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J Biol Chem, Vol. 275, Issue 12, 8397-8403, March 24, 2000


Hrs1/Med3 Is a Cyc8-Tup1 Corepressor Target in the RNA Polymerase II Holoenzyme*

Manolis Papamichos-ChronakisDagger §, R. Steven ConlanDagger , Niki GounalakiDagger , Tjana CopfDagger §, and Dimitris TzamariasDagger

From the Dagger  Institute of Molecular Biology and Biotechnology Foundation of Research and Technology and the § Department of Biology, University of Crete, Vassilika Vouton, P. O. Box 1527, GR-711 10 Heraklion, Crete, Greece

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Srb/Mediator, a multisubunit subcomplex of the RNA polymerase II (RNA pol II) holoenzyme has been proposed to function as a control panel regulating transcription in response to gene-specific activator proteins. In this report, we identify the Mediator subunit Hrs1/Med3 as a physical target for Cyc8-Tup1, a yeast transcriptional corepressor. Two-hybrid and glutathione S-transferase interaction assays show that Hrs1 can associate directly with Cyc8-Tup1. Moreover, affinity chromatography experiments, using yeast protein extracts, reveal that Cyc8-Tup1 co-purifies with Hrs1 and with additional Mediator subunits in a Hrs1-dependent manner. These observations suggest that Cyc8-Tup1 contacts the Mediator complex via its interaction with the Hrs1 subunit. Further on, genetic analysis indicates that increased Hrs1 dosage can alleviate Cyc8-Tup1-mediated repression, suggesting that Hrs1/Mediator's function is inhibited upon its interaction with Cyc8-Tup1. Finally, artificial holoenzyme recruitment assays support a model by which the contact between the corepressor and the Hrs1/Mediator may prevent pol II holoenzyme recruitment to the core promoter. These data, together with previous genetic evidence, establish a functional and physical interaction between the Cyc8-Tup1 corepressor and the RNA pol II holoenzyme and support a central role of the Mediator complex in transcriptional repression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic gene transcription is a complex and highly regulated process that involves interactions between promoter-specific regulatory proteins and components of the general transcription machinery. Substantial evidence indicates that many of these components, including a subset of general transcription factors, can be recruited to the promoter along with the 12 subunits of RNA pol II1 in the form of a preassembled multiprotein complex, the RNA polymerase II holoenzyme (1, 2). The yeast holoenzyme was originally characterized as a complex that contained nine Srb proteins (Srb2, Srb4-Srb11) associated with RNA pol II carboxyl-terminal domain (1, 3). Subsequently, Srb homologs were found in RNA pol II holoenzyme complexes purified from human cells (4-8). On the other hand, a subset of Srbs was independently isolated in a yeast protein complex, which was identified as a mediator of transcriptional activation in an in vitro transcription system (9-11). Mediator contains Srb2, Srb4, Srb5, Srb6, the proteins Sin4, Rgr1, Gal11, and seven additional subunits termed Med(s).

It is well established that RNA pol II holoenzyme forms play an important role in transcriptional control (2, 12). In particular, Srb/Mediator subunits have been identified as the physiologically relevant targets for activator proteins. The potent viral activator VP16 and the yeast activator Gal4 directly interact with specific Srbs in vitro, and these interactions are essential for transcription stimulation in vivo (13, 14). It is thought that activator-target interactions simply recruit the transcription machinery to the promoter (15-18), although they may regulate steps subsequent to pre-initiation complex formation (19-21). In contrast to transcriptional activators, little is known regarding the molecular function of transcriptional repressors. Specific Srb/Med subcomplexes have been implicated in negative regulation of transcription in both yeast and human systems (7-22), yet the mechanism by which the RNA pol II holoenzyme responds to gene-specific repressors and corepressors is poorly understood.

The yeast Cyc8(Ssn6)-Tup1 protein complex acts as a general corepressor inhibiting the transcription of a diverse set of genes (23). The corepressor does not bind DNA directly but is recruited to the various promoters via interactions with gene-specific DNA-binding repressor proteins. Recruitment is predominantly mediated by a domain of the Cyc8 subunit (TPR domain), while the repression function is performed by a specific domain of Tup1 (24, 25). An increasing amount of evidence strongly suggests that Tup1-mediated transcriptional repression is performed by two possibly complementary mechanisms. First, Tup1 establishes a repressive chromatin structure over the transcription start point, probably by interacting with histones H3 and H4 (26). Second, Tup1 interferes with the function of the RNA pol II holoenzyme; Tup1 represses transcription in the absence of chromatin in an in vitro transcription assay while genetic experiments implicate two distinct groups of holoenzyme subunits (Srb8, Srb9, Srb10, and Srb11; and Sin4, Rgr1, and Rox3) in Cyc8-Tup1-mediated repression (22, 27-31). These genetic and biochemical data established a functional connection between the Cyc8-Tup1 complex and the basic transcription machinery, suggesting that the corepressor may target specific component(s) of the RNA pol II holoenzyme.

In this paper, using a combination of genetic and biochemical methods, we demonstrate that Cyc8-Tup1 interacts physically with Hrs1/Med3, a subunit of the RNA pol II holoenzyme essential for activated transcription in vitro (11, 32). We also provide evidence suggesting that the Cyc8-Tup1 interaction with Hrs1 inhibits Mediator's function and may prevent holoenzyme recruitment to the core promoter.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Media-- The protease-deficient strain BJ5457 (34) was used for preparation of protein extracts; all other strains used are derivatives of FT5 (24, 31). The two-hybrid screening was performed as described previously (31). The hrs1Delta strain (hrs1Delta HIS3) was constructed by inserting the HIS3 gene between two internal BamHI sites (codons 95-342), by one-step gene replacement. A polymerase chain reaction-amplified Hrs1 fragment (from the ATG to the STOP codon) was used for the construction of the hrs1Delta HIS3 allele. Standard synthetic media were used; YP contained 2% glucose or 2% galactose, CS, or CS plus 0.6% casamino acids. pCBG-Cyc8-transformed BJ5457 cells were induced by 0.1 mM CuSO4 (42).

Plasmid Constructions-- HRS1 (NarI-HindIII genomic fragment) was cloned by complementation of the cold-sensitive growth phenotype of the hrs1Delta strain. NH2-terminal deletions of Hrs1 were generated by Bal31 exonuclease and were confirmed by sequencing. Full-length Hrs1 and NH2-terminal deletion derivatives were cloned in to pRSET vectors (Invitrogen). HA-Hrs1 cloned in Ycp91 and LexA-Hrs1 and LexA-Med2 cloned in Ycp91-LexA contain the entire Hrs1 or Med2 coding sequence, respectively (24). Hrs1-overexpressing plasmid contains the expression cassette of YCp91 in the multicopy plasmid YepLac181 (24). Glutathione S-transferase (GST) derivatives of Cyc8 and Tup1 have been described previously (25). The SUC2-CYC1 promoter contains the URSSUC2 (-542 to -392) element cloned in to the SmaI site of the pLGDelta 312S plasmid (23). SUC2-lop-His3 was kindly provided by P. Syntyhaki and contains URSSUC2 element in the BamHI site of VS12 (Lop-His3) (24).

Purification of His-tagged Proteins and in Vitro GST Interaction Assays-- Histidine-tagged Hrs1 and Cyc8 proteins were overexpressed in Escherichia coli and were purified by nickel-nitrilotriacetic acid chromatography in a buffer containing 50 mM HEPES, pH 7.9, 350 mM NaCl, 0,2 mM 4-(2-aminoethyl)-benzene-sulfonyl fluoride hydrochloride (Boehringer Mannheim), 0.1% Nonidet P-40, 4 mM imidazole, and 0.5% bovine serum albumin, and were eluted in a buffer containing 10 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.1% Nonidet P-40. Histidine-tagged proteins were incubated with 2 µg of agarose beads-immobilized GST or GST hybrids in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.01% Nonidet P-40, 0.25% bovine serum albumin for 2 h at 4 °C. Beads were washed in the same buffer, and proteins were analyzed by SDS-PAGE and immunoblotting using anti-His antiserum (Santa Cruz).

Preparation of Yeast Protein Extracts-- Cells harboring GST or GST-Cyc8 (pCBG-Cyc8) and HA-Hrs1 expression vectors were lysed, and proteins were extracted as described previously (31). The supernatant (~1 mg of protein) was incubated with glutathione-agarose beads, for 2 h, at 4 °C, and the beads were subsequently washed extensively in the same buffer. GST- or GST-Cyc8-bound proteins were eluted with 1× SDS-PAGE sample buffer and were analyzed by SDS-PAGE and immunoblotting using anti-HA antibody (Santa Cruz) and anti-Med2 and anti-Med4 polyclonal antisera, kindly provided by L. Myers.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Cyc8-Tup1 Corepressor Targets a Mediator Component-- The functional relationship of the Cyc8-Tup1 corepressor with the basic transcription machinery led us to the hypothesis that Cyc8-Tup1 might physically contact specific factors of the RNA polymerase II holoenzyme. We looked for such protein factors by employing the yeast two-hybrid method for defining protein-protein interactions (33). A chimera consisted of Tup1 and the DNA binding domain of the bacterial LexA protein was expressed in yeast cells along with a plasmid library of short genomic fragments (~500 base pairs) fused to the Gal4 activation domain. Library clones that stimulated transcription from two independent LexA operator-containing promoters were recovered and sequenced. One of the clones that activated transcription over 9-fold (Table I) turned out to contain a region of Hrs1/Med3 (codons 76-295, Fig. 1), a protein recently identified as a component of the Mediator complex (11, 34).

                              
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Table I
Two-hybrid assays for Cyc8-Tup1 Hrs1 interaction
beta -Galactosidase activity (average of three independent transformants) of wild-type (WT), cyc8Delta , and tup1Delta strains expressing the indicated combinations of LexA-hybrids along with Gal4-Hrs1 or Gal4 alone. The LacZ reporter contains four LexA operators upstream of the GAL1 TATA element. Values are normalized to A600 of cells and are accurate ±15%. -Fold activation represents the ratio of beta -galactosidase activity from strains containing the Gal4-Hrs1 versus those containing Gal4 activation domain.


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  		Fig. 1.   Structure and function of Hrs1 and Tup1 proteins. A, full-length Hrs1 (431 amino acids) and Hrs1 derivatives are indicated schematically along with their ability to interact with Cyc8 and Tup1 (data combined from Table I and Fig. 2). B, diagrammatic representation of full-length Tup1 (713 amino acids) and Tup1 deletion derivatives with different functional domains. The + or - sign indicates the ability of each protein to interact with Hrs1 or to repress transcription (data from Table I and Ref. 24, respectively). C-I, Cyc8 interaction domain; R, repression domain; WD, WD repeats.

To identify the domain of Tup1 that specifically interacts with the Hrs1 protein, and to determine whether this interaction requires Cyc8, various LexA-Tup1 deletion derivatives were tested for two-hybrid interaction with Hrs1 in a cyc8Delta yeast strain (Table I; Fig. 1). LexA-Tup1 and LexA-N200, a Tup1 derivative that contains the repression domain of the protein but lacks the WD40 domain for protein-protein interactions (24, 35), activate transcription when combined with the Gal4-Hrs1 (clone Hrs1 76-295, Fig. 1). In contrast, the Tup1 derivatives, LexA-N72, which lacks the repression domain and contains only the Cyc8-interaction domain (25), and LexA-C324, which comprises of the WD40 domain, failed to interact with Hrs1 as they did not activate transcription above the background level. The results of these two-hybrid assays suggest that Hrs1 interacts specifically with the repression domain of Tup1 and that this interaction does not require Cyc8. On the other hand, a LexA-Cyc8 hybrid protein together with Gal4-Hrs1 activated transcription of the reporter promoter in a tup1Delta strain (Table I), indicating that Cyc8 interacts independently with Hrs1 in vivo. Taken together, the above results suggest that the Cyc8-Tup1 corepressor targets the Mediator complex via independent interactions of its components with the Hrs1 protein.

Physical Association of Hrs1 with the Cyc8-Tup1 Protein Complex-- In order to determine whether the two-hybrid interactions reflected direct protein contacts, we tested whether Hrs1 associates with Tup1 and Cyc8 in the absence of any other yeast protein (Fig. 2A). Histidine-tagged Hrs1 protein (residues 1-431, Fig. 1) was purified from E. coli and was incubated with glutathione-Sepharose beads containing GST, GST-Cyc8/N405 (TPR domain), or GST-Tup1/N200 (Tup1 repression domain). In accordance with the results obtained in the two-hybrid assays, both GST-Cyc8 and GST-Tup1, but not GST alone, interacted with the Hrs1 protein in vitro. A deletion derivative of Hrs1, which lacks the first 83 NH2-terminal residues (C347, Fig. 1) but contains most of the Hrs1 76-295 sequence that was isolated in the two-hybrid screen, also interacted with GST-Tup1 and GST-Cyc8, whereas a smaller derivative (C243, Fig. 1), which lacks the first 196 residues, did not interact with either Tup1 or Cyc8 proteins. These results indicate that an internal region of Hrs1 (residues 84-196) is responsible for the interaction with the repression domain of Tup1 as well as with the TPR region of Cyc8.


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  		Fig. 2.   Hrs1-Tup1 and Hrs1-Cyc8 interactions in vitro. A, histidine-tagged full-length Hrs1 and derivatives C347 and C243 bound to glutathione-Sepharose beads immobilized GST-Cyc8 (TPR) and GST-Tup1 (repression domain). Input contains 20% of the amount of the proteins that was incubated with the beads. B, interaction of Hrs1 with a preformed Cyc8-Tup1 protein complex. Sepharose beads containing GST-Tup1 were incubated with histidine-tagged proteins: Cyc8 alone (lane 3) or first Cyc8 and subsequently Hrs1 (lane 4). Input lanes represent 20% of Cyc8 or Hrs1 that were incubated with the Sepharose beads. Proteins in A and B were detected by immunoblotting using anti-His antiserum. C, co-purification of Cyc8-Tup1 with HA-Hrs1, Med4, and Med2 proteins. Protein extracts from wild-type or hrs1Delta yeast cells expressing GST or GST-Cyc8 proteins were incubated with glutathione-Sepharose beads. Bound proteins (B) together with input (I) and flow-through fractions (FT) were subjected to SDS-PAGE and immunoblotting analysis. Hrs1, Med4, and Med2 proteins were detected using specific antibodies. Input lanes contain 1% of that used in the pull-down reaction.

To test whether Hrs1 can interact with the Cyc8-Tup1 protein complex, an excess amount of histidine-tagged Cyc8 (N405) protein (Fig. 2B, input lane 1) was pre-incubated with glutathione-Sepharose-bound GST-Tup1 and subsequently Hrs1 protein was added (input lane 2). GST-Tup1 associated with Cyc8 (lane 3) and Hrs1 bound stably to the pre-formed GST-Tup1/Cyc8 complex (lane 4). These results clearly show that, although both subunits can separately associate with the Hrs1 protein, strong interaction with Hrs1 is also accomplished by the functional form of the corepressor, the Cyc8-Tup1 protein complex.

Cyc8-Tup1 Co-purifies with Hrs1 and Additional Mediator Proteins-- In order to test whether Cyc8-Tup1 can contact the Hrs1 subunit when the latter is assembled in the Mediator complex, protein extracts were prepared from yeast cells expressing an influenza hemagglutinin (HA) epitope-tagged Hrs1 protein along with either GST or a GST-Cyc8 hybrid protein and were subjected to GST affinity chromatography. Proteins that co-purified with GST-Cyc8 or with GST alone were further analyzed by immunoblotting using a monoclonal anti-HA antibody and polyclonal antisera raised against the Mediator proteins Med4 and Med2 (a kind gift from L. Myers). As shown in Fig. 2C, a fraction of HA-Hrs1 was retained together with GST-Cyc8, but not with GST, in the glutathione-Sepharose column. Moreover, Med4 and Med2 proteins were also retained along with GST-Cyc8. In contrast, in extracts obtained from a hrs1Delta strain, GST-Cyc8 did not co-purify with either Med4 or Med2 proteins. Previous analysis (32) has revealed that Med2 and Hrs1, along with Sin4, Rgr1, and Gal11, form a Mediator subcomplex that is distinct from Med4 and additional Med/Srb subunits. Thus, it is possible that Cyc8-Tup1 contacts the entire Mediator complex via its interaction with the Hrs1 subunit.

Hrs1 Function Is Inhibited by Cyc8-Tup1-- Substantial lines of evidence indicate that the Mediator subunit Hrs1 plays a positive role in transcription. It is essential for activated transcription by Gcn4 or VP16 in vitro (32), and for transcriptional stimulation of various yeast genes in vivo (34). Interestingly, expression of Cyc8-Tup1-regulated genes is defective in hrs1Delta strains (34),2 indicating that Hrs1 is required for normal transcription of this class of genes as well.

We reasoned that the positive function of Hrs1 might be inhibited upon interaction with the Cyc8-Tup1 corepressor under repressive conditions. In such case, an increased Hrs1 concentration in vivo could possibly overcome Cyc8-Tup1-mediated repression function. In order to test this hypothesis, a specific SUC2 promoter element (URSSUC2) that contains binding sites for Mig1/Mig2, two Cyc8-Tup1-dependent DNA-binding repressor proteins (25, 36-38), was inserted upstream of a CYC1-LacZ reporter promoter and beta -galactosidase activity was analyzed in yeast strains expressing either normal or high levels of Hrs1 (Fig. 3A). SUC2-CYC1-LacZ expressed 10-fold lower beta -galactosidase activity than the control CYC1-LacZ reporter, indicating that URSSUC2 inhibits transcription. This inhibitory effect is mediated by Cyc8-Tup1, since deletion of the chromosomal TUP1 locus (tup1Delta strain) completely alleviated URSSUC2-dependent repression. High levels of Hrs1 (H-Hrs1) in a wild-type strain grown in glucose media (repression conditions) increased transcription from the SUC2-CYC1 promoter 5-fold, while expression of the CYC1-LacZ control reporter was not affected. In contrast, overexpression of Hrs1 in a tup1Delta strain did not further increase transcription from the SUC2-CYC1 promoter. We should note that Hrs1 overexpression does not significantly affect the corepressor's protein levels, as a GST-Cyc8 hybrid protein is equally stable in cells expressing either normal or high amount of Hrs1 protein (Fig. 3E).


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  		Fig. 3.   Hrs1 overexpression alleviates Cyc8-Tup1-mediated repression. A, beta -galactosidase activity (average of three independent transformants) from wild-type and tup1Delta mutant cells expressing normal or high levels of Hrs1 (H-Hrs1) and the truncated derivative N315 (H-N315). Both proteins are HA-tagged. LacZ reporter plasmids either lack (CYC1) or contain (SUC2-CYC1) the URSSUC2 element upstream of the CYC1 promoter. -Fold repression represents the ratio of activities in strains harboring the two different LacZ reporters. B, RNA from wild-type (WT) or tup1 strains, as well as from wild-type and tup1 strains that express high levels of Hrs1 (H-Hrs1), was fractionated in 1.4% agarose-formaldehyde gel, transferred to nylon membrane, and hybridized with 32P-labeled DNA probe specific for ANB1 and actin genes. Transcript 1 (tr1), which cross hybridizes with ANB1 probe, is not regulated by Cyc8-Tup1. Similarly, RNA from strains that express normal (WT) or high levels of Hrs1 (H-Hrs1) hybridized with DNA probe specific for FLO11 and actin genes. C, RNA from wild-type cells expressing either Hrs1 (H-Hrs1) or the Hrs1 truncated derivative N315 (H-N315) at high levels, was probed with ANB1 and actin probes. D, Total protein extracts obtained from the yeast cultures described in C were fractionated in 10% SDS-PAGE and Hrs1 or Hrs1-N315 proteins were detected by immunoblotting using anti-flu monoclonal antibody. E, total protein extracts obtained from ssn6Delta yeast cells expressing a GST-Cyc8 hybrid protein along with normal or high amount of Hrs1 protein (H-Hrs1, - and +, respectively) were fractionated in 10% SDS-PAGE and the GST-Cyc8 protein hybrid was detected by immunoblotting using anti-GST antiserum. Control lane contains protein extracts from cells that do not express GST-Cyc8 protein.

Hrs1 overexpression affected transcription of ANB1, another Cyc8-Tup1-repressible gene, in a similar manner (Fig. 3B). ANB1 is not expressed in wild-type cells but is constitutively de-repressed in cells carrying the tup1Delta mutation. Hrs1 elevated levels completely de-repressed ANB1 transcription in wild-type cells, whereas they had no further effect in cells carrying the tup1Delta mutation. Moreover, Hrs1-overexpressing cells exhibit slow growth and constitutive flocculation, phenotypes, which resemble (in some degree) to the ones of cyc8Delta or tup1Delta cells (data not shown). Consistent with this observation, Hrs1 overexpression increases transcription of FLO11, a distinct Cyc8-Tup1-regulated gene involved in flocculation and pseudohyphal growth (Fig. 3B). These observations indicate that large amounts of Hrs1 weaken the function of the corepressor on additional repressible genes. On the contrary, overexpression of Hrs1-associated Mediator subunits, such as Sin4 and Med2, did not affect the repression function of Cyc8-Tup1 (data not shown).

In order to see whether high levels of Hrs1 overcome repression by masking the Cyc8-Tup1 protein complex, we tested a truncated Hrs1 derivative (Hrs1-N315, Fig. 1), which lacks a small COOH-terminal region but contains the Cyc8-Tup1 interaction region. Overexpression of Hrs1-N315 did not de-repress SUC2 or ANB1 transcription (Fig. 3, A and C) and did not cause flocculent or slow growth phenotypes (data not shown), although it was stably expressed in vivo (Fig. 3D) and is capable to interact with the corepressor. Hrs1-N315 does not support normal activated transcription in vivo,3 indicating that de-repression of Cyc8-Tup1-regulated genes requires overexpression of a functional Hrs1 protein. Based on the above results, we consider it unlikely that Hrs1 overexpression de-represses transcription by simply masking the Cyc8-Tup1 corepressor.

To test whether Hrs1 overexpression increases transcription of Cyc8-Tup1-regulated genes specifically, we examined the expression level of three additional promoters that are not regulated by Cyc8-Tup1: GAL1, HIS3, and TPS2 genes, which are induced by galactose, amino acid depletion, and high concentration of salt, respectively (Fig. 4). In contrast to Cyc8-Tup1-regulated genes (SUC2, ANB1, or FLO11), overexpression of Hrs1 did not result in a further increase of either basal (white bars) or induced levels (black bars) of GAL1, HIS3, or TPS2 gene transcription.


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  		Fig. 4.   Hrs1 overexpression does not increase transcription from GAL1, HIS3, or TPS2 gene promoters. Histograms of beta -galactosidase activity from wild-type (WT) and HRS1-overexpressing (H-Hrs1) yeast strains that contain the GAL1 (A), HIS3 (B), or TPS2 (C) promoter-LacZ reporter constructs. Yeast cultures were grown under standard conditions (gray bars) or under inducing conditions (black bars), which require the presence of either 2% galactose, or 10 mM aminotriazole, or 0.4 M NaCl in the culture media for GAL1, HIS3, and TPS2 genes, respectively. LacZ values were normalized to A600 and are accurate ± 10%.

In conclusion, our results clearly indicate that Hrs1 is a limiting factor only for the transcription of Cyc8-Tup1-regulated genes and only upon repression (e.g. in a wild-type, not in a tup1Delta strain), suggesting that its function and consequently the function of the Mediator may be inhibited upon interaction with the Cyc8-Tup1 corepressor.

Direct Holoenzyme Recruitment Bypasses Repression by Cyc8-Tup1-- It was proposed that activation domains, via interactions with Srb/Med proteins, promote the rate of pol II holoenzyme recruitment to a promoter (14). We reasoned that Cyc8-Tup1, through its contacts with Hrs1, might antagonize the function of activators thereby preventing pol II holoenzyme recruitment. Therefore, we examined whether Cyc8-Tup1 repression could be bypassed by directly recruiting the pol II holoenzyme to the DNA template independently of activators.

It has been previously shown that tethering a holoenzyme component to a DNA-binding protein, such as LexA, is sufficient to activate transcription from a LexA operator containing reporter promoter, as the tethered holoenzyme protein apparently recruits the remaining transcription machinery to the core promoter (16). In agreement with these data, LexA fused to Hrs1 or Med2 proteins strongly activated transcription from Lop-His3, a LexA operator containing promoter (Fig. 5A, gray bars), in a manner comparable to the potent activation domain of a LexA-Gal4 hybrid protein.


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  		Fig. 5.   Artificial pol II holoenzyme recruitment bypasses Cyc8-Tup1 repression. A, beta -galactosidase activities (average of four independent transformants) from yeast cells transformed with the indicated LexA hybrids. The LacZ-expressing reporter promoters SUC2-Lop-His3 and Lop-His3 are also represented. Values are normalized to A600 and are accurate ±15%. -Fold repression represents the ratio of activities obtained by the Lop-His3 promoter versus those obtained by the SUC2-Lop-His3 one. B-D illustrate a model for Cyc8-Tup1 repression based on results from A. B, LexA-Gal4 interacts with Srb/Med subunits and recruits the RNA polII holoenzyme. C, Cyc8-Tup1 associated with a promoter specific DNA-binding protein (DBP) contacts the basic transcription apparatus through Hrs1, preventing pre-initiation complex formation. D, LexA-Med hybrids directly recruit the RNA pol II holoenzyme to the promoter bypassing the repression effect of Cyc8-Tup1.

Direct holoenzyme recruitment bypassed the repression effect of the Cyc8-Tup1 corepressor, as LexA-Hrs1 or LexA-Med2 hybrids strongly stimulated transcription from a SUC2-lop-His3 reporter promoter that contains the Cyc8-Tup1-repressible URSSUC2 element upstream of a LexA operator (Fig. 5, A and D, black bars). In contrast, activation by LexA-Gal4, which presumably recruits the RNA pol II holoenzyme through interactions with Mediator subunits (14), was inhibited by Cyc8-Tup1 (Fig. 5, B and C). Thus, we conclude that Cyc8-Tup1 may prevent pol II holoenzyme recruitment probably by precluding interactions between activator proteins and components of the Mediator complex.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we identified the Hrs1/Med3 subunit of the RNA pol II holoenzyme as a physical target of the general corepressor complex Cyc8-Tup1. We first isolated Hrs1 in a yeast two-hybrid screen, based on its ability to interact with Tup1. Furthermore, by using recombinant proteins and an in vitro interaction assay, we showed that Hrs1 associates physically with Cyc8-Tup1 in the absence of any other yeast protein.

It is thought that Hrs1 occupies a peripheral location in the Mediator complex (11, 32, 39); therefore, it is conceivable that such a location makes it accessible for interaction with promoter specific regulators as the Cyc8-Tup1 corepressor. Besides Hrs1, Cyc8-Tup1 co-purifies with at least two additional Mediator subunits, Med2 and Med4, but still in a Hrs1-dependent manner. It is noteworthy that Med2 associates with Hrs1, Sin4, Rgr1, and Gal11 in a Mediator subassembly distinct from Med4 and the remaining Srb/Med components (32). Thus, although interactions between Cyc8-Tup1 and additional Mediator subunits cannot be excluded, our data suggest that Cyc8-Tup1 contacts the Mediator complex primarily by a direct interaction with the Hrs1 subunit.

Two lines of evidence support the physiological relevance of the corepressor-Hrs1 interaction for transcriptional repression. First, Hrs1 is targeted by the Tup1 repression domain, which is essential for repression of all known Cyc8-Tup1-regulated genes (24). Second, Hrs1 overexpression either increases (SUC2-CYC1) or completely de-represses (ANB1) transcription of Cyc8-Tup1-repressible promoters under repressive conditions. Based on these observations and the fact that Hrs1 is essential for transcription of various yeast genes including Cyc8-Tup1-regulated ones (32, 34),2 we propose that the corepressor inhibits the positive function of the Hrs1/Mediator. We should note that, in contrast to the majority of the Mediator polypeptides that are present in roughly equal concentrations, Hrs1 and associated components form a distinct subassembly that is present in a lower and probably limiting amount (only 30-50%) relative to the remaining Mediator subunits (11). Thus, it is conceivable that higher Hrs1 dosage overcomes repression by Cyc8-Tup1 by increasing the Hrs1-containing fraction of the pol II holoenzyme that is competent for transcription initiation.

Based on artificial pol II holoenzyme recruitment assays, Fig. 5 illustrates a possible mechanism by which Cyc8-Tup1 exerts its repression function. Transcription activation by LexA-Gal4 (Fig. 5B), which predominantly functions by recruiting the RNA pol II holoenzyme to the promoter (16), is inhibited by the Cyc8-Tup1 corepressor (Fig. 5C). However, transcriptional activation by direct pol II holoenzyme recruitment (through LexA-Med hybrids; Fig. 5D) bypasses Cyc8-Tup1 repression. Thus, it is likely that Cyc8-Tup1 interferes with the recruitment of the pol II holoenzyme to the core promoter. One possibility is that attachment of Cyc8-Tup1 to the pol II holoenzyme via Hrs1 precludes specific interactions between Srb/Mediator subunits and activator proteins or between different holoenzyme subunits. In support to this notion, Mediator complexes lacking Hrs1 do not support Gal4-mediated activation in vivo (32, 34), although they retain the Srb4 polypeptide, the physical target for the Gal4 activator protein (14, 40). Alternatively, the corepressor-Hrs1 interaction might inhibit function(s) of the pol II holoenzyme Mediator complex as yet unidentified. For example, it has been reported that TATA-binding protein occupancy in vivo, which is eventually prevented by Cyc8-Tup1 and is stimulated by activator proteins, requires the concerted function of the RNA pol II holoenzyme (41). It is possible that Cyc8-Tup1 by contacting Hrs1 interferes with a functional interaction between the RNA pol II holoenzyme and transcription factor IID, thereby preventing TATA-binding protein binding to the TATA element.

We cannot exclude the possibility that Hrs1 and associated factors are functionally related to Srb10/Srb11, which are required for complete Cyc8-Tup1-mediated repression (30). However, Hrs1 overexpression alleviates Cyc8-Tup1 repression, even in a strain that lacks Srb10 kinase activity (data not shown) suggesting that Hrs1 and Srb10/Srb11 may independently contribute to transcriptional repression.

Tup1 contacts multiple targets; Roth and colleagues have shown previously that the Tup1 repression domain interacts in vitro with the NH2-terminal tails of histones H3 and H4 (26, 43), suggesting that Cyc8-Tup1 may establish a repressive chromatin structure. While it seems likely that multiple corepressor-target interactions cooperate to elicit high levels of repression, it is not yet clear whether the interactions observed with isolated proteins (Hrs1, H3, H4) are required for repression of all Cyc8-Tup1-regulated genes and whether they occur simultaneously during the repression process. It is possible that the Cyc8-Tup1 corepressor initiates repression by inhibiting the function of the pol II holoenzyme via its interaction with Hrs1, while it subsequently maintains this repressive state by organizing chromatin.

    ACKNOWLEDGEMENTS

We thank Maria Monastirioti, George Thireos, Despina Alexandraki, Iannis Talianidis, and Michael Strubin for critical reading of the manuscript; Popi Syntychaki and George Psakis for providing plasmid constructs; and L. Myers for Med antibodies.

    FOOTNOTES

* This work was supported by PENED and TMR research grants from the Greek Ministry of Development and the EU (to D. T.) and by EPEAEK fellowships by the Greek Ministry of Education (to M. P. C. and T. C.).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.

This article is dedicated to the memory of Nina Karamaliki-Tzamaria, a great teacher and mother.

To whom correspondence should be addressed. Tel.: 81-391162; Fax: 81-391101; E-mail: tzamarias@imbb.forth.gr.

2 D. Tzamarias, unpublished observations.

3 T. Copf and D. Tzamarias, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: pol II, polymerase II; GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; TPR, tetratrico peptide repeat.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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