., 2001; Naar et al., 2001; Clayton et al., 2006). Another complex that is recruited early in the process of gene activation is SWI/SNF (switchi ng/sucrose non-fermenti ng), which remodels the

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15 chromatin structure and facilita tes the accessibility of other me mbers of the transcriptional apparatus to the DNA (Gavin et al., 2001, Havas et al., 2000). After the promoter is made accessible, the TFIID complex is recruited to the TATA box in the promoter (Pugh, 2000). Activators also recruit Mediator complex which facilitates the formation of pol II PIC, which consists of RNA pol II and genera l transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) (Conaway et al., 2005). Next, TFIIH facilitates promoter melting a nd phosphorylates the CTD (carboxy-terminal domain) of the largest subun it of RNAP II (Jiang et al., 1996; Kim et al., 1994). This phosphorylation event is thought to be require d for promoter clearance and the start of transcriptional elonga tion (Dvir et al., 1997; Kugel and Goodrich, 1998; Kumar et al., 1998). After the synthesis of the initial transcript, most members of the PI C (with the exception of TFIIB and TFIIF) remain at the promoter and form a structure know as the scaffold that facilitates the reentry of RNA pol II, TFIIB and TFIIF for subsequent rounds of synthesis (Yudkovsky et al., 2000). In the process of reinitiation, the CTD of RNA pol II is dephosphorylated by a CTD phosphatase that is stim ulated by TFIIF (Friedl et al., 2003). This cycle of phosphorylation and dephos phorylation of the CTD is essent ial to the entry of RNA pol II to the PIC (with a hypophosphorylated CTD) and subsequent promoter clearance (hyperphosphorylated CTD) (Oelgeschlager, 2002). One of the key regulatory complexes involved in the process of promot er activation is the Mediator. This large assemblage of proteins (~ 2MDa) is conserved from yeast to humans and is composed of 25-29 subunits (Boube et al., 2002). It is becoming incr easingly clear that Mediator plays a critical role in both ac tivated and basal transcription me diated by RNA pol II in yeast and metazoans (Baek et al., 2002, Nair et al., 2005), b ecause it serves as a br idge between activators

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16 bound to the promoter and other ge neral transcription factors, as well as RNA pol II (Kornberg, 2005). Identification of the Mediator Complex in Yeast The presence of Mediator was proposed becau se of the discovery of activator inhibition in yeast. The activator GAL4-VP16 was found to repress the activatio n effect of another activator (a factor binding to a thymidine-rich DNA element) both in vivo and in vitro This phenomenon led to the hypothesis that the two ac tivators competed for a common intermediate factor. Activator inte rference was relieved in vitro with the addition of the fraction containing this intermediate factor, which was na med Mediator (Kelleher et al., 1990). The Mediator fraction from column chroma tography was shown to be required for GAL4-VP16 and GCN4-dependent gene transcription in an in vitro transcription system (Flanagan et al., 1991). This was th e initial direct evidence that Mediator was involved in gene transcription. The In vitro transcription system was reconsti tuted with purified RNA pol II and general transcription factors from yeast and wa s widely used for checking the presence of Mediator, thereafter. Three experimental approaches were originally used to identify proteins as Mediator subunits: 1) Identify the suppre ssors of RNA pol II CTD trunca tion mutations; 2) Isolate the fraction (RNA pol II holoenzyme) that can stim ulate the activator-dep endent transcription. Separate the proteins by electr ophoresis in a gel, and then identify the proteins by peptide sequencing; and 3) Identify the proteins th at co-immunoprecipitate with known Mediator subunits. The presence in the RNA pol II holo enzyme and support of activator-dependent transcription were two criteria that were used to confirm the identities of Mediator subunits. In total, 25 Mediator subunits have been identified in S. cerevisiae Nine Mediator subunits (Srb2, Srb4, Srb5, Srb6, Srb7, Srb8, Srb9, Srb10 a nd Srb11) were identified based on the

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17 suppression of S. cerevisiae RNA pol II CTD truncation mutations. All of these subunits were shown to be present in the holoenzyme (Thomp son et al., 1993; Kim et al., 1994; Koleske and Young, 1994; Liao et al., 1995; Hengartner et al ., 1995). Fifteen Mediator subunits (Med1, Med2, Pgd1 (Hrs1), Med4, Med7, Med8, Med11, Gal11, Si n4 Rgr1, Mtr32, Rox3, Nut1, Nut2, and Cse2) were detected in the R NA pol II holoenzyme and identified by peptide sequencing (Kim et al., 1994; Gustafsson et al., 1997; Gustafsson et al., 1998; Li et al., 1995; Myers et al., 1998). The mutant yeast strains for Gal11, Sin4 and Rg r1 showed similar mutant phenotypes, which suggested they may function in the same path way (Fassler et al., 1991, Jiang and Stillman, 1995, Suzuki et al., 1988; Chen et al., 1993, Sakai et al., 1990). More recently, Med31 was found to be a Mediator subunit in S. cerevisiae and S. pombe based on co-purifica tion with previously characterized Mediator subunits (Linder and Gustafsson, 2004). Identification of the Mediat or Complex in Human Cells Two methods were used to identify the Mediat or subunits in human cells: 1) Isolate the nuclear extract fraction that can stimulate the activator-dependent tran scription. Separate the proteins on the gel, and then identify the prot eins by peptide sequencing; and 2) Identify the proteins that co-immunoprecipita te with the transactivators (or their activation domains) or known Mediator subunits. Most id entified subunits are orthologs of the yeast Mediator subunits. However, various Mediator complexes with di fferent subunit compositions were isolated in different labs (Sato et al., 2004). A brief description of human Mediator types follows. TRAP Complex A thyroid hormone receptor-associated protei n (TRAP) complex was isolated based on co-precipitation with FLAG epitope-tagged hTRal phal (human thyroid hormone receptor alpha1) (Fondell et al., 1996).

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18 SMCC Complex The human SRB/MED Cofactor Comple x (SMCC) was purified by affinity chromatography of FLAG epitope-tagged hum an SRB proteins (Gu et al., 1999). DRIP Complex The DRIP (vitamin D receptor interacting prot ein) complex was isolated from the nuclear extract of human Namalwa B cells based on it s interaction with the VDR LBD (vitamin D3 receptor ligand-binding domain) in the presence of hormone. This complex contains 10 proteins and it can stimulate transcription by VDRRXR. It was shown that at least one of its subunits has histone acetyltransferase ac tivity (Rachez et al., 1998). ARC Complex The ARC (activator-recruited cofactor) comp lex was isolated by its affinity for the activation domains of SREBP-1a, VP16 and the p65 subunit of NF-kB, respectively, from HeLa cell nuclear extract (Naar et al., 1999). It can not only stim ulate transcription by activators such as SREBP-1a/Sp1, NF-kB/Sp1, Gal4-VP16/Sp1, but it also enhances basal transcription in vitro CRSP Complex The CRSP (cofactor required for Sp1 activa tion) complex was isolated from HeLa cell nuclear extract and shown to be required for Sp1dependent transcriptional activation (Ryu et al., 1999). This complex consists of 9 subunits and has a mass of approximately 0.7 MDa. PC2 Complex The PC2 (positive cofactor 2) complex was isolated from HeLa cell nuclear extracts based on its ability to stimulate HNF4 (hepatocyt e nuclear factor 4) and GAL4-AH dependent transcription (Malik et al., 2000). This complex consis ts of at least 15 subun its and is larger than 0.5MDa. The presence of these subunits w ithin the complex was confirmed by the

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19 co-immunoprecipitation of epit ope (FLAG and HA)-tagged MED 10. Both PC2 and CRSP were found to be subcompexes of ARC, DRIP, or TRAP/SMCC (Malik and Roeder, 2000). Despite being originally isolated by different approaches, some complexes found in human cells (ARC, DRIP, and TRAP/SMCC) were show n to be very similar in subunit composition (Naar et al., 1999; Mali k and Roeder, 2000). The finding that various closely related Mediator complexes have slightly different subunit composition raised the question of whether some of the proteins identified are true subunits, or just contaminants associated with a particular isolation strategy. Sato and colleagues (Sato et al., 2004) addressed this question by co-immunopreci pitation of human Mediator using six FLAG-tagged subunits to individually purify co mplexes for analysis of subunit composition by MudPIT (multidimensional protei n identification technology). Pr oteins present in all six independent Mediator preparations were consider ed to be true Mediator subunits. Their results support the conclusion that all prot eins identified previously are bona fide Mediator subunits. In addition, they identified the MED13L and the CDK8-like cyclin-dependent kinase CDK11 as putative Mediator-associated proteins. The inconsistency in Mediator subunit compos ition was thought to be due in part to the dissociation of Mediator subunits during chromatographic purificat ion and to insensitive protein detection methods. Another possibilit y is that the distinct Mediator types from different labs may have various functions, and theref ore, slightly different compos ition. For example, two distinct Mediator complexes were isolated using VP 16 and SREBP-1 (sterolresponsive enhancer binding protein) affinity resins, respectively (Taatjes et al., 2002) The larger one was named as ARC-L, which is almost identical to the TRAP/DRIP/ARC/SMCC complexes. The smaller complex was the CRSP complex. ARC-L and CRSP have many subunits in common, except that

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20 CRSP has a CRSP70 subunit not present in A RC-L and does not have the following four subunits present in the ARC-L: ARC240 /TRAP230/MED12, ARC250/ TRAP240/MED13, cdk8, and Cyclin C. In yeast, homologs (Srb8, -9, 10 and -11) of these four proteins comprise a distinct complex (Borggrefe et al., 2002), desi gnated as the CDK8 module. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active in a reconstituted Sp1/SREBP-dependent transcripti on system (Ryu et al., 1999). Mediator Interacts with Transactivators Many Mediator subunits, such as Med1, Med12, Med14, Med15, Med16, Med17, Med23, Med25, Med29, Cdk8 were found to interact with transactivators in human, yeast, or Drosophila cells (Table 1-1). Some transactivators, such as the glucocorticoid receptor (Hittelman et al., 1999) and differentiation-inducing factor (Kim et al., 2004), can interact with multiple Mediator subunits suggesting a mechanism for more efficiently recruiting the Mediator. The interaction between transactivators and Mediator subunits is important in transcriptional regulation Conditions that result in reduced levels of particular subunits may have a negative influence on transcription. For example, Med1 (TRAP220) was shown to interact with PPAR which is a nuclear receptor essential for adipogenesis (Zhu et al., 1997). In TRAP220 null mouse embryos, the adipogenesis markers and PPAR 2 target genes were not expressed in the embryonic fibroblasts (MEFs), and the MEFs failed to differentiate into adipocytes via the PPAR pathway (Ge et al., 2002). The aut hors also showed that activated transcription by PPAR can be greatly increased by the TRAP complex in a reconstituted transcription system. In addition, RXR another Med1 interacting pa rtner (Zhu et al., 1997), was shown to be able to enhance the effects of PPAR

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21 Mediator Interacts with RNA pol II Many lines of evidence indicate that Mediator interacts directly with the CTD of RNA pol II. Yeast Mediator, without the CDK8 module, and the human CRSP co mplex were isolated through CTD-affinity chromatography (Myers et al., 1998; Naar et al., 2002). RNA pol II lacking a CTD (Pol II CTD) functions just as well as WT enzyme in basal transcription in vitro when Mediator is absent. But contrary to th e WT polymerase, this mutant RNA pol II cannot respond to Mediator in basal tr anscription and in Gal4VP16 or GCN4 activated transcription (Myers et al., 1998). Precise structural information has revealed that the three module s of Mediator (head, middle and tail) wrap around the RNA pol II in the holoenzyme. RNA pol II makes multiple contacts with the head and middle modules and on e with the tail. These in teractions are centered on the RNA pol II Rpb3/Rpb11 heterodimer, but also involve Rpb1, Rpb2, Rpb6 and Rpb12 subunits. These contacts between Mediator a nd RNA pol II only account for 35% of the RNA pol II surface; however, th e remaining part is available for interaction with other PIC factors (Davis et al., 2002, Chad ick and Asturias, 2005). Phosphorylation of RNA Pol II CTD The cycle of phosphorylation and dephosphorylati on of RNA pol II CTD is significant for gene transcription. During transcri ption initiation, the recruitment of RNA pol II requires that the CTD be hypophosphorylated. The Medi ators isolated from Fleischmann's yeast (Kim et al., 1994), S. pombe (Spahr et al., 2000), S. cerevisiae (Myers et al., 1998) and mouse (Jiang et al., 1998) all stimulate the phosphorylation of the CTD by the TFIIH after PIC formation (Hengartner et al., 1998). This phosphorylation of the CTD happens during the transition from the transcriptional initiation to elongation a nd is thought to trigge r promoter clearance (Hengartner et al., 1998; Oelgeschlager, 2002). An additional role of the hyperphosphorylated

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22 CTD is to promote interaction of the mRNA capping enzyme with the nascent transcript (Cho et al., 1997). The Kin28 protein is a subunit of TFIIH in S. cerevisiae and is the primary kinase involved in the phosphorylation of RNA pol II CTD. Its ki nase activity can be st imulated by Mediator in vitro (Guidi et al., 2004). It was speculated that the Gal11 subun it of Mediator may regulate the phosphorylation activity of Kin28 du e to the interaction of Gal 11 with TFIIH (Sakurai and Fukasawa, 2000). The CDK8 module of Mediator in yeast contains Srb8, Srb9, Srb10, and Srb11 subunits and seems to exert a negative effect on transc ription (Song et al., 1996; Samuelsen et al., 2003). A plausible mechanism is provided by the ac tion of Srb10, which was shown to phosphorylate the CTD prior to PIC formation and, thus, preven t the entry of RNA pol II (Hengartner et al., 1998). Mediator Interacts with Coactivators Mediator has been shown to interact with other coactivators such as mammalian p300 and TFIID (Black et al., 2006; Koleske et al., 1992; Thompson et al., 1993; Johnson et al., 2002; Johnson and Carey, 2003). p300 is a coactivator that contains HAT activity, and in addition to histones, it can acetylate transc ription factors, as well as itself (Roth et al., 2001). The consequence of its interaction with Mediator is an elev ation in histone acetyl ation (Black et al., 2006), which makes chromatin more accessibl e to other factors (Roth et al., 2001). Autophosphorylation of p300 reduces its associati on with Mediator. The association of TFIID with Mediator competes with p300 binding and results in a displacem ent of p300 from the promoter. The joining of Mediator with TFIID contributes to the assembly of the PIC and activating of the promoter (Black et al., 2006).

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23 Mediator Promotes the Form ation of a Stable PIC In vitro and genetic evidence suggest that Mediator contributes to the formation of a stable PIC. It has been shown by a template commitment assay that Srb2 (Med20) is essential for the formation of the PIC (Koleske et al., 1992). In addition, mutations in Srb2 (Med20), Srb4 (Med17), or Srb5 (Med18) prevent the formation of the PIC (Ranish et al., 1999), and mutations in Sin4 (Med16) and Pgd1 (Med3) decrease both th e rate and amount of PIC formation in yeast (Reeves and Hahn, 2003). Mediator is Required in the Reinitiation Scaffold The association of Mediator with RNA pol II CTD, Gal11 with TFIIH (Sakurai and Fukasawa, 2000), and Srb2 with TFIID (Koleske et al., 1992) facilitate the formation of a stable PIC and maintain the reinitiati on scaffold (Nair et al., 2005). Reinitiation and then multiple rounds of transcription occur afte r RNA pol II, TFIIB, and TFIIF join the scaffold to re-form the PIC (Nair et al., 2005). Mutation of Pgd1 results in dissociation of Mediator from the scaffold after initiation and, thus, impairs reinitiation in ye ast (Reeves and Hahn, 2003). Mediator Stimulates both Basal and Activated Transcription The Mediator fraction from yeast has been shown to stimulate GAL4-VP16 or GCN4-dependent transcription in a reconstitute d system, and has also been shown to increase basal transcription by 8-fold (Kim et al., 1994). The ARC (activator-recruited cofactor) complex not only stimulates transcription by activat ors such as SREBP-1a/Sp1, NF-kB/Sp1 and Gal4-VP16/Sp1, but also enha nces basal transcription in vitro (Naar et al., 1999). Genome-wide expression analysis showed that only 7% of genes were expressed in the Med17 mutant of S. cerevisiae (Holstege et al., 1998). Dimi nished Mediator leads to the reduction of basal and activator-dependent transcription in yeast and HeLa cells, which can be restored by addition of purified Mediator complex in vitro (Baek et al., 2002, Nair et al., 2005).

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24 Model for Mediator Function in Activated Transcription Formation of the PIC starts with the bindi ng of transactivators to the DNA, which is followed by recruitment of TFIID, TFIIA and TFIIB to the promoter (Ranish et al., 1999, Reeves and Hahn, 2003; Woychik et al., 2002). The Mediator is recruited by trans activators and possibly by coactivators, such as p300 and TFIID (Koleske et al., 1992; Thompson et al., 1993; Johnson et al., 2002; Johnson and Carey, 2003; Black et al., 2006). Mediator and TFIID form a platform for the entry of the following factors. Mediator recruits the RNA pol II through interaction with the CTD. TFIIF may be enlisted together with RNA pol II. Then TFIIE and TFIIH enter the preinitiation complex (Thomas and Chiang, 2006) Next, the DNA helicase activity of TFIIH causes promoter melting (Jiang et al., 1996; Kim et al., 2000), an essential step before the synthesis of RNA can begin. Mediator greatly en hances the kinase activity of Kin28 of TFIIH, which hyperphosphorylates the RNAP II CTD (Gui di et al., 2004). Afte r CTD phosphorylation, RNA pol II leaves the promoter w ith TFIIF to start transcriptio nal elongation (Yan et al., 1999; Shilatifard et al., 2003). Mediator, TFIIA, TFIID, TFIIH and TFIIE stay on the promoter forming a platform that supports reinitiati on. This scaffold structure, in turn, recruits new TFIIB, TFIIF and RNA pol II repeatedly to support multiple ro unds of transcription (Yudkovsky et al., 2000). Hypothesis for a Mediator Complex in Arabidopsis Many of the basic mechanisms of transcrip tion are conserved in plants, metazoans and yeast (Reviewed in Gurley et al., 2006). The structures of many promoters in these three kingdoms contain a TATA box, CAAT box, transcription start site and cis -elements for the binding of general transcription factors and transactivators. In addition, RNA pol II and many general transcription factors ar e conserved between plants, f ungi and metazoans (Coulson and Ouzounis, 2003). Arabidopsis also has homologs of the subunits of some coactivators such as SAGA and other HAT containing complexes (Hsi eh and Fischer, 2005). This wide array of

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25 evidence for a high degree of conservation in the ba sic mechanisms of tran scription suggests that plants may also contain the Mediator coactivat or. This view is strongly reinforced by the presence of many putative Mediator subunits in Arabidopsis based on DNA sequence similarity (Gurley et al., 2006; Boube et al., 2002). A comp ilation of Mediator subunits from yeast, Drosophila and humans is presented in Table 12, along with putative subunits from Arabidopsis This provides the best estimate for Mediator s ubunit composition in plan ts and indicates that plants may have at least 20 Mediator subunits present in other eukaryotes. Despite evidence suggesting such a vital role for Mediator in gene expression, the precise subunit composition and function of Mediator has not been determined in plants. Up to now, two putative Mediator subunits in Arabidopsis thaliana have been studied. SWP (Struwwelpter) is the orthologue of Med14 and is involved in patt ern formation at the shoot apical meristem, as well as defining the duration of cell prolifera tion (Autran et al., 2002) PFT1 (phytochrome and flowering time 1) is the orthol ogue of Med25. It acts downstream of phyB to regulate the gene expression and induce flowering under low-li ght conditions (Cerda n and Chory, 2003). The important functions of these two putative Mediat or subunits hint at th e significance of the Mediator in plants. To unravel the mechanism of gene transcription in plants, it is important to identify the Mediator complex and characterize its function.

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26 Table 1-1. Interaction of the transactivators with the Mediator subunits in different organisms Transactivator Homo sapiens Saccharomyces cerevisiae Drosophila melanogaster ER and ER estrogen receptor (ER) Zhu et al., 1999; Burakov et al., 2000; Warnmark et al., 2001 GATA family of transcription factors Crawford et al., 2002 Breast cancer susceptibility gene 1 (BRCA1) Wada et al., 2004 Thyroid hormone receptor (TR TR 1) Yuan et al., 1998, Zhu et al., 1997 Androgen receptor Wang et al., 2002 Glucocorticoid receptor (GR) Hittelman et al., 1999 Peroxisome proliferator-activated receptors (PPAR and PPAR ) Zhu et al., 1997 Retinoic acid receptor (RAR ) Zhu et al., 1997 Retinoid-X-receptor for 9cis -retinoic acid (RXR ) Zhu et al., 1997 Vitamin D receptor (VDR) Rachez et al., 1999 Hepatocyte nuclear factor 4 (HNF-4) Malik et al., 2002 Farnesoid X receptor (FXR) Pineda et al., 2004 Retinoid-related orphan receptor (ROR ) Atkins et al., 1999 p53 Drane et al., 1997 Med1 Aryl hydrocarbon receptor (AHR) Wang et al., 2004 Med3 General control nondepressible factor 4 (GCN4) Park et al., 2000 SRY-box containing gene 9 (Sox9) Zhou et al., 2002 Med12 Replication and transcription activator (RTA) Gwack et al., 2003 Glucocorticoid receptor (GR) Hittelman et al., 1999 Hepatocyte nuclear factor 4 (HNF-4) Malik et al., 2002 Signal transducer and activator of transcription (STAT2) Lau et al., 2003 Med14 Sterol regulatory element-binding protein-1a (SREBP-1a) Toth et al., 2004

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27 Table 1-1. Continued. Transactivator Homo sapiens Saccharomyces cerevisiae Drosophila melanogaster Small mothers against decapentaplegic 2/3/4 (SMAD2, SMAD3, SMAD4) Kato et al., 2002 VP16 Lee et al., 1999; Park et al., 2000 General control nondepressible factor 4 (GCN4) Lee et al., 1999; Park et al., 2000 Med15 Gal4 Park et al., 2000 Med16 Differentiation-inducing factor (DIF) Kim et al., 2004 p53 Ito et al., 1999 VP16 Ito et al., 1999 Signal transducer and activator of transcription (STAT2) Lau et al., 2003 Differentiation-inducing factor (DIF) Kim et al., 2004 Med17 (Srb4) Heat-shock factor (HSF) Kim et al., 2004 Early region 1A (E1A) Boyer et al., 1999; Wang and Berk, 2002 ETS-like kinase protein-1 (Elk-1) Stevens et al., 2002 Epithelial-restricted with serine box (ESX) Asada et al., 2002 CCAAT/enhancer binding protein (C/EBP) Mo et al., 2004 Differentiation-inducing factor (DIF) Kim et al., 2004 Med23 HSF (heat-shock factor) Kim et al., 2004 Differentiation-inducing factor (DIF) Kim et al., 2004 Heat-shock factor (HSF) Kim et al., 2004 Med25 VP16 Mittler et al., 2003 Med29 Doublesex (dsxF) Garrett-Engele et al., 2002 Cdk8 Myc Eberhardy and Farnham, 2002

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28 Table 1-2. Mediator subunits in yeast, Arabidopsis Drosophila and humans (Gurley et al., 2006; Boube et al., 2002) Unified nomenclature (Bourbon et al., 2004) Saccharomyces cerevisiae Arabidopsis thaliana Drosophila melanogaster Homo sapiens MED1 Med1 Trap220 TRAP220-ARC/DRIP205 MED2 Med2 MED3 Med3 MED4 Med4 At5g02850 Trap36 TRAP36-ARC/DRIP36 MED5 Nut1 MED6 Med6 At3g21350 Med6 hMed6-ARC/DRIP33 MED7 Med7 At5g03220 Med7 ARC/DRIP34-CRSP33 MED8 Med8 Arc32 ARC32 MED9 Cse2/Med9 MED10 Nut2/Med10 At5g41910/ At1g26665 Nut2 hNut2-hMed10 MED11 Med11 Med21 HSPC296 MED12 Srb8 At4g00450 Kto TRAP230 ARC/DRIP240 MED13 Srb9 At1g55325 Skd/Pap/Bli TRAP240 ARC/DRIP250 MED14 Rgr1 At3g04740 (SWP1) Trap170 TRAP170-DRIP/CRSP150 MED15 Gal11 At1g15780 Arc105 ARC105 MED16 Sin4 Trap95 TRAP95-DRIP92 MED17 Srb4 At5g20170 Trap80 TRAP80-ARC/DRIP77 MED18 Srb5 At2g22370 P28/CG14802 p28b MED19 Rox3 CG5546 LCMR1 MED20 Srb2 At4g09070/ At2g28230 Trfp hTrfp MED21 Srb7 At4g04780 Trap19 hSrb7 MED22 Srb6 At1g07950/ At1g16430 Med24 Surf5 MED23 At1g23230 Trap150 hSur2/CRSP130 MED24 Trap100 TRAP/CRSP/DRIP100 MED25 At1g25540 (PFT1) Arc92 ARC92 MED26 At3g48060/ At3g48050 Arc70 CRSP70-ARC70 MED27 At3g09180 Trap37 TRAP37-CRSP34 MED28 Med23 Fksg20 MED29 Intersex Hintersex MED30 Trap25 TRAP25 MED31 Soh1 At5g19910 Trap18 hSoh1 CDK8 Srb10 At5g63610 CDK8 CDK8 CycC Srb11 At5g48640 CycC CycC

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29 CHAPTER 2 MATERIALS AND METHODS Plant Growth Conditions The ecotype of Arabidopsis thaliana used in this study was Co lumbia-0. The plants were grown in soil with continuous light from 40 W fluorescent bulbs at 27C. To examine the germination and root length, the seeds were gr own on vertical agar plates. The seeds were surface sterilized with 70% ethanol for 3-5 min, and then with 10% bleach for 15-20 min. After rinsing with sterile water (3 X 5 min), the seeds were plated in petri di shes containing 1/2 MS (Murashige & Skoog) medium supplemented w ith 1% sucrose, 0.5g/L MES (2-(N-morpholino) ethanesulfonic acid) and 0.8% agar. The plates were sealed with parafilm a nd placed vertically in a growth chamber with a 16h light / 8h dark cycl e provided by 40 W fluore scent bulbs at 22C. For dark treatment, the plates were wrapped in aluminum foil and placed ve rtically in a growth chamber at 22C. Genotyping of the T-DNA Insertion Lines The med31 T-DNA insertion mutants ( med31-1 and med31-2 ) were obtained from Arabidopsis Biological Resource Center (ABRC). For the genotyping of med31-1 Med31 -specific primer 5TGGATGTAAGTAGGATTGGCG -3 was paired with the T-DNA-specific primer LBb1 5-GCGTGGACCG CTTGCTGCAACT-3 to produce a 628 base pair (bp) fragment by polymerase ch ain reaction (PCR), or with another Med31 -specific primer 5GAACTTGTCTTGGCAAGTTGG -3 to produce a 975 bp fragment. For the genotyping of med31-2 Med31 -specific primer 5TGATGTACTCTGGT CGCTGC -3 was paired with the T-DNA-specific primer LBb1 5-GCGTGGA CCGCTTGCTGCAACT-3 to produce a 714 bp fragment, or with another Med31 specific primer 5-TTGCGGGGATTACAACATTAC-3 to

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30 produce a 1008 bp fragment. The T-DNA insertion sites were determined by sequencing the PCR products. RNA Analysis The leaves of 40-day-old plants grown on soil were collected and R NA was isolated with the Concert Plant RNA Reagent (Invitrogen). RNA bl ots were prepared as described by Cao et al. (1994) and probed with full-length Med31 cDNA. Microscopy A Zeiss Axiocam HRm camera was used to examine the subcellular localization of Med31-GFP fusion proteins in the root tip. GFP fl uorescence was monitored with the Zeiss filter set 10 (excitation, 450 to 490; dichro ic, 510 LP; emission, 515 to 565). DAPI (4',6-diamidino-2-phenylindole) fluorescence was monitored with Zeiss filter set 02 (excitation, 365; dichroic, 395 LP; emission, 420 LP). A Zei ss LSM 5 Pascal confocal laser scanning microscope was used to localize the Med31::GFP fusion proteins in the plant tissues with an Argon 488 nm laser and a Band Pass 505-530 filter. A Helium Neon 543 nm laser with a 560 nm filter was used to record chlorophyll autofluorescence. Plasmid Construction The pBI101sGFP(S65T) vector wa s provided by Dr. Robert Ferl (Manak et al., 2002). This vector was constructed by removing the GUS ( -glucuronidase) gene by digestion with restriction endonucleases XbaI a nd SacI, and then inserting th e sGFP(S65T) gene between the two restriction sites (Manak et al., 2002). The Med31 gene, including the 1.2 kb upstream sequence and the entire exon and intron region without the stop codon, was amplified from the genomic DNA by PCR using the primers 5 -tatTGTCGACTCTAATTA ATCAGTCTTGGTC-3 and 5agaTCTAGATATACCCTTCCTGACATTATATG ACT -3. The fragment was inserted in-frame to the 5 end of the sGFP(S65T) gene in the pBI101sGFP(S65T) vector using the SalI

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31 and XbaI sites. The Med31 1.2 kb upstream sequence was generated from the genomic DNA by PCR using the pimers 5-tatTGTC GACTCTAATTAATCAGTCTTGGTC-3 and 5-ttataTCTAGAGAACGAACG GAACCTGAAGC-3. This fragment was inserted in-frame to the 5 end of the GUS gene in the pBI101 vector using the SalI and XbaI sites. All of the PCR amplified fragments were confirmed by DNA sequencing. GUS Staining The tissues were immersed in GUS Staining Solution (1 M sodium phosphate (pH 7.0), 0.5 M EDTA (ethylenediaminetetraacetic acid), 50 mM K+ ferricyanide, 50 mM K+ ferrocyanide, 10% Triton X-100 and 2 mM X-gluc) and vacuum infiltrated for 20 min. The samples were incubated at 37 C until blue color appeared. As a final step, 70% ethanol was used to clear the tissue. Agrobacterium Transformation Technique The binary vector was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation and the T-DNA transferred to Arabidopsis plants via the standard floral dip protocol (Clough and Bent, 1998). Agrobacterium starter cultures were grown in 30 ml LB (Loria broth) liquid culture medium with 25 g/ml gentamicin, 50 g/ml rifampicin and 50 g/ml kanamycin with shaking (250rpm) at 28 C overnight. A 15 ml aliquot of the starter culture was added to 150 ml of LB liquid medium containing 25 g/ml gentamicin, 50 g/ml rifampicin and 50 g/ml kanamycin, and the culture was in cubated with shaking (250 rpm) at 28 C until an OD600 of 0.8 was reached. The cells were collected by centrifugation (5000 g, 30 min) and resuspended in 150 ml of 5% sucrose. After addition of 30 l of Silwet L-77 detergent, the 3-week-old Arabidopsis plants were dipped in the Agrobacterium solution for several sec, with gentle agitation. The plants were covered overnight to keep high humidity. Transformants

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32 were selected by germinating the seeds on plat es containing 1/2 MS medium with 50 mg/L kanamycin. Chromatin Immunoprecipitation The putative Mediator subunits were ma pped to promoter DNA using chromatin immunoprecipitation (ChIP) according to Ge ndrel and colleagues (2005), with minor modifications. The aerial parts of Arabidopsis plants were harvested (1.5-2.0 g) and rinsed with water. The sample was then placed in 37 ml of 1% formaldehyde for cross-linking and vacuum infiltrated for 15 min at room temperature. Th e reaction was quenched by the addition of 2.5 ml of 2 M glycine, and the sample was placed under vacuum for an additional 5 min. The tissue was rinsed thoroughly, frozen in liquid nitrogen a nd stored at -80 C until further treatment. Chromatin was extracted by grinding the fro zen samples in 30 ml of Extraction Buffer 1 (0.4 M sucrose, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM -mercaptoethanol, 0.1 mM PMSF (phenylmethylsulphonyl fluoride), 1 X pr otease inhibitor). (To make 200 X Protease Inhibitor, dissolve 0.16 g TPCK (tosyl phenyl alanyl chloromethyl ketone) and 0.16 g TLCK (tosyl-L-lysine chloromethyl ketone) in 5 ml of DMSO (dimethyl sulfoxide), then dissolve in 10 ml of 0.2 M PMSF in isopropanol.) Next, the sample solution was f iltered with Miracloth (CalBiochem) and then centrifuged at 3000 X g at 4 C for 20 min. The pellet was dissolved with 1 ml of Extraction Buffer 2 (0.25 M sucros e, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1% Triton X-100, 5 mM -mercaptoethanol, 0.1 mM PMSF, 1 X protease inhibito r) and centrifuged at 12,000 X g at 4 C for 10 min. After that, the pellet was resuspended with 300 l of Extraction Buffer 3 (1.7 M sucrose, 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 0.15% Triton X-100, 5 mM -mercaptoethanol, 0.1 mM PMSF, 1 X prot ease inhibitor), placed on another 300 l of extraction buffer 3, and centrifuged at 14,000 X g at 4 C for 1 hr. The pellet was resuspended with 300 l of Nuclei Lysis Buffer (50 mM Tris-H Cl (pH8.0), 10 mM EDTA, 1% SDS (sodium

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33 dodecyl sulfate), 1 X protease inhibitor), and the chromatin was sheared to a size of 150 bp to 750 bp by sonication (10 times for 15 sec each at an amplitude setting of 20 using a Tekmar Sonicator). The sample was centrifuged at 12,00 0 X g for 10 min and ChIP Dilution Buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris -HCl (pH 8.0), 167 mM NaCl) was added to the supernatant to make a final volume of 3 ml. The solution was divided into three tubes and 40 l of protein A-agarose (Santa Cr uz) was added to each tube of sa mple for pre-clearing at 4 C for 1 hr with gentle agitati on. The protein A-agarose was re moved by centrifugation (12,000 X g at 4 C for 30 sec), and the supernatan t was transferred to fresh tubes. A 60 l aliquot was saved at -20 C as the Input DNA control. The immunoprecipitation was set up as follows: 10 l of IgG (Immunoglobulin G) Sepharose (Amersham Biosciences) and 10 l of c-Myc (cellular myelocytomatosis oncogene) antibody (Santa Cruz) were added, respectively, to two tubes to precipitate the TAP-tagged Mediator subunits. No antibody was added to the third sample, which served as the no antibody control. The tubes were incubated at 4 C overn ight with gentle agitati on. In order to purify Mediator-bound complexes, 50 l of protein A-agarose beads we re added to the tubes with c-Myc antibody and no antibody cont rol, respectively, and the thr ee tubes were incubated at 4 C for 1 hr with gentle agita tion. The agarose beads were pell eted by centrifugation at 3800 X g at 4 C for 30 sec and washed sequentially with Low Salt Wash Buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris -HCl (pH 8.0)), High Salt Wash Buffer (500 mM NaCl, 0.1% SDS, 1% Trit on X-100, 2 mM EDTA, 20 mM Tris -HCl (pH 8.0)), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% DOC ( 21-hydroxyprogesterone), 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)) and TE buffer. In order to ex tract the immune complex from the beads, 250 l of elution buffer (1% SDS, 8.4 mg/ml NaHCO3) was added to each sample, followed by

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34 incubation at 65 C for 15 min with gentle agita tion. The elution step was repeated once to reach a final volume of 500 l. Elution buffer (440 l) was also added to the 60 l of the Input DNA control. After adding 20 l of 5 M NaCl to each sample, the cross-linking was reversed by incubation at 65C overnight. The pr oteins in the sample were di gested by incubation with 10 l of 0.5 M EDTA, 20 l of 1 M Tris-HCl (pH6.5), and 2 l of 10 mg/ml proteinase K at 45C for 1 hr. Then the proteins were removed from the DNA by phenol/chloroform extraction, and the DNA was precipitated with the a ddition of ethanol (2.5 volume), sodium acetate (1/10 volume, pH5.2) and 20 g of glycogen. The DNA was resuspended with 50 l of 10 mM Tris-HCl (pH7.5). PCR Analysis of Chromatin Immunoprecipitation The immunoprecipitated fraction was analyzed by PCR amplification to determine if the DNA fragments from various prom oters were present. The 25 l of PCR reaction system contained 12.5 pmol of each primer, 5 nmol of dNTP, 3 l of DNA sample, 2.5 l of 10 X PCR Buffer I and 1 unit of AmpliTaq Gold (Applied Biosystems, Foster Cit y, CA, USA). The cycling conditions were 8 min of thermal activation at 95 C, followed by 50 cycles of 94 C (30 sec), 55 C (30 sec), and 72 C (3 min). The prim ers used for PCR were as follows: 5cgtggcctagaatacaaagaag -3 and 5tcaaacaataagaaagaccatga ca -3 were used for the amplification of the CCA1 promoter; 5-agattgttgacattctcggaaatttagtgccaactgt-3 and 5-aaatgctcctttttctaaaacc ttcgcttggagtct-3 for the amplification of Hsp18.2 promoter; 5-acaccacggcgtgaccat-3 and 5-attatccagtcga catctgta-3 for the amplification of Adh1 promoter; and 5TGTTTCTTCCCTTTAAGCAACC-3 and 5AACATTTCTTTAGAACATTGAC TTGG-3 were used to amp lify the intergenic region between At2g32950 and AT2G32960

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35 Bioinformatics Multiple protein sequence alignments were pe rformed with AlignX, which is a component of Vector NTI Advance 10.3.0 from Invitrogen. The accession numbers at the NCBI (National Center for Biotechnology Information) for the Med31 homologs are XP_307924 ( Anopheles gambiae ), NP_197491 ( Arabidopsis thaliana ), BQ583133 ( Beta vulgaris ), CD834180 ( Brassica napus ), NP_492413 (Caenorhabditis elegans), EAK92332 ( Candida albicans ), DY287612 ( Citrus clementina ), CX051496 ( Citrus sinensis ), EAU91557 ( Coprinopsis cinerea ), XP_626881 ( Cryptosporidium parvum ), DR063080 ( Cycas rumphii ), XP_638330 ( Dictyostelium discoideum ), NP_649483 ( Drosophila melanogaster ), CAD25946 ( Encephalitozoon cuniculi ), DV154959 ( Euphorbia esula ), BM892402 ( Glycine max ), DT547393 ( Gossypium hirsutum ), CO126156 ( Gossypium raimondii ), NP_057144 ( Homo sapiens ), DW049205 ( Lactuca saligna ), DW126099 ( Lactuca sativa ), CF393635 ( Loblolly pine ), BQ147110 ( Medicago truncatula ), NP_080344 ( Mus musculus ), DY336178 ( Ocimum basilicum ), CA902198 ( Phaseolus coccineus ), CF808645 ( Phytophthora sojae ), DR501487 ( Picea sitchensis ), CV015282 ( Rhododendron catawbiense ), NP_011388 ( Saccharomyces cerevisiae ), NP_587859 ( Schizosaccharomyces pombe ) and CAD21541 ( Taenia solium ). The accession numbers for the Med6 homologs are XP_319180 ( Anopheles gambiae ), NP_188772 ( Arabidopsis thaliana ), NP_504791 ( Caenorhabditis elegans ), EAK97077 ( Candida albicans ), XP_638621 ( Dictyostelium discoideum ), NP_731403 ( Drosophila melanogaster ), XP_965884 ( Encephalitozoon cuniculi ), NP_005457 ( Homo sapiens), NP_081489 ( Mus musculus ), NP_001057150 ( Oryza sativa ), NP_011925 ( Saccharomyces cerevisiae ) and Q9US45 ( Schizosaccharomyces pombe ).

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36 CHAPTER 3 RESULTS Med31/Soh1 is a mediator subunit that has be en identified in humans (Gu et al., 1999), Drosophila (Park et al., 2001), S. pombe and S. cerevisiae (Linder and Gustafsson, 2004). Soh1 (suppressor of hpr1) was first id entified as a suppressor of the S. cerevisiae hpr1 mutant which is temperature-sensitive for growth and can reduce the hyperrecombination phenotype (Fan and Klein, 1994). Yeast two hybrid analysis showed Soh1 interacts with the Rad5p protein, and a Soh1 mutation exacerbated the DNA repair defect of a rad5-535 mutant (Fan et al., 1996). The Soh1 orthologue Sep10 in S. pombe was identified in screening mu tants for both sterility and for defects in cell separation. Sep10 mutants are temperature-sensitive. At a non-permissive temperature (36 C), the mutants formed multip le, ill-organized septa (Grallert et al., 1999). Med31/Soh1 was identified in the Mediator complex in humans (Gu et al., 1999) and Drosophila (Park et al., 2001), but its functi on has not been determined, and the exact relationship between mutations in Med31 and phenotype is still not clear. Analysis of Arabidopsis Med31 Gene by Multiple Sequence Alignments The Med31 homologs are pres ent in the protists ( Cryptosporidium parvum Dictyostelium discoideum Encephalitozoon cuniculi ), fungi ( Candida albicans Coprinopsis cinerea Saccharomyces cerevisiae Schizosaccharomyces pomb ), metazoans ( Anopheles gambiae Caenorhabditis elegans Drosophila melanogaster Homo sapiens Mus musculus Taenia. solium ) and plants ( Arabidopsis thaliana Oryza sativa ) (Figure 3-1). Based on sequence homology, we identified AT5G19910 in Arabidopsis as the putative Med31 gene (AtMed31), which contains 6 exons and en codes a protein of 196 amino acids (aa), with a calculated molecular mass of 22.8 kDa. There is a conserved block of 70 aa (aa R30 to R99 of AtMed31) (Figure 3-1) that shows high simila rity to Med31 in other species ( D. melanogaster 58.3%

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37 identity and 76.4% similarity; H. sapiens 61.1% identity and 72.2% similarity; S. cerevisiae 45.2% identity and 56.2% similarity; S. pombe 59.7% identity and 77.8% similarity), where its functional identity has been demonstrated. Linder and Gustaf sson (2004) showed that this region is required for its assembly within the Mediator complex in S. cerevisiae which suggests this conserved domain is important for inte raction with other Mediator subunits. A search of the NCBI EST database using TBLASTN (Altschul et al., 1997) for AtMed31 identified the Med31 homologs in many other plan t species (Figure 3-2). There is a conserved block of 139 aa (aa M1 to V139 of AT5G19910) between these plant Med31 homologs, which includes the 70 aa domain conserved between differe nt species (Figure 3-1). The C-termini of the Med31 homologs are less conserved compared with their N-termini (Figure 3-1 and Figure 3-2), and often contain regions that resemble transcriptional activation domains which have glutamine-rich or serine/proline-rich blocks. Since these domains function in transactivator proteins to make contact with ta rget transcription factors, it se ems reasonable to assume that the C-terminal region of Med31 containing these activa tor domain-like blocks may be at the outside of the complex and provides surface for interaction with transactivators or other transcriptional machinery. Phenotype Characterization of med31 Mutants The Mediator plays a vital role for RNA pol II-m ediated transcription; therefore, disruption of the highly conserved Med31 subunit is predicte d to have a disruptive effect on the expression of a large number of genes, some leading to abnormal phenotypes. To test this hypothesis and study the function of this putative subunit, we searched the SIGnAL (signal.salk.edu) T-DNA insertion collection for mutants (Alonso et al., 2003). Four T-DNA insertion lines were identified and ordered from the Arabidopsis Biological Resource Cent er (ABRC). The T-DNAs of Salk035522 and Salk051025 lines insert into the promoter and 3 UTR (untranslated region) of

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38 Med31 respectively, but we did not observe any mutant phenotype for these two lines. The med31-1 (Salk145479) mutant line has the T-DNA inse rtion in the promoter region, and the T-DNA of med31-2 (Salk 143815) mutant line is located wi thin the 5 UTR (Figure 3-3). The insertion sites for all Salk lines were confirmed by DNA sequencing. In contrast to the previo us two mutant lines, both med31-1 and med31-2 plants showed abnormalities in growth. Under our experiment al conditions, the germination rate of med31-1 seeds was 17% compared with 100% for the wild type (WT). In addition, their root length was 41.6% of the WT root length (Figure 3-4). The seeds of med31-2 plants germinated as well as the WT seeds; however, their root length was 47.7% of the WT root length (Figure 3-5). These differences in growth were not present under dark conditions, where the med31-2 seedlings grew as well as the WT seedlings (F igure 3-6). This result suggests that the function of Med31 during seed initial development may be dependent on light. Some of the med31-2 plants had aberrant patterns of co tyledon development, such as three cotyledons and three first true leaves, a single cotyledon, or fork ed cotyledons (Figure 3-7). However, these mutant phenotypes were only inherited by some of their progeny, and the med31-2 mutants with normal cotyledons also pr oduced progeny with abnormal cotyledons. The seedlings with abnormal cotyledons segregated 26% (5/19) progeny with abnormal cotyledons; whereas the seedlings with normal cotyledons se gregated 30% (5/20) progeny with abnormal cotyledons. In addition, the overall sizes of the med31-2 mutants were reduced, their leaves were smaller, and they had fewer rosette leaves compared with WT plants (Figure 3-8). Med31 Expression in the med31-2 Plants In med31-2 plants, the T-DNA inserts into the 5 UTR of the gene and may, therefore, influence the expression of Med31 gene at either the transcrip tional or translational level. Northern blotting was used to examine the expression of Med31 in the WT and med31-2 plants.

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39 In med31-2 mutant plants, the corresponding mRNA was mo re abundant than that in WT plants (Figure 3-9). The mutant phenotype of med31-2 plants may be caused by the overexpression of Med31 protein, which possibly sequesters the adj acent Mediator subunits or other components of the transcriptional apparatus. Alternatively, Me d31 translation may be inhibited due to the missing, or changed nucleotides at the 5 end of the transcript. Subcellular Localization and Tissue Expression Pattern of Med31::GFP Fusion Proteins To investigate the subcellu lar localization of Med31, the 1.2 kb upstream sequence and the entire exon and intron region of Med31 (without the stop codon) was amplified from the genomic DNA by PCR. The resulting DNA was ligated in-frame to the 5 end of the sGFP(S65T) gene in the pBI101 sGFP(S65T) vector. We obser ved that the Med31::GFP fusion proteins were expressed in the tip of primary roots and that the signal was c onfined to the nucleus (Figure 3-10). In addition to being expre ssed in the lateral root tips, pr imordia (Figure 3-11) and root hairs (Figure 3-12), the Med31::GFP signal was also found to be pres ent in the aerial portions of the plants including leaves (Fi gure 3-13), trichomes (Figure 314) and petioles (Figure 3-15). Free GFP has been shown to be present in both th e nucleus and cytosol (Li et al., 2001; Ye et al., 2002; Zhong et al., 2005). To further complicate anal ysis, GFP has been shown to move to other cells and tissues via the plasmodesmata (Crawfor d and Zambryski, 2001). However, in each type of tissue we observed, signal from the Med31::G FP fusion protein was almost exclusively found in the nucleus. The nuclear local ization exhibited by Med31::GFP is consistent with its presence in the nucleus being a property conferred by the Med31 portion of th e protein, as contrasted with the more general subcellular localiza tion previously shown for GFP alone. Tissue Expression Pattern of Med31 Promoter::GUS Fusions To investigate the tissue expres sion pattern of Med31 protein, the Med31 promoter was fused to the 5 end of the -glucuronidase (GUS) gene and transferred to Arabidopsis The GUS

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40 signal was detected in the shoot apexes (Figure 3-16A) and lateral root primordia (Figure 3-16B) of young seedlings (16 days old) of transformed plants. It was also de tected in the whole young inflorescences (Figure 3-17A), anthers (Figure 317B) and stigmas (Figure 3-17C) of adult plants (46 days old) and in developing seeds (Figur e 3-17D). This pattern differs from where Med31::GFP signal was detected in that no GUS si gnal was detected in the primary root tips, leaves and petioles. Two possibl e explanations for this appare nt inconsistency are that GUS staining sensitivity may be less than that of GFP. Alternatively, the promoter DNA alone as present in the Med31::GUS cons truct (without the e xons, introns and untranslated regions) is insufficient to fully reproduce the expression pattern of the endogenous Med31 gene. Co-immunoprecipitation Maps Med6 and Med31 to Promoter DNA Chromatin immunoprecipitation (ChIP) is a powerful tool to explore in vivo protein-DNA interactions. The ChIP assays conducted here involves the cross-linking of proteins and DNA in chromatin, followed by co-immunoprecipitation of DNA fragments associated with the epitope-tagged Mediator subunits. After the pr oteins have been removed, the pool of DNA fragments can be queried by PCR amplification for the presence of specific promoter regions. Mediator associates with pr omoter DNA indirectly by binding with transactivators and RNA pol II. Previous studies in yeast using th e ChIP technique (Andrau et al., 2006, Zhu et al., 2006) showed that Mediator could not only bind to the core promoters and upstream activating sequences, but also to the coding regions of ma ny genes, as well. It can associate with the promoters of both active and some inactive genes, but genes with higher transcriptional activity usually have higher promoter occupancy by Mediator It is thought that th e presence of Mediator at inactive promoters may be required for quick response to environmental changes. If Med31 is a genuine mediator subunit, it should be found associated with promoter DNA. We used the ChIP assay to test this hypot hesis. In addition, we checked if another Arabidopsis

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41 putative Mediator subunit, Med6 (AT3G21350), was also localized to promoter DNA. As with Med31, the assignment of Arabidopsis Med6 as a putative subunit of Mediator was based strictly on protein sequence homology (Figure 3-18). Ther e is a conserved block of 129 aa (aa M33 to S161 of AtMed6) that shows similar ity to Med6 in other species ( D. melanogaster 36.4% identity and 51.2% similarity; H. sapiens 42.6% identity and 55.0% similarity; S. pombe 31.1% identity and 48.1% similarity) where its functi onal identity has been demonstrated. It was predicted to be localized in the nucleus by two web tools (Hua and Sun, 2001; Nair and Rost, 2002). Our prediction is that both proteins we have tentativel y identified as AtMed31 and 6, respectively, should be associated with the pr omoter regions of a wide array of genes. The Med6 and Med31 cDNAs were introduced in to the pC-TAPa vectors (Rubio et al., 2005) and individually transformed into Arabidopsis by Dr. Kevin OGra dy (Gurley laboratory, University of Florida). Their C-termini were fused with nine repeats of the myc epitope, followed by six histidine residues, the 3C protease cleavage site and two copies of the protein A IgG binding domain. The fusions of Med6 or Me d31 with the epitope tags were confirmed by Western blots. Immunoglobulin G Sepharo se and c-Myc antibody were used to immunoprecipitate the tagged Med6 or Med31 proteins, respectivel y, in the ChIP experiment. ChIP Analysis for Med31 Immunoglobulin G Sepharose was used to immunoprecipitate Med31-DNA complexes from the T1 generation of Med31-pC-TAPa transgenic Arabidopsis plants. Primer pairs for the promoters of CCA1 (AT2g46830), Hsp18.2 (AT5g59720), Adh1 (AT1g77120) and a fragment in the intergenetic region (betw een AT2g32950 and AT2g32960) were used to test if Med31 binds to these sequences. The CCA1 ( circadian clock associated 1 ) gene encodes a MYB-related transcription factor and its expression oscillat es with a circadian rhythm (Wang and Tobin, 1998). Hsp18.2 ( heat shock protein 18.2 ) is a heat inducible gene. Adh ( alcohol dehydrogenase ) is also

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42 an inducible gene regulated by environmental stresses, such as low oxygen, dehydration, and low temperature (Dolferus et al., 1994). Both Hsp18.2 and Adh genes are expressed at low levels under normal conditions (Volkov et al., 2003; Dolferus et al., 1994). Mining of Arabidopsis EST database failed to find the transcripts of th e intergenetic region (>4kb) between At2g32950 and AT2G32960, suggesting this region is not transcribed. Therefore, we used this region as a negative control which Mediator may not bind. The promoters of CCA1 Hsp18.2 and Adh1 were all co-immunoprecipitated with the epitope tagged Med31 prot ein by IgG Sepharose (Figure 3-19), demonstrating the localization of Med31 to th ese promoters. As pred icted, the intergenetic region was not co-immunoprecipitated with the tagged Med31 by IgG Sepharose. ChIP Analysis for Med6 Immunoglobulin G Sepharose and c-Myc an tibody were used individually to immunoprecipitate Med6DNA complexe s from the T2 generation of Med6-pC-TAPa transgenic plants. The same set of primer pairs were used for the amplification from the DNA pool derived from co-immunoprecipitation with epit ope tagged Med6. The promoters of CCA1 Hsp18.2 and Adh1 were all co-immunoprecipita ted with Med6 by both IgG Sepharose and c-Myc antibody (Figure 3-20), demonstrating the localization of Med6 with these promoters. Again, as predicted, the intergenetic region was not co-immunoprecipitated with Med6. It should be noted that both Med6 and Med31 were independently found to be localized to the promoters of three unrelated genes, CCA1 Hsp18.2 and Adh1, a finding consistent with both proteins belonging to a Mediator complex. Wild type plants were also included in ChIP experiment as a negativ e control to check if IgG Sepharose and c-Myc anti body can immunopr ecipitate the CCA1 promoter in the absence of epitope tagged Mediator subunits. No PCR product of this promot er was amplified (Figure 3-21),

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43 validating the conclusion that ou r ChIP protocol serves as a re liable indicator that Med6 and Med31 can specifically immunopr ecipitate promoter DNA. Conclusion Two T-DNA insertion lines in either the Med31 promoter or 5 unt ranslated region were identified. The germination rate of med31-1 plants was lower, and th eir root length was much shorter than that of WT pl ants. The root length of med31-2 plants was also shorter than that of WT plants. The med31-2 mutants exhibited a dwarfed phenotype with fewer rosettes leaves than the WT plants, and some of them had aberrant pa tterns of cotyledon development, such as three cotyledons and three first true leaves, a single cotyledon, or fork ed cotyledons. These mutant phenotypes imply that Med31 plays an important role in many aspects of plant development, such as germination, root elongation and cotyledon development. Using Med31::GFP constructs, we found that the Med31 protein was localized in the nucleus. The Med31::GFP signal was detected in all the tissues that were examined, including roots, root hairs, leaves trichomes and petioles. In another experiment, the Med31 promoter was fused to GUS gene to study its tissue expression pattern. The Med31 promoter::GUS reporter was detected in the shoot apexes and lateral r oots of young seedlings (16 days old) and in the young inflorescences, anthers, stigma s of the adult plants (46 days old) and in developing seeds. The promoters of three unrelated genes ( CCA1 Hsp18.2 and Adh1 ) were all co-immunoprecipitated with Med31 by IgG Sepha rose and with Med6 by both IgG Sepharose and c-Myc antibody. These results demonstrate the localizatio n of Med6 and Med31 to these promoters, which is consistent with the function of Mediator. Taken together, this study provides eviden ce that Med6 and Med31 are both Mediator subunits because 1) Med31 was localized in the nucleus; 2) Med31 was e xpressed in every type of tissue that were examined; 3) Disruption of Med31 resulted in abnormal plant growth; and 4)

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44 Both Med6 and Med31 proteins were localized to promoters. Th ese data strongly support our hypothesis that the Mediator complex found in fungi and metazoans is also present in plants.

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45 1 120 10 20 30 40 50 60 70 80 90 100 110 (1) M A S P E E M G D D A S E I P S P P K N T Y K D P D G G R Q R F L L E L E F I Q C L A N P T Y I H Y L A Q N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q NArabidopsis_thaliana(1) M E E A E A R P A P P D P N D A R Q R F L L E L E F I Q C L A N P T Y I H Y L A Q N R Y F E D E A F I G Y L K Y L K Y W Q R P E Y I K Y I M Y P H C L F F L E L L Q NOryza_sativa(1) M S S S S P I N E N D N G N I E N N N E T N I T E N G D N G E S I D K K D D N I V L P Y E N D E E E A N Y L R F I M E L E F I Q C L S N P R Y L N Y L A Q N R Y F Q D K A F V N Y L V Y L Q Y W K K P E Y A K F I V Y P Q S L Y F L D L L Q EDictyostelium_discoideum(1) M L S E G E S E L L I E D E N P I A R F S L E L E F V Q C L S N P D Y L Q W L S K E G Y F E D E S F V N Y L K Y L L Y W C E F P Y V K Y I S Y P H C I K M L R L L Q ICryptosporidium_parvum(1) L R F Q V E L E F V Q C L A N P N Y L H F L A Q R G Y F K D A A F V N Y L K Y L L Y W K E P E Y A K Y L K F P M C L Y F L D L L Q YAnopheles_gambiae(1) M A K M Y G K G K T A I E S E E L Q K R R W Q I E L E F V Q C L S N P N Y L N F L A Q R G F F K D Q S F I N Y L K Y L Q Y W K E P D Y A K Y L M Y P M C L Y F L D L L Q YDrosophila_melanogaster(1) M A A A V A M E T D D A G N R L R F Q L E L E F V Q C L A N P N Y L N F L A Q R G Y F K D K A F V N Y L K Y L L Y W K D P E Y A K Y L K Y P Q C L H M L E L L Q YHomo_sapiens(1) M A A A V A M E T D D A G N R L R F Q L E L E F V Q C L A N P N Y L N F L A Q R G Y F K D K A F V N Y L K Y L L Y W K E P E Y A K Y L K Y P Q C L H M L E L L Q YMus_musculus(1) M E S V E S E K T R F E V E C E F V Q A L A N P N Y L N F L A Q R G Y F K E E Y F V N Y L K Y L L Y W K D P Q Y A R C L K F P Q C L H M L E A L Q SCaenorhabditis_elegans(1) M Q N R P K S V L T P A R L G T S G V V R N T L E D P W V R F Q I E L E F V Q S L G N P D Y L T F L A Q Q G C F D K P E F I N Y L S Y L Q Y W K S P S Y S R F I T Y P F C L H M L D L L Q STaenia_solium(1) M S A Q T D Q P I T E Q Q K K E Q E Q Y T N L I N S L P T R W E I E L E F V Q S L S N I P Y V N Y L A Q N N Y F N D E N F I N Y L N Y L Q Y W T Q P E Y S K F L V Y P N C L H I L K L L Q DCandida_albicans(1) M S S T N G N A P A T P S S D Q N P L P T R F E V E L E F I Q S L A N I Q Y V T Y L L T Q Q Q I W K S P N F K N Y L K Y L E Y W C N P P Y S Q C I V Y P N C L F I L K L L N GSaccharomyces_cerevisiae(1) M S G S R F E R E L E F V Q L L C N P D Y L R W L T R E G H F E S E E F R S Y L R Y L E Y W R S P E Y S R F L T Y P Q C L A V L E H L N SEncephalitozoon_cuniculi(1) M E T K W L L S K V P D D K S R F E I E L E F V Q M L S N P W Y L N F L A Q H K Y F E D E A F L Q Y L E Y M E Y W R E P E Y V K F I I Y P T C L H M L T L L K NSchizosaccharomyces_pombe(1) M S A H P G Q T P G V S A P T D P K S A N R A R F E L E L E F V Q A L A N P Y Y L H S L A Q Q N I L E K P A F V N Y L K Y L L Y W K D K D Y A R F I H Y P H A L H H L E L L Q NCoprinopsis_cinerea(1) R R F L E L E F V Q C L A N P Y L N F L A Q G Y F D A F V N Y L K Y L Y W K E P E Y A K F I Y P C L H M L E L L Q Consensus(1) 121 240 130 140 150 160 170 180 190 200 210 220 230 (121) P N F R T A M A H P A N K E L A H R Q Q F Y Y W K N Y R N N R L K H I L P R P L P E P V P P Q P P V A P S T S L P P A P S A T A A L S P A L S P M Q Y N N M L S K N D T R N M G A T G I D R R K R K K G I -Arabidopsis_thaliana(96) A N F R N A M A H P A S K E V A H R Q Q Y F F W K N Y R N N R L K H I L P R P P P E P T P T P A P A P A A V P P S A S V P S T V V P P V A A P P S A L L P M S A A G A S A M S P M Q F A G T P G T N I P K N D M R N V M G G Q G G R K R K I GOryza_sativa(84) E R F R Q E L N H S Q S T D F I H E Q Q F Y H W Q Y Y R N N R M S I K E Q E L Q Q Q Q Q Q Q Q Q Q Q V Q P P T T V -Dictyostelium_discoideum(120) E D F R K N L S K E E V I Q I I R E Q Q T Y Q W I Y S D I K K E H L K L -Cryptosporidium_parvum(84) E H F R R E I V S A Q C C K F I D D Q A I L L W Q H Y T R R R T R L T A L G T T S L T G L A V G G Q P V G -Anopheles_gambiae(67) E H F R R E I V N S Q C C K F I D D Q A I L Q W Q H Y T R K R I K L I E N V T A A Q Q Q Q Q Q L Q Q Q Q Q Q A N G M E A A T G G E S A A P T P N V N G S A S T A D S Q Q T S S A L Q P V Q A Q P G N P Q Q Q Q Q I N G V A S G A N I K L E L NDrosophila_melanogaster(86) E H F R K E L V N A Q C A K F I D E Q Q I L H W Q H Y S R K R M R L Q Q A L A E Q Q Q Q N N T S G K -Homo_sapiens(82) E H F R K E L V N A Q C A K F I D E Q Q I L H W Q H Y S R K R V R L Q Q A L A E Q Q Q Q N N T A G K -Mus_musculus(82) Q Q F R D S M A Y G P S A K F V E D Q V V L Q W Q F Y L R K R H R L C M M P D E G Q E L E E S E D E A D I R Q K D T E D E D D E E T M K K P D A D T A E K N S T T S T V S K K E K -Caenorhabditis_elegans(75) P D F R R E V A H E S V T R F I D D Q M L L H W K N Y L R K R A E M V N K H V Q S L D A M A T P G P G P S S -Taenia_solium(95) E N F R K N I I N Q D F M N S L M N D M V K R W Q S N A N D Q D E N K E K E E N K E V P E V R I N G T N -Candida_albicans(95) F M E S A I V N E D G L L E G L D E L P K I I Q L Q G P Q W M N E M V E R W A N -Saccharomyces_cerevisiae(88) E N I N D M L S D E N F F L A L G E Q Q Y F I W L N K H K E G W N K -Encephalitozoon_cuniculi(70) P Q F R N D I S R A D L S K Q V N D E I Y Y E W L G K G L Q Q Y G S A D D A T L S Q P Q Q E E D E K K V D V K K E N E -Schizosaccharomyces_pombe(81) A Q F R A A L K K D E F L R D Y L Q Q K Q F D H W R T W R D P K H L N P S T S N T S N E A P A D E T A A D K Q Q Q A I -Coprinopsis_cinerea(89) E F R L K I E Q Q W Y R L Consensus(121) Figure 3-1. Multiple sequence alignments of Med31 homologs in different species. Identical amino acids are indicated in yellow, cons ervative amino acids in light blue and similar amino acids in green.

PAGE 46

46 1 120 10 20 30 40 50 60 70 80 90 100 110 (1) M A S P E E M G D D A S E I P S P P K N T Y K D P D G G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QArabidopsis_thalina(1) M I F R L R L F S P G D Y S Q P S P W A C L P S S V S V S S M A S P E E M V D A S E T P S T P K S T Y K D P D V G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QBrassica_napus(1) N S R V C R F C S C S E S T N S M A S K I E S E N S T D T S P S S P K N I Y K D P D D G Q Q R F L L E L E F V Q C L A N P T Y I H Y L A QGlycine_max(1) V P S S N P A N Y I S L N R C V F V L V V E D S V S M A S K T E S G S P R D T P P S P P K S I Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QMedicago_truncatula(1) M A S K I E S E N S T D T S P S S P K N I Y K D P D D G Q Q R F L L E L E F V Q C L A N P T Y I H Y L A QPhytophthora_sojae(1) I D L G L I F I Q R R E Q G R I A V L T P P D F V F H T L C W F C S E S T D S M A S K N E S D N S T D T S P S S P K N I Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QPhaseolus_coccineus(1) Q T A I S V N L P I D I I F N F S I C K K N K M A A S K D N E E A S D A P S S P K K V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QCitrus_clementina(1) F S I C K K N K M A A A K D G E E A S D A P S S P K K V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QCitrus_sinensis(1) I R H E G F F P V L G F I T S M A S N Q E T D A S A N T P S S P K N V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QRhododendron_catawbiense(1) R X X X N A R A G F H H P X X S S K E S D S A P D T P S S P K S L Y K D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QEuphorbia_esula(1) R I S F K P F C T V T I E F L R F N C F V I S R V G V L K S L R D S L N F T V W H S T D Q P L K L N H R L F S V F H I V K V V V S M A S T K E S D N A S D T P S S P K N V Y K D P D D G R Q R F L L E L E F L Q C L A N P T Y I H Y L A QGossypium_raimondii(1) V D F L S I L S N F L G L N F M I S T V N R F Q E L C K F A A R Q V K V D S S S E F I C L M A S S N D A D D T S N S P S L T Q N V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QBeta_vulgaris(1) G G A D R W W I L R Q R L L P R T R R E S N L P S E I C N S M A S S H E D D D S S N T H S S P K K V Y Q D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QLactuca_saligna(1) G N N D A D R W I L R R L L P R T V T G V Y S L L K Q R R E S N L P S E V A N S M A S S H E D D D S S N T H S S P K K V Y Q D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QLactuca_sativa(1) H P F R C R I G N E D S M E I P S P P P S P P K T V Y K D P D D G R Q R F F L E L E F V Q C L A K P T Y I H Y L A QOcimum_basilicum(1) G T R K G G M D L L L A P S I P K E P Y N D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QCycas_rumphii(1) G K G S S D S S P L P S I P K E P Y K D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QPicea_sitchensis(1) C L S R T F N Q I R R R D L D L V E D D W C N K S G R R T V Q S R C I G A R N R P E Y F V R P K D I E V T R H F Q A K N M E P G K G N S D S S P H P S I P K E P Y K D P D D G R Q R F L L E L E F I Q C L A N P T Y I H Y L A QPinus_taeda(1) M A S E D D A S T P S S P K V Y K D P D D G R Q R F L L E L E F V Q C L A N P T Y I H Y L A QConsensus(1) 121 240 130 140 150 160 170 180 190 200 210 220 230 (121) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N P N F R T A M A H P A N K E L A H R Q Q F Y Y W K N Y R N N R L K H I L P R P L P E P V P P Q P P V A P S T S L P P A P S A T A A L S PArabidopsis_thalina(54) N R Y F E D E A F I E Y L K Y L Q Y G Q R P E Y I K F I M Y P H C L Y F L E L L Q N P N F R S A M A H P A N K E L A H R Q Q F Y Y W K N Y R N N R L K H I L P R P L P E P V A P Q P P P V P S S S L P P A P P A T A A PBrassica_napus(83) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L A H R Q Q F Y F W K N Y R N N R L K H I L Q R -Glycine_max(70) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L T H R Q Q F Y F W K N Y R N N R L K H I L P R S L A E P S A A L P A P A S T Q P Q P P V P A L P P V P A T S V A V T T S S S QMedicago_truncatula(80) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R S L P E L S A T P A A P A S T S S Q A P V S A L P P V P A T S V A V T A T P S QPhytophthora_sojae(54) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P T N K E L A H R Q Q F Y F W K N Y R N N R L K H I X P R S L P E P S A T S A V P A P V S T T -Phaseolus_coccineus(93) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E P S E A P P P A A A P P L P P A P P V L T P V T A A P G PCitrus_clementina(76) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E P A E A P P P A A A P P L P P A P P V P T P V T A A S G PCitrus_sinensis(61) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N S N F R N A M A H P G N K E L A H R Q Q F Y Y W K N Y R N N R M K H I L P K P P P E P V A A P P A S V P P P P P I P P S T I P V S A V P P P Q P A P S PRhododendron_catawbiense(68) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E P A P A A P V S A P P P P V Q P M P P V P P T T I G G P A G S A SEuphorbia_esula(66) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E V A H R Q Q F F F W K N Y R N N R L K F I L P K P P P E E V P T P A P L P P A S A P P Q Q S L P A S N I A M T T A P P A P A SGossypium_raimondii(118) N R Y F D D E A F I G Y L K Y L Q Y W Q Q P E Y I K F I M Y P H C L F F L E L L Q N A N F R N A M A H P G S K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E P D P -Beta_vulgaris(98) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K Y I M Y P H C L Y F L E L L Q N A S F R N A M A H P A N K E L T H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E T T A P P P S N A V P P P P T T T I A A A S S G G P V A V P PLactuca_saligna(83) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K Y I M Y P H C L Y F L E L L Q N A S F R N A M A H P A N K E L T H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E T T A P P P S N A V P P P P T T T L L L L L L V V L W L C R Q Y S R L CLactuca_sativa(93) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y L K F I M Y P H C L F F L E L L Q N P N F R N A M A H P A N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P K P L P E S S T T A T S A S V A P L A L P P T T V P A A V S N I P P A P P P QOcimum_basilicum(59) N R Y F D D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L F F L E L L Q N A N F R T A M A H P A N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E A A P P P P L L A -Cycas_rumphii(52) N R Y F D D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L F F L E L L Q N A N F R S A M A H P T N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E A A P A P P P A G A A T A P A P A A A A L P V P P T A V A V S S S Q K TPicea_sitchensis(50) N R Y F D D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L F F L E L L Q N A N F R S A M A H P A N K E L A H R Q Q F F F W K N Y R N N R L K H I L P R P L P E A A P A P S P A V A A T A P A P A A A A L P A P Q T A V A V S S A Q K TPinus_taeda(113) N R Y F E D E A F I G Y L K Y L Q Y W Q R P E Y I K F I M Y P H C L Y F L E L L Q N A N F R N A M A H P A N K E L A H R Q Q F Y F W K N Y R N N R L K H I L P R P L P E P A P P A P P A Consensus(121) 241 360 250 260 270 280 290 300 310 320 330 340 350 (241) A L S P M Q Y N N M L S K N D T R N M G A T G I D R R K R K K G I -Arabidopsis_thalina(164) S P S P M Q Y N N M L A K N E T R N M V S A G I D R R K R K K G P A Y L A L K Q T P W D L A Y A S C V -Brassica_napus(191) -Glycine_max(150) A P S P M P Y G I P P G S G I A K N D M X N T S A D -Medicago_truncatula(200) A P S P M P Y G M P P G S G L A K N D M R N P T V D N R R K R K L Y N T T C K L I I K A M Q W W L D Q A I S L F F L -Phytophthora_sojae(174) -Phaseolus_coccineus(191) A L S P M Q Y G I P P G S A L M K N D M R S S S I D R R K R K K D G I G I T F L C V K L M R N A Y Y K D Y K G Y I R A Y E W R S F F R P I I Y L S R G F H F G R W E A R N H H I L T P P N S Y F S Q G Q R I V N F W S A L P C W Citrus_clementina(189) A L S P M Q Y G I P P G S A L M K N D M R S S S I D R R K R K K D G I G I T F L C L K L M R S F L F Q S V Y V I R G N S I V I Y S I C I D F E I V L A A S R S N M S F L F G Y F V K L I SCitrus_sinensis(174) A L S P M Q Y A I P H G S A L P K N D P R T S G G D R -Rhododendron_catawbiense(186) A L S P M P Y G M P A G S T L A K N D M R N T G M D R R K R K K E G P N I R S S E S N W P N I S -Euphorbia_esula(183) T H S P M P Y G L P S G S A L A K N D M R N S G I D R R K R K H E R S L N P T I Y -Gossypium_raimondii(237) -Beta_vulgaris(185) V L S P M Q Y G V P S G P P L K S D P R S G I D R R K R K K D G F D L R Y S C N T S W L X E H K T R G L V F P F V V L S K F Y I L L L A M R A -Lactuca_saligna(198) S M V Y L L V H L K V T L G V G L I E E R E S K I F L S E R W I V F E V F V Q Y F M V E R T N S R F S V S F C S V V V L Y F T I G N E S L I F C L C V F F Y I K Y T F V F V Y I T L K S W I Y R E I M Y P L M M -Lactuca_sativa(213) V P S P M Q Y G I G S G S T F V K N D P R N S G V E K R K R K S L L T S A V N F D I L K F K F L V N F Y N -Ocimum_basilicum( 177) -Cycas_rumphii(145) E N T R G S T G E R R K R K Y N N L L K P Y F L D V V I L F I V S E N Y L C F R F C L L S S G S G V F A T E I T Q I C S V P L S G G G -Picea_sitchensis(170) E N T R G S A V E R R K R K Y I I D Y R R T F -Pinus_taeda(233) A S P M Y G I G L K N D R I D R R K R K Consensus(241) 361 4 62 370 380 390 400 410 420 430 440 450 (361) -Arabidopsis_thalina(197) -Brassica_napus(242) -Glycine_max(150) -Medicago_truncatula(226) -Phytophthora_sojae(232) -Phaseolus_coccineus(191) A S V V F I F V C L S R S T Y I G M C A L S P S C I D L L K I L F S N L S P T G Q I Q N R N D L N Q S N R G S F G G K R Y P G G K R E R F E F Q I L A L N S L A F S N L G K P P L F S F P P C P K I K L K Citrus_clementina(301) L F N L A L F F L K K K K K -Citrus_sinensis(267) -Rhododendron_catawbiense(213) -Euphorbia_esula(231) -Gossypium_raimondii(278) -Beta_vulgaris(185) -Lactuca_saligna(269) -Lactuca_sativa(317) -Ocimum_basilicum(230) -Cycas_rumphii(145) -Picea_sitchensis(237) -Pinus_taeda(256) Consensus(361) Figure 3-2. Multiple alignments of AtMed31 with the deduced amino acid sequences of its homologs in other plant species. Identical amino acids are indicated in yellow, conservative amino acids in light blue and similar amino acids in green.

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47 Figure 3-3. Diagrammatic representation of the insertions of the T-DNA in med31-1 and med31-2 The Med31 gene contains six exons, whic h are represented by red boxes. UTR regions are indicated by blue boxes. The position of triangle represents the T-DNA insertion site. Figure 3-4. Germination rate a nd root length of WT and med31-1 seedlings (9-day-old). A) WT. B) med31-1 The size bars represent 0.5 cm. Figure 3-5. Nine-day-old WT and med31-2 seedlings grown under cont inuous light. A) WT. B) med31-2 The size bars represent 0.5 cm.

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48 Figure 3-6. Nine-day-old WT and med31-2 seedlings grown under dark. A) WT. B) med31-2 The size bars represent 0.5 cm. Figure 3-7. Ten-da y-old WT and med31-2 seedlings. A) WT. B) med31-2 with three cotyledons and three first true leaves. C) med31-2 with forked cotyledon. D) med31-2 with a single cotyledon. Figure 3-8. Comparison of adult WT plants and med31-2 plants. A) 30-day-old WT and med31-2 plants. B) 55-day-old WT and med31-2 plants. In both panels, the left plant is med31-2 and the right plant is WT.

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49 Figure 3-9. Northern blot analysis of Med31 expression in WT and med31-2 plants. Figure 3-10. Subcellula r localization of Med31::GFP fusion pr oteins in the root tip of a 35-day-old plant. A) Image of G FP. B) Image of DAPI staining.

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50 Figure 3-11. Expression of Med31::G FP fusion proteins in lateral ro ots. A) A late ral root. B) A lateral root primordium. Figure 3-12. Expression of Med31::G FP fusion proteins in a root ha ir. A) Image of GFP signal. B) DIC image. C) Overlay of the DIC and GFP images.

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51 Figure 3-13. Expression of Med31::G FP fusion proteins in a leaf. A) Image of GFP signal. B) DIC image. C) Image of autofluorescence. The chloroplasts are red because of autofluorescence of chlorophyll. D) Overla y of the GFP and autofluorescence images.

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52 Figure 3-14. Expression of Med31::G FP fusion proteins in a trichom e. A) Image of GFP signal. B) DIC image. C) Image of autofluorescen ce. The chloroplasts are red because of autofluorescence of chlorophyll. D) Overla y of the GFP and autofluorescence images.

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53 Figure 3-15. Expression of Med31::G FP fusion proteins in a petiole. A) Image of GFP signal. B) DIC image. C) Image of autofluorescence. The chloroplasts are red because of autofluorescence of chlorophyll. D) Overla y of the GFP and autofluorescence images.

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54 Figure 3-16. Med31 promoter directed GUS tissue e xpression pattern in young plants (16-day-old). GUS signal was detected in A) A shoot apex. B) Lateral root primordia and tips. Figure 3-17. Med31 promoter directed GUS tissue expression pattern in adult pl ants (46-day-old). GUS signal was detected in A) A young inflor escence. B) Anthers. C) A stigma. D) Developing seeds.

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55 1 100 10 20 30 40 50 60 70 80 90 (1) M D S S L L S A A T A D T F N G N A A D Q I P P P L Q P P G T D M T G I S F R D Q L W I N S Y P L D R N Y I F D Y F A L S P F Y D TArabidopsis_thalina(1) M S G T P L P P P A L P P P P G T D M T G I C F R D Q L W L N T Y P L D R N L V F D Y F A L S P F Y D LOryza_sativa(1) M E D F N N D D P M N F D K D D M I N N N N D N N N N N N D N D N N N E N N E D S N N N S N L E N M I S D T S T P R I L E E E P D L T Q V M W R D P L W L Q M Y P L N P Q T I L Q Y F S Y S Q F Y D K Dictyostelium_discoideum(1) L A P L L Q E N P L W I S W H D S N W I P V L N P G N V M D Y F S E K S N P F Y D R Anopheles_gambiae(1) M A S R Q M T N D H L R L S W H D T Q M M A T L S P Q T V M D Y F C R K S N P F Y D HDrosophila_melanogaster(1) M A A V D I R D N L L G I S W V D S S W I P I L N S G S V L D Y F S E R S N P F Y D R Homo_sapiens(1) -Mus_musculus(1) M M P R M G P P A A A R Q D N P L H V S F R N P Q W P P N F I N K D N V L D Y F C N Q A N A F Y E M Caenorhabditis_elegans(1) M E S L D E I Q W K S P E F I Q E R G L N T N N V L E Y F S L S P F Y D R Candida_albicans(1) M N V T P L D E L Q W K S P E W I Q V F G L R T E N V L D Y F A E S P F F D K Saccharomyces_cerevisiae(1) M A S G A P P S V D L T S I Q W R M P E W V Q S M G G L R T E N V L E Y F S Q S P F Y S HSchizosaccharomyces_pombe(1) M E E R E E S I S F V D Q R F L G S K P L D D T N V L E Y F S G S P F Y D K Encephalitozoon_cuniculi(1) L I S W R D W I Q L V Y D Y F S S P F Y D K Consensus(1) 101 200 110 120 130 140 150 160 170 180 190 (101) T C N N E I L R R R S I H P L D L S H L S K M T G L E Y M L T D A T E P N L F V F R K Q K R D G P E -Arabidopsis_thalina(67) T C N N E S L R S R Q I H P L D M S H L T K M T G M E Y V L S D V M E P H L F V I R K Q R R E S P E -Oryza_sativa(53) N C N N E Q L K M Q R L D L S A L K N M D G L E Y E L I K F V E P S F F L I A K Q T R I S P T -Dictyostelium_discoideum(100) T C N N E I V R M Q R Q S L E L L N N M T G V E Y I P L H V Q D P I L Y V I R K Q H R H S P T -Anopheles_gambiae(43) M C N N E T V R M Q R L G P E H L H N M I G L E Y I L L H V A E P I L Y V I R K Q H R H N P S -Drosophila_melanogaster(44) T C N N E V V K M Q R L T L E H L N Q M V G I E Y I L L H A Q E P I L F I I R K Q Q R Q S P A -Homo_sapiens(44) M Q R L T L E H L N Q M V G I E Y I L L H A Q E P I L F I I R K Q Q R Q S P A -Mus_musculus(1) N S C N Q Q I R M Q N I V N R T V E E C L R T M P G I Q Y V L W Y S Q P P L F I I C K Q R R N N V T -Caenorhabditis_elegans(51) T S N N Q V L M M Q F Q Y Q Q I Q I P P G V S F H Q Y F Q S R L S E M T G I E F V I A Y T K E P D F W I I R K Q K R Q D P Q N Candida_albicans(38) T S N N Q V I K M Q R Q F S Q L N D P N A A V N M T Q N I M T L P D G K N G N L E E E F A Y V D P A R R Q I L F K Y P M Y M Q L E E E L M K L D G T E Y V L S S V R E P D F W V I R K Q R R T N N S G Saccharomyces_cerevisiae(40) K S N N E M L K M Q S Q F N A L D L G D L N S Q L K R L T G I Q F V I I H E R P P F L W V I Q K Q N R L N E N -Schizosaccharomyces_pombe(46) S C N N E I L K M Q T Q F R G L D Q K S K L F S M V G I F Y E V E S S N H E K T L F V I R K A Y N H G D T -Encephalitozoon_cuniculi(39) T C N N E I L K M Q R L L S L M G I E Y V L L H E P L F V I R K Q R P T Consensus(101) 201 300 210 220 230 240 250 260 270 280 290 (201) K V T P M L T Y Y I L D G S I Y Q A P Q L C S V F A A R V S R T I Y N I S K A F T D A A S K L E T I R Q V D T E N Q N E P A E S K P A S E T V D L K E M K R V Arabidopsis_thalina(117) K S N A M L A Y Y I L D G S I Y Q A P Q L C S V F A S R I S R A M H H I S K A F T T A C S K L E K I G H V E T E P D T A A S E S K T Q K E A I D L K E L K R V Oryza_sativa(103) D V L I N T L Y Y V I N G N I Y Q A P E L H V V F K S R V S Q S I S H L S E A F N S I S S I V N W D I V N G Y S L N L D P S N Q Q E K S K L A A Y S R K Q I E D T K R LDictyostelium_discoideum(147) E A T P M A D Y Y I I A G T V Y Q A P D L A S V F N S R I L S T V H H L Q T A F D E A S S Y S R Y H P S K G Y S W D F S S N K A I A E K T K T Q T K K E A P V K E E P S S I F Q R Q R V Anopheles_gambiae(90) E A T P I A D Y Y I I G G T V Y K A P D L A N V I N S R I L N T V V N L Q S A F E E A S S Y A R Y H P N K G Y T W D F S S N K V F S D R S K S D K K D A N S A K D E N S G T L F Q K Q R V Drosophila_melanogaster(91) Q V I P L A D Y Y I I A G V I Y Q A P D L G S V I N S R V L T A V H G I Q S A F D E A M S Y C R Y H P S K G Y W W H F K D H E E Q D K V R P K A K R K E E P S S I F Q R Q R V Homo_sapiens(91) Q V I P L A D Y Y I I A G V I Y Q A P D L G S V I N S R V L T A V H G I Q S A F D E A M S Y C R Y H P S K G Y W W H F K D H E E Q E K V K P K A K R K E E P S S I F Q R Q R V Mus_musculus(40) N V S P I A Y Y Y V I N G S V H Q A P D M Y S L V Q S R L L G A L E P L R N A F G E V T N Y S R Y N T A K G Y Y W E F K N K P N V K K R E E E K K E D E E E K L E D R S T N F Q K T R TCaenorhabditis_elegans(101) T V T L Q D Y Y I I G A N V Y Q A P R I Y D V L S S R L L A S V L S I K N S T D L L N D M T S Y H I S D G G H S Y I N S I H G S S S K P S Q S S A V S K P S S T N T G T N A T T T PCandida_albicans(101) V G S A K G P E I I P L Q D Y Y I I G A N I Y Q S P T I F K I V Q S R L M S T S Y H L N S T L E S L Y D L I E F Q P S Q G V H Y K V P T D T S T T A T A A T N G N N A G G G S N K S S V R P T G G A Saccharomyces_cerevisiae(139) E V K P L T V Y F V C N E N I Y M A P N A Y T L L A T R M L N A T Y C F Q K A L T K I E K F P Q Y N P Q E G Y T Y P K L S N D N L E V D H S N T N E P A D E N K Schizosaccharomyces_pombe(101) A E T L G M Y Y I I H G H V Y A A P T N Y S I Y R C R M G D S M W Q L N S F I D R M M E K R R F N P F S P P K G R R L A K S L E D S K DEncephalitozoon_cuniculi(92) V P L A D Y Y I I A G I Y Q A P D L S V I N S R V L A V H L Q S A F D E A S Y R Y P S G Y W K S K S I R V Consensus(201) 301 3 77 310 320 330 340 350 360 (301) D V I L T S L Y R K L A P P P P P P P F P E G Y V S Q E A L G E K E E L G T Q G G E S Q P P Q V D P I I D Q G P A K R M K F Arabidopsis_thalina(196) D H I L M S L Q R K L Q P A P P P P P F P E G Y V P S E Q E K A S D D L L A S E A L P P Q V D P I I D Q G P A K R P R F Q Oryza_sativa(182) D Q L I N S L F I K F P A I N R P V E N P Q Q M G G G I T P Q Q Q P S Q P Dictyostelium_discoideum(231) D M L L G D L L R K F P L P L P Q M T N N P T G G N P S D T A N A S N N H G G A A G D S D H V G A D A T L I K Q E P T E G G V Anopheles_gambiae(182) D M L L A E L L R K F P P P I P P M L Q N L Q Q P P P A G D D L N T A R N A S E M N N A T G P L D I K T E G V D M K P P P E K K S K Drosophila_melanogaster(184) D A L L L D L R Q K F P P K F V Q L K P G E K P V P V D Q T K K E A E P I P E T V K P E E K E T T K N V Q Q T V S A K G P P E K R M R L Q Homo_sapiens(178) D A L L I D L R Q K F P P R F V Q Q K S G E K P V P V D Q A K K E A E P L P E T V K S E E K E S T K N I Q Q T V S T K G P P E K R M R L Q Mus_musculus(127) M M L L N Q L F S E M P A E D A L E R E E K E E V E E E E E E T L K T E E P T T S T D E P K F A E P T A R T T S K Q Caenorhabditis_elegans(193) I T L T T P S G A T V P S T V S N G I S T S T E I A S G V F D T L L N D V V M N D D H L Y I D E I P L Y G E G S T L E R L G L K G N K D A G L S L Candida_albicans(191) N M A T V P S T T N V N M T V N T M G T G G Q T I D N G T G R T G N G N M G I T T E M L D K L M V T S I R S T P N Y I Saccharomyces_cerevisiae(237) N Q S I E N A D Y S F S P E D F S V V R A F M Q S L H S S K E A P D V K Schizosaccharomyces_pombe(181) L D F M M E I F N D F K K E Q A E S Encephalitozoon_cuniculi(160) D L L L K F P P M V K P K R Consensus(301) Figure 3-18. Multiple sequence alignments of Me d6 homologs in different species. Identical amino acids are indicated in yellow, conser vative amino acids are indicated in light blue, and similar amino acids are indicated in green.

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56 Figure 3-19. Med31 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region. The promoters used ar e indicated above the gels. Lane M was loaded with 100 bp DNA Ladder from New En gland Biolabs. The templates for each PCR are as follows: Lane 1: Genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic DNA from Med31-pC-TAPa transgenic plants); Lane 3: Negative control (chromatin ex tract without antibody immunoprecipitation from Med31-pC-TAPa transgenic plants); Lane 4: Chromatin immunoprecipitated with IgG Sepharose from Med31-pC-TAPa transgenic plants. Figure 3-20. Med6 associates with the promoters of CCA1 Hsp18.2 and Adh1 but not with the intergenetic region. The promoters used ar e indicated above the gels. Lane M was loaded with 100 bp DNA Ladder from New En gland Biolabs. The templates for each PCR are as follows: Lane 1: genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic DNA from Med6-pC-TAPa transgenic plants); Lane 3: Negative control (chromatin extrac t without antibody immunoprecipitation from Med6-pC-TAPa transgenic plants); Lane 4: Ch romatin immunoprecipitated with IgG Sepharose from Med6-pC-TAPa transgenic plants. Lane 5: Chromatin immunoprecipitated with c-Myc antibody from Med6-pC-TAPa transgenic plants.

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57 Figure 3-21. Immunoglobulin G Sepharose and c-Myc antibody cannot immunoprecipitate the CCA1 promoter from WT Arabidopsis The templates for each PCR are as follows. Lane M was loaded with 100 bp DNA Ladder from New England Biolabs. Lane 1: genomic DNA from wild-type plants; Lane 2: Input DNA control (sonicated genomic DNA from wild-type plants); Lane 3: Negative control (c hromatin extract without antibody immunoprecipitation from wild-t ype plants); Lane 4: Chromatin immunoprecipitated with IgG Sepharose from wild-type plants. Lane 5: Chromatin immunoprecipitated with c-Myc an tibody from wild-type plants.

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58 CHAPTER 4 DISCUSSION Phenotype Characterization of med31 Mutants The Med31 promoter or 5 UTR were disrupted by T-DNA insertion in med31-1 and med31-2 lines. Both mutant lines had shorter root s than WT plants under our experimental conditions. In addition, seeds were examined in pr eliminary studies (data not included) for their responses to a variety of hormones. The med31-2 seedlings were insensitive to ABA, kinetin, and 2, 4-D, compared with WT seedlings. A pos sible cause for the mutant phenotypes of these two insertion lines is due to the disruption of eith er transcriptional or tr anslational expression of Med31 The T-DNA in med31-1 breaks a GT-1 cis -element (identified by AthaMap web tools; www.athamap.de), which has been shown in other prom oters to play a role in the gene regulation by light, pathogens and salt (Vil lain et al., 1994; Park et al ., 2004). Likewise, the T-DNA in med31-2 breaks the CCAAT BOX1 (identified in the PLACE database; www.dna.affrc.go.jp/PLACE), which has been repor ted to be involved in transcriptional expression by heat stress (Rieping and Scho ffl, 1992; Haralampidis et al., 2002). T-DNA insertions not only disrupt the inserted cis -elements, but also impede the function of the cis -elements upstream of the insertion sites. Th e location of the two T-DNA insertions found in med311 and -2 are predicted to strongly interfere with th e regulation of Med31 gene expression. Med31 is a subunit of Mediator complex, which is important in gene transcription mediated by RNA pol II. Defective Med31 expression has the potential to influence the binding of the transactivators to Med31 subunit, alter the structure of the medi ator, hinder the entry of other subunits into the Mediator, or the entry of general transc ription factor or RNA pol II into the PIC, and thus, cause pleiotropic effects by impeding RNA pol II-dependent transcription. Consistent

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59 with this hypothesis, our preliminary data show ed multiple aspects of plant development were influenced for the med31-2 mutant. Evidence for a Mediator Complex in Arabidopsis The transcription apparatus of plants, metazoans and yeast are conserved (Gurley et al., 2006). Many of the promoters in these three ki ngdoms contain the TATA motif and CAAT box for the binding of RNA pol II and general transcription factors. Transactivators generally bind the upstream cis -elements to regulate gene expression. The RNA pol II in a ll the three kingdoms contains 12 conserved subunits. In addition, plants possess the genes coding for all the general transcription factors (TFIIA, B, D, E, F, and H) that are presen t in metazoans and fungi (Coulson and Ouzounis, 2003). Arabidopsis also has the homologs of th e subunits of some coactivators (Hsieh and Fischer, 2005), such as the SAGA (S pt-Ada-Gcn5-acetyltransfe rase) (Stockinger et al., 2001) and SWI/SNF complexes (Brzeski et al ., 1999; Eshed et al., 19 99; Ogas et al., 1999). This high degree in conservation of the transcription machinery suggests that the plants may also have the Mediator coactivator which has been shown to play an essential role in RNA pol II-mediated transcription in other eukaryotes. Iden tification of the homologs of most of the yeast and metazoan Mediator subunits in Arabidopsis strongly supports this hypothesis (Gurley et al., 2006; Boube et al., 2002). The experiments described here explore va rious aspects of gene expression for two putative Mediator subunits from Arabidopsis Med31 and Med6. By all measures tested, these two proteins behaved as expected for bona fide members of plant Mediator. AtMed31 was localized in the nucleus, and was widely e xpressed throughout the plants. Both AtMed6 and AtMed31 were localized to the prom oters of three unrelated genes: CCA1 Hsp18.2 and Adh1 Together with the sequence homology between Arabidopsis proteins and known Mediator subunits from other eukaryotes, these data str ongly support the presence of a Mediator complex

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60 in Arabidopsis, and higher plants in gene ral, that shows strong conservation in both form and function with analogous complexes in fungi and metazoans.

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73 BIOGRAPHICAL SKETCH Wei Pan received his Bachelor of Scien ce degree in biology from Northeast Normal University in Changchun, China, and then a Mast er of Science degree in biophysics from the Chinese Academy of Agricultural Sciences in Be ijing, China. His current research interests are centered on genetics, devel opment and molecular biology.