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J. Biol. Chem., Vol. 276, Issue 40, 37020-37026, October 5, 2001
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,From the National Creative Research Initiative Center for Genome Regulation, Department of Biochemistry, 134 Sinchon-dong, Seodaemoon-ku, Yonsei University, Seoul 120-749, Korea
Received for publication, June 18, 2001, and in revised form, July 10, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The yeast Mediator is composed of two
subcomplexes, Rgr1 and Srb4, known to be required for diverse aspects
of transcriptional regulation; however, their structural and functional
organizations have not yet been deciphered in detail. Biochemical
analyses designed to determine the subunit composition of the Rgr1
subcomplex revealed that the regulator-interacting subcomplex has a
modular structure and is composed of the Gal11, Med9/Cse2, and
Med10/Nut2 modules. Genome-wide gene expression and Northern analyses
performed in the presence or absence of the various Mediator modules
revealed a distinct requirement for the Gal11, Med9/Cse2, and
Med10/Nut2 modules in transcriptional repression as well as activation.
GST pull-down analysis revealed that the transcriptional repressor Tup1
binds to distinct but overlapping regions of the Gal11 module that were
shown previously to be transcriptional activator binding sites.
These data suggest that competition between transcriptional activators
and repressors for a common binding site in the Mediator and distinct
conformational changes in the Mediator induced by repressor binding may
underlie the mechanism of transcriptional repression in eukaryotes.
The Mediator complex was identified first as a coactivator
essential for transcriptional activation in the yeast
Saccharomyces cerevisiae (1, 2). Mediator associates tightly
with RNA polymerase II (Pol
II)1 to form a Pol II
holoenzyme (h-Pol II) and plays a pivotal role in diverse aspects of
transcriptional regulation. The search for homologous Mediator
complexes in the mammalian system identified a number of protein
complexes that contain several yeast Mediator homologs that function as
both positive and negative regulators of transcription in
vitro (3-7).
The Mediator complex is required in general for the regulation of Pol
II transcription. However, certain Mediator subunits function in an
activator-specific manner to modulate the expression of distinct
subsets of genes (8-10), suggesting the presence of functional modules
within the Mediator complex. Differential dissociation of Mediator
components by high urea treatment (11) and compositional analysis of
mutant h-Pol II complexes (8, 12, 13) revealed that Mediator
subunits with similar genetic properties form distinct modular
subassemblies. Recently, the modular structure of Mediator was
visualized by low resolution structural analysis (14). Electron microscopy and image processing of single particles of Mediator and
h-Pol II revealed that Mediator alone assumes a compact structure; however, at high pH or in the presence of Pol II, Mediator displays an
extended conformation with three domains (head, middle, and tail)
forming an envelope that wraps around Pol II (14).
Because purified yeast h-Pol II in conjunction with basal transcription
factors can support activated transcription in a well defined yeast
transcription system (1, 2), it was hypothesized that gene-specific
activators communicate either directly or indirectly with Mediator to
recruit Pol II to the promoter. This hypothesis was substantiated by
the following observation. First, h-Pol II was shown to interact with
the functional activation domains of the VP16 and Gcn4 transcriptional
activator proteins (8, 15, 16). Subsequently, we demonstrated that a
distinct Mediator module (the Gal11 module) is required for
transcriptional activator binding to h-Pol II (8).
In addition to the essential role of Mediator complex in
transcriptional activation, several lines of evidence indicate the involvement of Mediator in transcriptional repression as well. Genetic
analyses revealed that several Mediator genes (such as SIN4,
RGR1, and GAL11) play a role in the
transcriptional repression of the HO, SUC2, and
IME1 genes (17-19). Alleles of SIN4 and
GAL11 were identified as suppressors of (i) an
NC2 (Dr1/DRAP1) repressor mutation and (ii) defective
silencing at the HMR mating-type locus in yeast (20). In addition,
yeast cells lacking the MED1 gene, which encodes a subunit
of the Rgr1 subcomplex, are viable but show a complex phenotype that
includes partial defects in both repression and induction of
GAL gene transcription (21). Finally, deletion of the
HRS1 gene, which encodes a subunit of the Gal11 module,
causes derepression of the GAL1, PHO5, and
HSP26 genes (22), and Hrs1 protein was shown to interact
with Tup1 protein in vitro (23). Taken together, these
results suggest that several proteins in the Rgr1 subcomplex are
required for transcriptional repression.
To decipher the modular structure of Mediator and identify the
functions associated with each module, we used a urea-induced disruption assay and reconstitution experiment with recombinant proteins to analyze the protein-protein interactions among the Mediator
subunits and examined the role of each subunit in transcriptional regulation with genome-wide expression analysis.
Construction of Repressor Expression System--
The
copper-inducible LexA-tagged yeast expression vector was constructed as
follows. The CUP1 promoter region from RNA Preparation and Northern Analysis--
Various Mediator
mutant cells (10, 27) containing the copper-inducible repressor
expression system and the reporter construct (Table
I) were grown in an appropriate medium to
early exponential phase at 30 °C (A 600 = 0.5), divided into two aliquots, and then allowed to grow for an
additional 2.5 h in the presence or absence of 0.1 mM
Cu2+ ion. After the copper induction, the cells were
shifted to 37 °C and then allowed to grow for an additional 2.5 h. Total RNA was prepared as described previously (28). To prepare the
probe for Northern blot analysis, the BamHI-SacI
DNA fragment of LacZ and the LexA DNA binding region were isolated and
32P-labeled by Klenow fragment in the presence of random
hexanucleotide. Specifically hybridized signals were quantified using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and the associated
software.
Construction of Recombinant Baculoviruses--
Recombinant
baculoviruses expressing each Mediator component were created using the
Bac-to-Bac baculovirus expression systems (Life Technologies, Inc.). To
construct a baculovirus that expresses an N-terminal HA-tagged Mediator
protein, the HA epitope sequence (YPYDVPDY,
5'-TACCCATACGACGTGCCAGACTAC-3') was inserted into the NcoI
and EcoRI sites in pFASTBACHTb (Life Technologies, Inc.) to
construct pHAFASTBACHTb. All the Mediator genes were cloned by in
vivo gap repair, verified by nucleic acid sequencing, and introduced into pFASTBACHTb and pHAFASTBACHTb expression vectors in-frame. Recombinant baculovirus clones for each of the Mediator proteins were isolated and further amplified until a titer of >1 × 108 plaque-forming units/ml was achieved according to
the baculovirus expression system manual (Life Technologies, Inc.).
Expression and Purification of Mediator Module Complexes from
Sf9 Cells--
Sf9 cells were cultured in Grace's
medium containing 10% fetal calf serum (Hyclone, Logan, UT). Each
recombinant virus was used for infection at a multiplicity of infection
of 2. Multiple recombinant viruses were infected together at the same
multiplicity of infection for coexpression experiments; Sf9
cells were infected with virus mixtures A (HA-Med1, Med4, and
Med9/Cse2) and B (HA-Srb7, Med7, and Med10/Nut2) to make Med9/Cse2 and
Med10/Nut2 modules, respectively. The cells were harvested by
centrifugation for 5 min at 500 × g after 48 h of
incubation, washed once with a phosphate-buffered saline solution,
resuspended in 10 ml of IP300 buffer (20 mM Tris acetate
(pH 7.9), 10% glycerol, 0.1 mM EDTA, 0.2% Nonidet-400, and 300 mM potassium acetate), and incubated on ice for 30 min. Next, the lysate was disrupted by Dounce homogenization (20 strokes with a "B" pestle) and centrifuged at 100,000 × g for 1 h at 4 °C. The whole-cell extract was
diluted with the same volume of IP buffer (same as IP300 buffer with
the exclusion of 300 mM potassium acetate) and then applied
to a Q-Sepharose FF column (10 ml) equilibrated with D150 buffer (20 mM Tris acetate (pH 7.9), 0.1 mM EDTA, 0.01% Nonidet P-40, 10% Glycerol, plus 150 mM potassium
acetate). The column was washed with 2 volumes of D300 buffer and then
eluted with D600 buffer. Fractions containing recombinant Mediator
proteins were identified by immunoblot analysis with anti-Med6
antibodies (29), dialyzed against IP150 buffer, and then incubated for 8 h at 4 °C with 100 µl of HA.11 Ab affinity matrix (BabCO).
After binding, the resin was washed three times for 5 min at 4 °C
with 500 µl of IP1500 buffer. The proteins bound to the resin were eluted by washing twice with IP150 buffer containing 0.5 mg/ml HA
peptide (YPYDVPDYA, BabCO) at 4 °C for 30 min. The purity of each
eluate was analyzed by SDS-polyacrylamide gel electrophoresis followed
by silver staining and immunoblotting.
Genome-wide Gene Expression High Density Oligonucleotide Array
(HDA) Analysis--
Wild-type and mutant yeast strains were grown to
an A600 of 0.5 in YPD at 30 °C. For
temperature-sensitive (ts) mutants, the cultures were
diluted with an equal volume of YPD and allowed to grow for 45 min at
37 °C. Total RNA was isolated, and the genomic expression profile of
each strain was determined using four Affymetrix GeneChip arrays.
Poly(A) mRNA was isolated, converted to biotin-cRNA, and hybridized
to the GeneChips, which were then washed, stained, and scanned as
described previously (9). Five poly(A)-tagged control RNAs were added
to equal amounts of total RNA from each preparation. The levels of
these controls were then used to normalize each wild-type and mutant
expression profile to total RNA. Two independent experiments were
performed for each wild type versus mutant comparison.
Individual mRNA levels were scored if the computer algorithm
analyzing the scanned results (30) returned a "Present" call in
both of the two wild-type and the two mutant expression profiles for
that gene or if the expression levels of the gene changed in the same
direction and were greater than background levels in both wild-type and
mutant comparisons. A decrease was called if an mRNA dropped more
than 2-fold in both comparisons. An increase was called if an mRNA
increased more than 2-fold in both comparisons.
Modular Structure of the Mediator Complex--
The observations
that the Mediator has a structure that is composed of two functionally
distinct subcomplexes (Srb4 and Rgr1) (11) and that the Gal11, Med2,
Hrs1, and Sin4 proteins form a functional module that bind to the
C-terminal region of Rgr1 (12) prompted us to examine whether Med9/Cse2
and Med10/Nut2 also form modular structures with distinct functions. To
this end, we first purified h-Pol II from wild-type yeast and mutant yeast strains that carried either the med9
To confirm the above result, we immobilized the Mediator complex with
anti-Med9/Cse2 Ab-coupled agarose beads and analyzed the polypeptides
retained after a urea wash. The 1 M urea wash removed
mainly the polymerase subunits associated with the Mediator complex,
whereas a 2 M urea wash removed the Srb4 subcomplex from the beads without affecting the association of the Rgr1 subcomplex with
the beads (Fig. 1B). When the beads were washed extensively at an even higher urea concentration (3 M), only Med1 and
Med4 (in addition to Med9/Cse2) were retained on the beads.
To determine whether direct interactions exist among Med9/Cse2, Med1,
and Med4, Sf9 cells were co-infected with baculoviruses that
contained Med4, Med9/Cse2, and HA-tagged Med1 expression constructs.
The Sf9 lysate was then analyzed by immunoprecipitation with
anti-HA monoclonal Ab-coupled agarose beads. Immunoblot and silver-staining analysis of the immunoprecipitate showed that Med4 and
Med9/Cse2 proteins co-precipitated with the HA-Med1 protein via direct
interaction, whereas GST protein added to the extracts remained in the
supernatant (Fig. 2A and data
not shown). These observations demonstrate that Med9/Cse2, Med1, and
Med4 also form a stable modular structure, as do the components of the
Gal11 module.
Although the existence of the Gal11 and Med9/Cse2 modules was
demonstrated with several experimental techniques, we were unable to
observe a Med10/Nut2 containing module either by compositional analysis
of med10ts/nut2ts h-Pol II or using the urea
dissociation method (because of the relatively low binding affinity of
anti-Med10/Nut2 Ab; data not shown). Therefore, we examined the
physical interactions in Sf9 cells of Med10/Nut2 with the rest
of the polypeptides (Med7 and Srb7) in the Rgr1 subcomplex.
Immunoprecipitation of Sf9 cell extracts in which Med7,
Med10/Nut2, and Srb7 were coexpressed with anti-Med10 Ab showed that
Med7 and Med10/Nut2 associated directly with Srb7 in the absence of
other Mediator proteins, thus demonstrating the presence of a
Med10/Nut2 module (Fig. 2B). Therefore, Mediator appears to
contain at least three modular structures (the Gal11, Med9/Cse2, and
Med10/Nut2 modules) that are tethered independently to the Rgr1 protein
in the Rgr1 subcomplex.
Differential Requirement for Each Mediator Subunit in
Transcriptional Activation and Repression--
To identify the
distinct roles of the Med9/Cse2 and Med10/Nut2 modules in
transcriptional regulation, we analyzed the genome-wide gene expression
patterns in yeast strains that contained mutations in the
MED9/CSE2 and MED10/NUT2
genes using HDAs for 6181 yeast genes. Total RNA was isolated from
med9
To examine whether the Med9/Cse2 module is involved in transcriptional
repression as suggested by the HDA analysis, we used Northern
analysis to examine the effect of med9
In addition to the MED9/CSE2 and GAL11
results shown here, certain alleles of other Mediator components
(SIN4 and RGR1) have been implicated in
transcriptional repression (18, 19, 22, 31, 32). However, whether the
Mediator proteins are involved directly in transcriptional repression
or indirectly by controlling the expression of a specific
transcriptional repressor protein has not yet been demonstrated
clearly. To determine whether specific Mediator subunits are involved
directly in transcriptional repression, we measured the transcriptional
repression of a reporter gene (LacZ) that contained a
binding site for the LexA repressor protein. We expressed LexA-Ssn6 or
LexA-Srb10 repressor fusion proteins in various Mediator mutant
backgrounds and monitored reporter gene expression (Fig.
4A). In the absence of copper,
LexA-Srb10 and LexA-Ssn6 were expressed at a barely detectable level
from the CUP1 promoter; therefore a moderate level of basal
expression of the reporter gene (LacZ) was detected in all
of the strains tested (Fig. 4B). Upon induction with copper,
the highly expressed repressor fusion proteins were recruited to the
reporter gene promoter (CYC1 promoter fused to
LacZ), which resulted in a 9-fold repression of
LacZ transcription in wild-type cells. The inactivation of
MED10/NUT2 activity did not affect the
transcriptional repression of LacZ by either of the
repressor fusion proteins. However, when the transcriptional repressors
were overexpressed in the absence of the Med9/Cse2 or Gal11 proteins,
neither LexA-Srb10 nor LexA-Ssn6 was able to repress LacZ
expression. These results suggest that Med9/Cse2 and Gal11, but not
Med10/Nut2, are required directly for transfer of the repression signal
from gene-specific transcriptional repressor proteins to the basal
transcription machinery. Therefore, the Med9/Cse2 and Med10/Nut2
modules seem to play major roles in the transcriptional repression and
activation processes, respectively, whereas the Gal11 module is
required for both types of gene regulation.
Transcriptional Repressor Binding Module of Mediator--
Our
previous studies (8, 10) and the genome-wide gene expression analysis
presented here show that two Mediator modules (the Gal11 and
Med10/Nut2) are required for distinct aspects of transcriptional
activation. The Gal11 module functions as binding sites for
transcriptional activator proteins, whereas the Med10/Nut2 module
functions at the post-activator binding stages to modify and relay the
activation signal to the basal transcription machinery (8). The
observation that transcriptional repression also requires two different
Mediator modules (the Gal11 and Med9/Cse2 modules) suggests that a
similar mechanism may work to mediate the signal between repressors and
the basal transcription machinery. To test this idea, we analyzed the
physical interactions between transcriptional repressor Tup1 and
Mediator modules using a GST pull-down assay. The Med9/Cse2 and
Med10/Nut2 modules and the Srb4 subcomplex were purified with anti-HA
Ab-coupled beads, and the HA-agarose eluates were applied to GST- or
GST-Tup1-agarose beads. The protein bound to the GST and GST-Tup1 beads
were subjected to immunoblot analysis with anti-HA antibodies that
recognize the HA-tagged versions of Med1, Srb7, and Srb4 in the
Med9/Cse2 and Med10/Nut2 modules and the Srb4 subcomplex, respectively.
This analysis revealed that none of the Mediator components tested
bound to the Tup1 protein (Fig.
5A). However, when we tested
Rgr1 and each of the Gal11 module proteins for Tup1 interaction using
the GST pull-down assay, we detected strong Tup1 binding to the Rgr1
and Med2 in addition to the previously reported Hrs1 (23) but not to
the Sin4 and Gal11 proteins (Fig. 5B). Therefore, Tup1
interacts with the Mediator mainly through the Rgr1 and Gal11 module
proteins, as do certain transcriptional activator proteins (VP16, Gal4, and Gcn4).
Although some of the individual Mediator subunits required for Tup1
interaction (Rgr1 and Med2) are distinct from those for the
transcriptional activator proteins we tested (VP16, Gal4, and Gcn4),
the fact that both types of transcriptional regulators interact with
Mediator through the Gal11 module is intriguing. In particular, the
interaction of Hrs1 both with Gcn4 and Tup1 suggests that certain
transcriptional activator and repressor proteins may share Mediator
binding sites. To investigate this possibility, we deciphered the
transcriptional regulator binding regions of Hrs1 using a series of
GST-fusion proteins that contained various Hrs1 fragments in GST
pull-down experiments. The GST pull-down assays revealed amino acids
180-343 of Hrs1 interact specifically with Gcn4, and amino acids
83-179 interact specifically with Tup1. The N-terminal region of Hrs1
that includes amino acids 1-82 interacted with both Gcn4 and Tup1
proteins (Fig. 5C). These results demonstrate that Gcn4 and
Tup1 each utilize distinct but overlapping regions of Hrs1 as the
Mediator binding targets.
Disruption of Mediator Complex by a Transcriptional Repressor
Protein--
The observation that the Mediator binding sites for a
transcriptional activator (Gcn4) and a transcriptional repressor (Tup1) partially overlap suggests that competition between the two types of
transcriptional regulators for a common binding site in the Mediator
complex may play a significant role in mechanisms of transcriptional
regulation. To test this hypothesis, we challenged the Gcn4-Mediator
interaction with increasing amounts of Tup1 protein or bovine serum
albumin as follows. Purified Mediator complex was first bound to
GST-Gcn4 fusion protein beads. After extensive washing, increasing
amounts of Tup1 or bovine serum albumin protein were added to the
beads, and the amount of Mediator complex retained in the beads or
released to the supernatant was monitored by immunoblot assay with
anti-Srb4 Ab. No detectable change in the amount of Mediator bound to
GST-Gcn4 was observed by the addition of bovine serum albumin protein
(Fig. 6). In contrast, the addition of a
10-fold excess of Tup1 protein caused a significant reduction in the
amount of Mediator bound to the GST-Gcn4 beads along with a significant
increase in the amount of Mediator proteins released to the
supernatant. This result suggests a mechanism in which a
transcriptional repressor blocks the access of a transcriptional activator protein to Mediator complex to repress transcription.
Modular Structure of Mediator Complex--
Mediator was identified
first as a coactivator that enables the basal transcription machinery
to respond to traditional gene-specific transcriptional activator
proteins (33, 34). Subsequent biochemical purification of the Mediator
complex revealed its role as an intermediary molecule between
transcriptional regulators and Pol II (1). The intermediary role of
Mediator was also shown by genetic studies that revealed the existence
of two classes of Mediator genes: one that encodes Pol II-interacting
proteins and another that interacts with gene-specific transcriptional
factors (11, 18, 19, 31, 35).
The structural organization of the Mediator complex also reflects the
two aspects of Mediator function, as suggested by several experiments,
some of which are in this study. First, the co-disappearance of several
polypeptides from the Mediator complex in
med9
Among the modular structures of the Mediator complex, only two
components of the middle domain (the Rgr1 and Med10/Nut2 module proteins) and Med6 of the head domain are conserved evolutionarily (37). Even in the Rgr1 protein, only the N-terminal region of the
protein is conserved. The C-terminal region, where the species-specific tail domain is connected, shows a high sequence divergence (5). Therefore, the head and tail domains of the Mediator complex seems to
have evolved to accommodate the diversity and specificity of higher
eukaryotic transcriptional regulators and Pol II, whereas the conserved
middle domain contains the basic architecture of the Mediator complex.
Function of Repressor-specific Modules of Mediator--
The
modular structure of the Mediator complex suggests that each module is
responsible for distinct functions. Transcriptional activation requires
the Gal11 and Med10/Nut2 modules for the activator binding and
post-activator binding processes, respectively. Similarly, the Tup1
repressor also requires two separate modules, one for binding to
Mediator and the other for post-repressor binding processes (transfer
of the repressive signal to inhibit Pol II transcription of target
genes). Intriguingly, the Gal11 module seems to be a common binding
site for transcriptional activator and repressor proteins. Therefore, a
simple competition for Mediator-binding sites may occur between
transcriptional activator and repressor proteins and may ultimately
determine the mode of transcriptional regulation. In addition to the
Tup1-Mediator interaction shown here, biochemical analysis showed that
Sfl1 coimmunoprecipitates from yeast cell extracts with Srb9, Srb11,
Sin4, and Rox3 (38). Sfl1 is a known transcriptional repressor and
regulates SUC2 transcription through a repressor-binding
site located immediately 5' to the TATA box. Therefore, the direct
interaction between Sfl1 and Mediator may be the main mechanism of
transcriptional repression for SUC2.
If both transcriptional activator and repressor proteins interact with
Mediator through the Gal11 module, how can activators and repressors
regulate transcription in opposite directions? One of the mechanisms by
which regulatory proteins activate or inhibit transcription is through
their interaction with additional coactivator or corepressor complexes.
In addition to its interaction with Mediator, the Ssn6-Tup1 repressor
complex interacts with histones H3 and H4 in chromatin to form a
repressive chromatin structure over the promoter region, and the
interaction of Ssn6-Tup1 with Mediator may block the access of
transcriptional activator proteins to the promoter (39). Therefore, it
would be interesting to determine whether this repressor-Mediator
complex interacts with additional repressor complexes such as the
histone deacetylation complex to repress target gene expression.
Our results suggest another mechanism for transcriptional repression.
Although transcriptional activator and repressor proteins may share
some binding sites in the Mediator complex, the precise collection of
Mediator binding surfaces required for each type of regulatory protein
are distinct. Therefore, in addition to the competition between
activator and repressor proteins for binding to Mediator, the binding
of a transcriptional repressor to a distinct surface of the Gal11
module may cause a specific conformational change in the Mediator
complex that results in transcriptional repression. This may result in
the dissociation of the Mediator complex from other transcriptional
factors, which in turn destabilizes the preinitiation complex formation
at the promoter region as shown by the decreased occupancy of
TATA-binding protein and h-Pol II at the Tup1-bound
promoters (40).
Contrary to the most common function of the Gal11 module in the binding
of both types of transcriptional regulators, the Med9/Cse2 module seems
to be involved mainly in transcriptional repression. Because
transcriptional repressor proteins we tested do not interact directly
with the Med9/Cse2 module, we hypothesized that it functions at the
post-repressor binding stages, as does the Med10/Nut2 module during
transcriptional activation, or Med9/Cse2 may serve as a binding site
for a different type of transcriptional repressor protein.
Identification of the precise function of the Med9/Cse2 module will be
necessary to determine the mechanism by which Mediator complex
participates in the transcriptional repression process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 to 1000 (translation initiation site is +1) was amplified using the polymerase chain reaction (PCR) (XhoI and HindIII sites are
in the 5'- and 3'-end primers, respectively). A DNA fragment containing
the LexA DNA binding domain, a multicloning site, and the
ADH1 terminator was isolated from pEG202
(CLONTECH, Palo Alto, CA) by using
HindIII and XbaI. These fragments were introduced
into the XhoI and XbaI sites of pRS313 (24) and
named pCE1. The SSN6 and SRB10 coding regions
were cloned by in vivo gap repair (25) and inserted in-frame
into the multicloning site of pCE1 to create pCESSN6 and pCESRB10,
respectively. pAJ201 (26), which had four LexA binding sites in the
upstream region of the CYC1 core promoter, was used as a
reporter construct. The TUP1 open reading frame was cloned
by in vivo gap repair and then introduced into pGEX-4T-1 in-frame to construct the GST-Tup1 fusion construct. GST-Tup1 was
expressed in bacterial strain DH5
and then purified. The Tup1 open
reading frame was also cloned into pHAFASTBACHTb (see "Construction
of Recombinant Baculoviruses") in-frame to construct an N-terminal
hemagglutinin (HA) epitope-tagged Tup1 protein, and the virus
expressing the HA-tagged Tup1 was screened as described in the
baculovirus expression system manual (Life Technologies, Inc.).
The HA-tagged Tup1 was purified with Q-Sepharose FF and HA.11 Ab matrix
(BabCo, Richmond, CA).
Yeast strains used in this study
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(MED9/CSE2 deletion) or med10ts
(temperature-sensitive) mutations. Subsequent immunoblot analysis of
h-Pol II purified from the wild-type and mutant yeast strains revealed
that the composition of h-Pol II from the med10ts strain was
indistinguishable from that of wild type (data not shown). However, the
h-Pol II from the med9 strain was completely devoid of
Med9/Cse2 and Med1, deficient in Med4, and retained all the other
Mediator components but at a slightly reduced level (Fig.
1A). This result indicates
that Med9/Cse2, Med1, and probably Med4 associate to form a modular
structure.
View larger version (33K):
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Fig. 1.
Identification of Med9/Cse2 module.
A, immunoblot analysis of the polypeptide composition of
h-Pol II purified from wild-type (WT) yeast and
med9/cse2 null
(med9 /cse2) strains. Antibodies used for
the assay are indicated at the left side. B,
differential dissociation of Mediator components by urea treatment.
h-Pol II was immobilized on anti-Med9/Cse2 antibody beads. The proteins
that remained bound to the beads after washing with IP150 buffer
containing no urea (lane 1) or 1 (lane 2), 2 (lane 3), or 3 M (lane 4) urea were
visualized by silver staining of SDS-polyacrylamide gels
(left) and immunoblot analysis with antisera specific to the
Mediator component indicated (right).
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[in a new window]
Fig. 2.
Reconstitution of Mediator modules.
Coimmunoprecipitation (IP) of Med9/Cse2 (A) and
Med10/Nut2 (B). Mediator modules from Sf9 cells that
were infected with the recombinant viruses indicated at the
top by immunoblot analysis. Immunoprecipitation of the
module was carried out with the antibody indicated at the
top (anti-HA and anti-Med10/Nut2 antibodies) in the presence
(+) or absence ( ) of the corresponding Mediator subunit (HA-Med1 and
Med10/Nut2). Antibodies used for the immunoblot analysis are indicated
at the left.
/cse2 and
med10ts/nut2ts mutant yeast strains and their
wild-type counterparts 45 min after a shift to the nonpermissive
temperature, and these RNA preparations were hybridized to the
HDAs.2 Among the 5376 yeast genes, the expression levels of which were determined for the
med10ts mutant strain, 2899 genes (54%) were down-regulated
more than 2-fold under restrictive conditions, whereas only 56 gene
were up-regulated (1%) (Table II). In
contrast, the number of genes, the expression of which was affected in
the med9 mutant strain was quite small (384 of 4806 genes
down- or up-regulated); however, a significant number of genes (209 of 384 genes affected) were up-regulated (Tables II and
III). Therefore, MED10/NUT2 seems to be required for the
transcriptional activation of a large number of genes, whereas
MED9/CSE2 is generally dispensable for
transcriptional activation but required for the mediation of
transcriptional repression for a subset of genes.
DNA chip analysis of Mediator mutants
/cse2 mutant yeast strains and their
wild-type counterparts 45 min after a shift to the nonpermissive
temperature were hybridized to four Affymetrix GeneChip arrays that
contain probes for 6181 yeast genes.
Negative and positive requirement of Med9 for transcription
mutation on the expression of genes that are repressed under a rich growth condition (FLO1, SUC2, SOL4, and
HXK1) and compared their effect with that of the
gal11 mutation. When the cells were grown in YPD,
expressions of the SOL4, SUC2, and
HXK1 genes were repressed completely (SUC2) or at
least several fold (SOL4 and HXK1) in wild-type
cells. However, the med9/cse2 mutant was
defective in transcriptional repression of SOL4 and
HXK1 (4- and 3.5-fold increases in transcripts,
respectively), whereas gal11 mutants showed defects in
the transcriptional repression of the SOL4 and
SUC2 genes (12- and 10-fold increases, respectively; Fig.
3). In addition, Med9 is required
for the transcriptional repression of FLO1, but Gal11 is
dispensable for the FLO1 repression (Fig. 3). Therefore,
Gal11 and Med9 proteins are required for transcriptional repression of
distinct group of genes and seem to constitute repressor interaction
modules of the Mediator complex.
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Fig. 3.
Transcriptional repression defects in
Mediator mutant yeast strains. Northern blot analysis of Mediator
mutants is shown. To determine the effect of Mediator mutations on the
transcriptional repression, wild-type (wt),
med9/cse2 null, and gal11 null strains
were grown in YPD at 30 °C to the early exponential phase and then
shifted to 37 °C for another 2.5 h. Nucleic acid probes for
Northern blotting are shown at the right.
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Fig. 4.
Effect of Mediator mutations on
transcriptional repression by artificially recruited repressor
protein. A, LexA-fusion repressor expression
construct (effector plasmid) and reporter construct. Expression of the
LexA-repressor fusion proteins was controlled by a copper-inducible
promoter (CUP1). The effect of repressor binding on
CYC1 promoter activity was monitored. B, Mediator
mutant strains containing the copper-inducible LexA-Srb10
(YSJW10, YSJ910, YSJG1110, and YSJ1010) or LexA-Tup1 (YSJWS6, YSJ9S6,
YSJG11S6, and YSJ10S6) expression systems along with a LacZ
reporter gene construct driven by the CYC1 core promoter and
a LexA binding site were cultured in the presence (+) or absence ( )
of 0.1 mM copper ion and then shifted to the nonpermissive
temperature. mRNA was prepared from each strain and analyzed by
Northern blot with the specific probe indicated at the
right. As a control, the ACT1 transcript
(AIP1) level is shown. wt, wild type.
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Fig. 5.
Identification of the Tup1 binding site of
Mediator. A, GST pull-down analysis of Mediator modules
and subcomplex with GST-Tup1 fusion protein. The Med9/Cse2 and
Med10/Nut2 modules and the Srb4 Mediator subcomplex each containing a
HA-tagged component indicated at the right were incubated
with GST-Tup1 protein bound to agarose beads. The bead-bound proteins
are shown by immunoblot analysis with anti-HA antibodies. B,
GST pull-down analysis of Gal11 module proteins and Rgr1 binding to
GST-Tup1. The components of the Gal11 module proteins were purified and
incubated with GST-Tup1 protein agarose beads. The proteins bound to
the beads were visualized by immunoblot analysis with anti-Gal11 Ab
(Gal11) and anti-HA Ab (HA-tagged proteins). C, mapping of
the Tup1 interaction domain of Hrs1. Affinity-purified Gcn4 and Tup1
proteins were incubated with a series of GST-fusion proteins each
containing a different fragment of Hrs1, as indicated at the
left of the gel panels. The amounts of Gcn4 and Tup1 bound
to each of the GST-Hrs1 fusion proteins are shown by immunoblot
analysis. Fragments of the Hrs1 polypeptide fused to GST are
represented as solid bars, and the amino acid residues
contained in each fragment are indicated.
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Fig. 6.
Competition between transcriptional activator
and repressor proteins for Mediator binding. Mediator complex
bound to GST-Gcn4 protein beads were subjected to competition with
increasing amounts of a control protein (bovine serum albumin
(BSA)) or a transcriptional repressor protein (Tup1). The
amounts of Mediator complex remaining on the beads (Bound)
and appearing in the supernatant (Sup) are shown by
immunoblot analysis with the anti-Srb4 Ab.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/cse2, and gal11 strains
indicates that these subunits are connected to the rest of the complex
via the Med9/Cse2 or Gal11 proteins. Second, high concentration urea
treatment of Mediator complex immobilized on beads revealed not only
the Srb4 and Rgr1 subcomplexes but also the Med9/Cse2 module. Third,
coimmunoprecipitation from Sf9 cells of Mediator subunits that
belong to the same module demonstrates that the Mediator modules or
subcomplexes can be assembled with recombinant proteins. Lastly,
electron microscopy and image processing of Mediator revealed the
modular structure of the complex, which is composed of head, middle,
and tail domains (14). In particular, the absence of the tail domain in
the sin4 Mediator indicates that the tail domain
corresponds to the Gal11 module. Because the Gal11 module is connected
to the C-terminal region of Rgr1, the tail and middle domains must
constitute the Rgr1 subcomplex. Therefore, the Srb4 subcomplex
appears to form the head structure, which is connected to Pol II. An
extensive genome-wide two-hybrid screen in yeast (36) also identified physical interactions among the subunits of each Mediator subcomplex (Srb5-Med8 and Srb6-Med11 interactions for the Srb4 subcomplex; Med7-Med4, Med7-Srb7, and Med9/Cse2-Med4 interactions for the Rgr1
subcomplex), which are in good agreement with the physical interactions
identified in this study.
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ACKNOWLEDGEMENTS |
|---|
We thank Frank Holstege and Richard Young for their devoted effort in HDA analysis of Mediator mutants and kindly providing all the data related to the HDA analysis in this study. We thank A. D. Johnson for providing plasmid constructs, Kelly LaMarco for careful reading of the manuscript, and our colleagues in the Kim lab for helpful discussion.
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FOOTNOTES |
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* This work was supported by Creative Research Initiatives Program Grant 99-C-CT-01-C-48 from the Korean Ministry of Science and Technology (to Y.-J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Molecular and Cellular Biology, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030.
§ To whom correspondence should be addressed. Tel.: 82-2-312-8835; Fax: 82-2-312-8834; E-mail: yjkim@yonsei.ac.kr.
Published, JBC Papers in Press, July 24, 2001, DOI 10.1074/jbc.M105596200
2 S. J. Han, J.-S. Lee, J. S. Kang, and Y.-J. Kim, unpublished data.
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ABBREVIATIONS |
|---|
The abbreviations used are: Pol II, RNA polymerase II; h-Pol II, Pol II holoenzyme; GST, glutathione S-transferase; HA, hemagglutinin; Ab, antibody; YPD, yeast extract-peptone-dextrose medium; ts, temperature sensitive; HDA, high density oligonucleotide array.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608 |
| 2. | Koleske, A. J., and Young, R. A. (1994) Nature 368, 466-469 |
| 3. | Gu, W., Malik, S., Ito, M., Yuan, C. X., Fondell, J. D., Zhang, X., Martinez, E., Qin, J., and Roeder, R. G. (1999) Mol. Cell 3, 97-108 |
| 4. | Sun, X., Zhang, Y., Cho, H., Rickert, P., Lees, E., Lane, W., and Reinberg, D. (1998) Mol. Cell 2, 213-222 |
| 5. | Jiang, Y. W., Veschambre, P., Erdjument-Bromage, H., Tempst, P., Conaway, J. W., Conaway, R. C., and Kornberg, R. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8538-8543 |
| 6. | Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R. (1999) Nature 398, 828-832 |
| 7. | Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M., Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999) Nature 398, 824-828 |
| 8. | Lee, Y. C., Park, J. M., Min, S., Han, S. J., and Kim, Y. J. (1999) Mol. Cell. Biol. 19, 2967-2976 |
| 9. | Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998) Cell 95, 717-728 |
| 10. | Han, S. J., Lee, Y. C., Gim, B. S., Ryu, G. H., Park, S. J., Lane, W. S., and Kim, Y. J. (1999) Mol. Cell. Biol. 19, 979-988 |
| 11. | Lee, Y. C., and Kim, Y. J. (1998) Mol. Cell. Biol. 18, 5364-5370 |
| 12. | Li, Y., Bjorklund, S., Jiang, Y. W., Kim, Y. J., Lane, W. S., Stillman, D. J., and Kornberg, R. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10864-10868 |
| 13. | Myers, L. C., Gustafsson, C. M., Hayashibara, K. C., Brown, P. O., and Kornberg, R. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 67-72 |
| 14. | Asturias, F. J., Jiang, Y. W., Myers, L. C., Gustafsson, C. M., and Kornberg, R. D. (1999) Science 283, 985-987 |
| 15. | Drysdale, C. M., Jackson, B. M., McVeigh, R., Klebanow, E. R., Bai, Y., Kokubo, T., Swanson, M., Nakatani, Y., Weil, P. A., and Hinnebusch, A. G. (1998) Mol. Cell. Biol. 18, 1711-1724 |
| 16. | Hengartner, C. J., Thompson, C. M., Zhang, J., Chao, D. M., Liao, S. M., Koleske, A. J., Okamura, S., and Young, R. A. (1995) Genes Dev. 9, 897-910 |
| 17. | Jiang, Y. W., and Stillman, D. J. (1992) Mol. Cell. Biol. 12, 4503-4514 |
| 18. | Covitz, P. A., Song, W., and Mitchell, A. P. (1994) Genetics 138, 577-586 |
| 19. | Song, W., Treich, I., Qian, N., Kuchin, S., and Carlson, M. (1996) Mol. Cell. Biol. 16, 115-120 |
| 20. | Kim, S., Cabane, K., Hampsey, M., and Reinberg, D. (2000) Mol. Cell. Biol. 20, 2455-2465 |
| 21. | Balciunas, D., Galman, C., Ronne, H., and Bjorklund, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 376-381 |
| 22. | Piruat, J. I., Chavez, S., and Aguilera, A. (1997) Genetics 147, 1585-1594 |
| 23. | Papamichos-Chronakis, M., Conlan, R. S., Gounalaki, N., Copf, T., and Tzamarias, D. (2000) J. Biol. Chem. 275, 8397-8403 |
| 24. | Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19-27 |
| 25. | Muhlrad, D., Hunter, R., and Parker, R. (1992) Yeast 8, 79-82 |
| 26. | Keleher, C. A., Redd, M. J., Schultz, J., Carlson, M., and Johnson, A. D. (1992) Cell 68, 709-719 |
| 27. | Fassler, J. S., and Winston, F. (1989) Mol. Cell. Biol. 9, 5602-5609 |
| 28. | Lee, T. I., Wyrick, J. J., Koh, S. S., Jennings, E. G., Gadbois, E. L., and Young, R. A. (1998) Mol. Cell. Biol. 18, 4455-4462 |
| 29. | Lee, Y. C., Min, S., Gim, B. S., and Kim, Y. J. (1997) Mol. Cell. Biol. 17, 4622-4632 |
| 30. | Wodicka, L., Dong, H., Mittmann, M., Ho, M. H., and Lockhart, D. J. (1997) Nat. Biotechnol. 15, 1359-1367 |
| 31. | Jiang, Y. W., Dohrmann, P. R., and Stillman, D. J. (1995) Genetics 140, 47-54 |
| 32. | Sakai, A., Shimizu, Y., Kondou, S., Chibazakura, T., and Hishinuma, F. (1990) Mol. Cell. Biol. 10, 4130-4138 |
| 33. | Kelleher, R. J. d, Flanagan, P. M., and Kornberg, R. D. (1990) Cell 61, 1209-1215 |
| 34. | Flanagan, P. M., Kelleher, R. J. d., Sayre, M. H., Tschochner, H., and Kornberg, R. D. (1991) Nature 350, 436-438 |
| 35. | Koleske, A. J., Buratowski, S., Nonet, M., and Young, R. A. (1992) Cell 69, 883-894 |
| 36. | Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. M. (2000) Nature 403, 623-627 |
| 37. | Kwon, J. Y., Park, J. M., Gim, B. S., Han, S. J., Lee, J., and Kim, Y. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14990-14995 |
| 38. | Song, W., and Carlson, M. (1998) EMBO J. 17, 5757-5765 |
| 39. | Jabet, C., Sprague, E. R., VanDemark, A. P., and Wolberger, C. (2000) J. Biol. Chem. 275, 9011-9018 |
| 40. | Kuras, L., and Struhl, K. (1999) Nature 399, 609-613 |
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