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MOLECULAR ANALYSIS OF TWO PUTATIVE MEDIATOR SUBUNITS INTArabidopsis
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
To my family
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
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...
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
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
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
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
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
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
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
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.
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,
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
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.
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).
The human SRB/MED Cofactor Complex (SMCC) was purified by affinity
chromatography of FLAG epitope-tagged human SRB proteins (Gu et al., 1999).
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).
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
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.
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
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
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.,
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
ERa and ER 13 estrogen receptor (ER)
GATA family of transcription factors
Breast cancer susceptibility gene 1
Thyroid hormone receptor (TRu, TRBl)
Glucocorticoid receptor (GR)
receptors (PPAR aand PPARy)
Retinoic acid receptor (RARu)
Retinoid-X-receptor for 9-cis-retinoic
Vitamin D receptor (VDR)
Hepatocyte nuclear factor 4 (HNF-4)
Farnesoid X receptor (FXR)
Retinoid-related orphan receptor
Aryl hydrocarbon receptor (AHR)
Me3 General control nondepressible factor 4
Zhu et al., 1999;
Burakov et al.
et al., 2001
Crawford et al.
Wada et al., 2004
Yuan et al., 1998,
Zhu et al., 1997
Wang et al., 2002
Hittelman et al.,
Zhu et al., 1997
Zhu et al., 1997
Zhu et al., 1997
Rachez et al.,
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
Zhou et al., 2002
Gwack et al., 2003
Hittelman et al.,
Malik et al., 2002
Lau et al., 2003
Toth et al., 2004
Glucocorticoid receptor (GR)
Hepatocyte nuclear factor 4 (HNF-4)
Medl4 Signal transducer and activator of
Sterol regulatory element-binding
protein-l a (SREBP-la)
Table 1-1. Continued.
Kato et al., 2002
Small mothers against
decapentaplegic 2/3/4 (SMAD2,
General control nondepressible factor
Medl6 Differentiation-inducing factor (DIF)
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
CCAAT/enhancer binding protein
Differentiation-inducing factor (DIF)
HSF (heat-shock factor)
Differentiation-inducing factor (DIF)
Med25 Heat-shock factor (HSF)
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
Stevens et al.
Asada et al., 2002
Mo et al., 2004
Kim et al., 2004
Kim et al., 2004
Kim et al., 2004
Kim et al., 2004
Mittler et al., 2003
Med29 Doublesex (dsx )
Table 1-2. Mediator subunits in yeast, Arabidopsis, Drosophila and humans (Gurley et al., 2006;
Boube et al., 2002)
(Bourbon et al.,
TRAP3 7-CRSP3 4
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
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.
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.
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.
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
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
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
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.
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).
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
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
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.
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
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
Latuass alna (269) --
Cyru cas rumnzphl5) --~ r CS S Y GCLS SILKLSLE~
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
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.
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
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.
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.
ii r ,~flr*~",~'!
d"i..* *e ..":~?*Y~1
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.
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.
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
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)
(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
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
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--
Drosoph la~melanogaster(184) -----P-PI P~N LCPEG~NARA MNEG LDIKTEGVDMKPPPEKKSK--
Caenorhabdits~elegans(193) --------TEAEREKEVEEE TSTDEPEPTTRTSQ
Saccharomyces~cerev slae(237) -----------LMVTSIRSTPNYI--RTG GNMG
Sch izosaccharomyces~po mbe(181) ---------------SW AF US HSS-KEAPD~K --
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.
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.
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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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.