Genetic Analysis of the Role of Pol II Holoenzyme Components in Repression by the Cyc8-Tup1 Corepressor in Yeast

Corresponding author: Kevin Struhl, Department of Biological Chemistry, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115., kevin{at}hms.harvard.edu (E-mail)

Communicating editor: M. HAMPSEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Cyc8-Tup1 corepressor complex is targeted to promoters by pathway-specific DNA-binding repressors, thereby inhibiting the transcription of specific classes of genes. Genetic screens have identified mutations in a variety of Pol II holoenzyme components (Srb8, Srb9, Srb10, Srb11, Sin4, Rgr1, Rox3, and Hrs1) and in the N-terminal tails of histones H3 and H4 that weaken repression by Cyc8-Tup1. Here, we analyze the effect of individual and multiple mutations in many of these components on transcriptional repression of natural promoters that are regulated by Cyc8-Tup1. In all cases tested, individual mutations have a very modest effect on SUC2 RNA levels and no detectable effect on levels of ANB1, MFA2, and RNR2. Furthermore, multiple mutations within the Srb components, between Srbs and Sin4, and between Srbs and histone tails affect Cyc8-Tup1 repression to the same modest extent as the individual mutations. These results argue that the weak effects of the various mutations on repression by Cyc8-Tup1 are not due to redundancy among components of the Pol II machinery, and they argue against a simple redundancy between the holoenzyme and chromatin pathways. In addition, phenotypic analysis indicates that, although Srbs8–11 are indistinguishable with respect to Cyc8-Tup1 repression, the individual Srbs are functionally distinct in other respects. Genetic interactions among srb mutations imply that a balance between the activities of Srb8 + Srb10 and Srb11 is important for normal cell growth.

THE yeast Cyc8-Tup1 corepressor complex is required for repressing diverse classes of genes that are expressed only under specific, but distinct, conditions of environmental challenge (DERISI et al. 1997 ). Although Cyc8-Tup1 does not bind DNA, it is targeted to promoters by DNA-binding proteins that repress promoters in specific pathways: 2, cell type (KELEHER et al. 1992 ; KOMACHI et al. 1994 ); Mig1 and Nrg1, glucose (TREITEL and CARLSON 1995 ; TZAMARIAS and STRUHL 1995 ; PARK et al. 1999 ); Rox1, oxygen (DECKERT et al. 1995 ); Crt1, DNA damage (HUANG et al. 1998 ); Acr1, osmolarity (PROFT and SERRANO 1999 ); and Rtg3, mitochondrial function (CONLAN et al. 1999 ). Cyc8 and Tup1 are differentially important for recruitment by pathway-specific DNA-binding repressors; Cyc8 is important for recruitment by Mig1 and Rox1 (TZAMARIAS and STRUHL 1994 , TZAMARIAS and STRUHL 1995 ), whereas Tup1 is important for recruitment by 2 (KOMACHI et al. 1994 ). Tup1 is sufficient to mediate transcriptional repression in the absence of Cyc8, and short, nonoverlapping regions of Tup1 with minimal sequence similarity can independently mediate repression, suggesting that the Tup1 repression domain functions through protein-protein interactions (TZAMARIAS and STRUHL 1994 ). Unlike the Sin3-Rpd3 corepressor complex, which possesses histone deacetylase activity (RUNDLETT et al. 1996 ; KADOSH and STRUHL 1997 , KADOSH and STRUHL 1998 ), Cyc8-Tup1 has no known enzymatic function.

Two models, not mutually exclusive, have been proposed for the mechanism of repression by Cyc8-Tup1, one involving an effect on chromatin structure and the other involving direct action on the Pol II machinery. In support of the chromatin model, the Tup1 repression domain overlaps a region that interacts directly with histone H3 and H4 N-terminal tails in vitro (EDMONDSON et al. 1996 ), mutations in histone H3 and H4 tails can mildly reduce repression by 2 (ROTH et al. 1992 ; EDMONDSON et al. 1996 , EDMONDSON et al. 1998 ; HUANG et al. 1997 ), and Cyc8-Tup1 can affect chromatin structure of some, but not all, repressed genes (ROTH et al. 1990 ; MATALLANA et al. 1992 ; COOPER et al. 1994 ; HUANG et al. 1997 ). However, the altered chromatin structure caused by Cyc8-Tup1 can be reversed by loss of Swi/Snf function, suggesting that ordering of nucleosomes at Cyc8-Tup1-repressed promoters occurs independently of the corepressor (GAVIN and SIMPSON 1997 ). In support of a chromatin-independent model involving a direct effect on the Pol II machinery, Cyc8-Tup1 weakly represses transcription in vitro on purified DNA templates (HERSCHBACH et al. 1994 ; REDD et al. 1997 ), and mutations in components of Pol II holoenzyme (Srb8, Srb9, Srb10, Srb11, Sin4, Rgr1, Rox3, and Hrs1) partially alleviate repression by Cyc8-Tup1 (SAKAI et al. 1990 ; CHEN et al. 1993 ; BALCIUNAS and RONNE 1995 ; KUCHIN et al. 1995 ; WAHI and JOHNSON 1995 ; SONG et al. 1996 ; KADOSH and STRUHL 1997 ; KUCHIN and CARLSON 1998 ; CONLAN et al. 1999 ; PAPAMICHOS-CHRONAKIS et al. 2000 ). Sin4, Rox3, Hrs1, and Rgr1 are part of the same holoenzyme subcomplex (LI et al. 1995 ; LEE et al. 1999 ), whereas Srbs8–11 are present in a distinct subcomplex that is found in some holoenzyme preparations (KOLESKE and YOUNG 1994 ; LIAO et al. 1995 ), but not in others (LEE et al. 1997 ; MYERS et al. 1998 ).

It is important to note that all of the existing mutations in either the chromatin or holoenzyme pathways cause very modest effects on repression by Cyc8-Tup1. Similarly, Cyc8-Tup1 repression in vitro (typically 2- to 4-fold) is far less pronounced than repression of natural promoters in vivo (typically 15- to 50-fold). These observations suggest that Cyc8-Tup1 repression involves redundant functions, within and/or between the chromatin and holoenzyme pathways. In addition, repression by Cyc8-Tup1 might involve other, as yet undefined, molecular mechanisms. Here, we analyze Cyc8-Tup1 repression of natural yeast promoters in strains containing multiple mutations. Our results argue against functional redundancies among components of the Pol II machinery and against a simple redundancy between the holoenzyme and chromatin pathways. In addition, we provide evidence for distinct functions within the Srb8–11 module.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains:
The initial SLY strains were kindly provided by the laboratory of Rick Young, and derivatives of these strains are described in Table 1. Several of the SLY strains had srb alleles containing a hisG::URA3::hisG cassette (ALANI et al. 1987 ), and we removed URA3 sequences by selecting for cells on medium containing 5-fluoroorotic acid (5-FOA). The histone N-terminal mutant strains were the generous gift of the laboratory of Michael Grunstein (KAYNE et al. 1988 ; MANN and GRUNSTEIN 1992 ) and derivatives of these strains are also described in Table 1. Disruptions of the SRB genes and of SIN4 were performed using standard protocols, with srb9 and srb11 alleles being generated by two-step gene replacement; the resultant deletion alleles are termed LC. It is extremely unlikely that the resulting strains have accumulated genetic modifiers that affect temperature sensitivity or growth on galactose, because all gene replacement events were performed on glucose medium at 30°, conditions where the strains grow well, and because the same phenotypes were observed in independent strains. The construct for disrupting SRB9, pJZ991 (kindly provided by Jianhua Zhang and Rick Young), contains the SRB9 locus with a deletion of the entire open reading frame. The SRB11 disruption construct (pML2042) deletes the N-terminal 129 amino acids of the coding region (between the translational start site and an internal SphI site), with no remaining in-frame start codons until the C-terminal 45 amino acids. The srb10 and sin4 alleles were generated by a one-step disruption construct in which the open reading frame of these genes was replaced with LEU2 or URA3. MAT strains were derived from MATa strains by transient expression of the HO endonuclease. Strains were transformed with a URA3-marked centromeric plasmid carrying the HO gene, and colonies were picked when visible to the naked eye and immediately streaked to FOA to cure the HO expression plasmid; the resulting cells were examined for mating type using thr4 tester strains.

Transcriptional analyses:
RNA levels for Cyc8-Tup1-regulated and control genes were assayed by Northern blotting. In general, strains were grown in complete casamino acid medium containing 2% glucose and harvested at an OD₆₀₀ below 0.5. For induction of SUC2, cells were grown as above, harvested by centrifugation, washed in an equal volume of complete casamino acid medium lacking glucose, and resuspended in the same medium containing 0.1% glucose for 1 hr. Total RNA from cells was prepared by hot acid phenol extraction (IYER and STRUHL 1996 ), quantitated by OD₂₆₀, and tested for integrity by agarose gel electrophoresis and ethidium bromide staining. The ³²P-labeled probes were generated by random hexamer labeling of the following DNAs: a 1.2-kb HindIII SUC2 fragment; a 1.5-kb SmaI-BamHI frag-ment containing ANB1 (which also hybridizes with the tr1 transcript); a 1-kb HindIII fragment from MFA2; and a 250-bp PstI-EcoRI fragment of TUB2. ß-Galactosidase assays for transcriptional repression by LexA hybrid proteins (TZAMARIAS and STRUHL 1994 ) and for Gal4-dependent activation (SINGER et al. 1990 ) were carried out as described previously.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Very modest role of Srbs8–11 in transcriptional repression of natural promoters by Cyc8-Tup1:
Strains containing individual or multiple mutations in SRB8, SRB9, SRB10, and SRB11 were analyzed for transcriptional repression of natural promoters representing four regulatory pathways regulated by Cyc8-Tup1: cell type (MFA1, MFA2), glucose (SUC2), oxygen (ANB1), and DNA damage (RNR2). Deletion of any combination of these SRB genes, including the quadruple mutant, exhibits an identical effect on the expression of all genes tested (Fig 1). Unexpectedly, SUC2 is the only message that is affected, with srb mutant strains showing 3-fold higher levels of expression than the wild-type strain. This effect is modest in comparison to a tup1 deletion strain, which shows a 50-fold increase. The low level of SUC2 transcription in srb mutant strains is not due to a concurrent defect in transcriptional activation, because all srb mutant strains are fully competent for SUC2 induction in response to conditions of low glucose. Furthermore, deletion of tup1 in the background of the srb quadruple mutant results in levels of SUC2 transcription that are indistinguishable from that observed in a tup1 mutant strain (data not shown).

View larger version (138K): In this window In a new window Download PPT slide   Figure 1. Effects of srb mutations on Cyc8-Tup1-repressed genes. (A) Transcription from the SUC2, ANB1, and RNR2, and TUB2 genes in strains containing the indicated srb mutations. tr1 RNA crossreacts with the ANB1 probe and is not regulated by hypoxia or Cyc8-Tup1. Although a lower signal is observed for the wild-type strain upon SUC2 induction, the level of induction is comparable to the other strains when normalized to the TUB2 control in the same experiment (data not shown). (B) Effect on MFA2 repression in MAT strains of the indicated genotypes.

Transcription of genes representing the other three pathways repressed by Cyc8-Tup1 are not detectably affected by mutations in srbs8–11. Our inability to see an effect on MFA2 RNA levels is in apparent contrast to the observation that srb8 and srb10 mutant strains can result in increased expression of an integrated MFA2-LacZ reporter (WAHI and JOHNSON 1995 ). This apparent discrepancy might be due to an inability to detect very low RNA levels, although there are increasing numbers of examples in which LacZ reporter assays and RNA measurements give different results. However, even in the case of the experiments involving the MFA2-LacZ fusions, srb8 and srb10 strains show robust, although somewhat weakened, repression. In any event, the combined results indicate that Srbs8–11 have a minimal or very modest effect on Cyc8-Tup1 repression of natural promoters.

We also examined potential redundancy among Srbs8–11 under conditions where the Cyc8-Tup1 complex is artificially recruited to promoters via a LexA DNA-binding domain (Table 2). Specifically, LexA-Cyc8 and LexA-Tup1 fusion proteins were analyzed on ß-galactosidase reporters driven by promoters that do or do not contain four LexA-binding sites upstream of the CYC1 UAS and TATA element. In accord with previous results (KADOSH and STRUHL 1997 ; KUCHIN and CARLSON 1998 ), repression by either of these LexA fusion proteins is reduced three- to fivefold in srb10 and srb11 strains. As noted previously (KUCHIN and CARLSON 1998 ), repression by artificial recruitment of Cyc8-Tup1 is quite modest (two- to threefold) in srb mutant strains, which is in contrast to the situation with natural promoters where Cyc8-Tup1 repression is robust. A similar effect on repression by LexA-Cyc8 and LexA-Tup1 is observed in srb8 and srb9 strains as well as strains lacking all four proteins. Taken together, these observations indicate that Srbs8–11 play indistinguishable roles in repression by Cyc8-Tup1 and that the minimal or modest effects of Srbs8–11 on natural promoters are not due to redundant functions within this module.

Functional distinctions among components of the Srb8–11 module:
Although the entire panel of srb8–11 disruption strains behaves indistinguishably with respect to repression by Cyc8-Tup1, we found that certain srb mutant strains are unable to grow at 37° (Fig 2). Among the single mutants, only the srb11 strain exhibited temperature-sensitive (ts) growth. However, strains deleted for either srb8 or srb10 in combination with the srb11 deletion were viable at 37° as was the srb8, srb10, srb11 triple mutant. Deletion of srb9 did not alter the ts phenotype of any of these strains. Given that Srb10 and Srb11 encode a kinase/cyclin pair (KUCHIN et al. 1995 ; LIAO et al. 1995 ), we introduced an srb11 deletion allele into a strain carrying srb10-D290A, which encodes a version of Srb10 with a point mutation in the kinase active site (LIAO et al. 1995 ). The resulting strain was phenotypically identical to the srb10, srb11 strain, indicating the involvement of the kinase activity in this genetic interaction. One interpretation of these results is that Srb11 might regulate the activity of Srb8 and Srb10 and that the ts phenotype is due to hyperphosphorylation by Srb10. However, the srb8, srb10 double mutant exhibits a ts phenotype that is suppressed by an srb11 deletion, indicating that the ts phenotype is not simply related to Srb10 kinase function.

View larger version (55K): In this window In a new window Download PPT slide   Figure 2. Genetic interactions among the srb mutations. Approximately 105 cells of the indicated genotypes were spotted on plates containing YPD (30°, 37°) or synthetic minimal medium containing 2% galactose.

A related, though nonidentical, distinction between components of the Srb8–11 module is observed when strains are grown on galactose. It has been observed previously that srb10 mutants are significantly defective for Gal4-dependent activation (LIAO et al. 1995 ; KUCHIN and CARLSON 1998 ), which is likely due to Gal4 being a substrate for Srb10 kinase (HIRST et al. 1999 ). Although the srb10 and other single mutant srb strains grow on synthetic minimal medium containing galactose as the sole carbon source, a strain deleted for both srb8 and srb10 fails to grow. This Gal^- phenotype is unaffected by an srb9 deletion, but it is suppressed by an srb11 deletion. In accord with these growth phenotypes, srb8, srb10 strains are unable to activate a Gal4-respon-sive ß-galactosidase reporter, whereas single mutant strains and the srb8, srb10, srb11 triple mutant strain show only mildly reduced levels of activity (Fig 3). Taken together, these genetic interactions imply that a balance between the activities of Srb8 + Srb10 and Srb11 is important for normal growth of the cell, and that individual components of the Srb8–11 module have different functions.

View larger version (25K): In this window In a new window Download PPT slide   Figure 3. Gal4-dependent transcriptional activation in srb mutant strains. ß-Galactosidase activities in the indicated srb mutant strains containing Ycp86-Sc3801 (SINGER et al. 1990 ), a Gal4-dependent LacZ reporter that was initially grown in casamino acid medium with 2% raffinose (noninducing condition) and then induced for 12 hr by addition of 2% galactose.

Srb8–11 does not have redundant functions with Sin4 or histones H3/H4 with respect to Cyc8-Tup1 repression:
Having demonstrated the absence of redundancy between Srbs8–11, we tested for redundancy between these Srbs and Sin4. As mentioned in the Introduction, although Sin4 and Srbs8–11 have been implicated in Cyc8-Tup1 function, Sin4 appears to be an integral component of the Pol II holoenzyme, while Srbs8–11 appear to be in a distinct and more loosely associated subcomplex. If distinct subcomplexes with Pol II holoenzyme represent redundant targets for Cyc8-Tup1 action, simultaneous loss of Sin4 and Srbs8–11 might be expected to cause a dramatic loss of Cyc8-Tup1 repression. Moreover, Pol II holoenzyme in sin4 deletion strains also lacks Hrs1, Med2, Gall1, and perhaps other components (LI et al. 1995 ; MYERS et al. 1999 ). However, under repressing conditions, a strain lacking both Sin4 (and associated holoenzyme components) and Srbs10 or 11 shows only a weak increase in SUC2 expression that is comparable to that observed in strains lacking either Sin4 or Srbs8–11 (Fig 4). It should be noted that the SUC2 induction in low glucose medium is significantly compromised in sin4 deletion strains (Fig 4, lanes 2–4). However, a sin4, tup1 double deletion strain (Fig 4, lane 5) displays levels of ANB1 and SUC2 that are comparable to tup1 strain (Fig 4, lane 6). For this reason, it appears unlikely that the absence of synergistic loss of repression in the sin4, srb double deletion strains can be attributed to an activation defect caused by the sin4 mutation. Instead, we conclude that Srbs8–11 and Sin4 are not redundant for Cyc8-Tup1 repression.

View larger version (74K): In this window In a new window Download PPT slide   Figure 4. Expression of Cyc8-Tup1-repressed genes in strains containing sin4 and srb10 or srb11 mutations on Cyc8-Tup1-repressed genes. Northern blots of ANB1, SUC2 (induced and repressed), and TUB2 mRNA levels in wild-type (lane 1), sin4 deletion (lane 2), sin4, srb10 double deletion (lane 3), sin4, srb11 double deletion (lane 4), sin4, tup1 double deletion (lane 5), and tup1 deletion (lane 6) strains. For the SUC2 experiments, the panel for induced expression is exposed for a shorter time than the panel for repressed expression, and lanes 5 and 6 of the panel for repressed expression are underexposed in comparison to lanes 1–4. The faint band seen below the tr1 band in lanes 2–4 is of a different size than the band for ANB1 and was not reproducible in repeated trials.

Because mutation of the N-terminal tails of histones H3 and H4 can partially interfere with Cyc8-Tup1 repression (EDMONDSON et al. 1996 , EDMONDSON et al. 1998 ), we examined potential redundancy between holoenzyme and chromatin mechanisms. In apparent contrast to a previous report involving RNR2- and 2-dependent LacZ reporter constructs (EDMONDSON et al. 1996 ), we did not observe any effect of the histone tail mutations on the ANB1 promoter (Fig 5). This apparent discrepancy might reflect differences in sensitivities of the assays and/or differences between LacZ reporters and natural promoters, although the reported effects of the histone tail mutations appear to be roughly comparable to the modest effects of the srb mutations. In any event, strains containing mutations in the histone H3 or H4 tails as well as Srbs8–11 do not result in a synergistic loss of Cyc8-Tup1 repression; indeed, repression of the ANB1 promoter is virtually unaffected (Fig 5). This result suggests that the mechanisms involving histone tails and Srbs8–11 do not represent redundant pathways for repression by Cyc8-Tup1.

View larger version (26K): In this window In a new window Download PPT slide   Figure 5. Expression of ANB1 in strains containing srb10 or srb11 and histone H3 and H4 tail mutations.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic analysis from many laboratories has identified a number of proteins that appear to have some involvement in repression by Cyc8-Tup1 (SAKAI et al. 1990 ; CHEN et al. 1993 ; BALCIUNAS and RONNE 1995 ; KUCHIN et al. 1995 ; WAHI and JOHNSON 1995 ; SONG et al. 1996 ; KUCHIN and CARLSON 1998 ; PAPAMICHOS-CHRONAKIS et al. 2000 ). These include components of the Srb8–11 and Sin4-Rgr1-Rox3 subcomplexes of Pol II holoenzyme (KOLESKE and YOUNG 1994 ; LI et al. 1995 ; LIAO et al. 1995 ; LEE et al. 1997 #2530, 1999; MYERS et al. 1998 ; HAMPSEY and REINBERG 1999 ), Ctk1, which phosphorylates the C-terminal tail of Pol II in a manner distinct from Srb10 (HENGARTNER et al. 1998 ), and the N-terminal tails of histone H3 and H4 (EDMONDSON et al. 1996 , EDMONDSON et al. 1998 ). Importantly, all of the existing mutations in either the chromatin or holoenzyme pathways cause modest effects on repression by Cyc8-Tup1. Indeed, when RNA levels of natural genes are assayed, the effects of many of these mutations are modest on the SUC2 gene and undetectable on the ANB1, RNR2, and MFA2 genes.

There are several possible explanations, not mutually exclusive, for why the above mutations have a modest effect on Cyc8-Tup1 repression. First, the mutations might not completely abolish the functions of the proteins or complexes. This is unlikely to be the case for the individual proteins, as the mutations analyzed are drastic disruptions or complete deletions, but it might account for the modest effects of the histone tail mutations. It is not possible to analyze yeast strains lacking all histone tails, as such strains are inviable. Second, Cyc8-Tup1 might function through multiple targets with the Pol II machinery and/or distinct holoenzyme and chromatin pathways defined by the mutations. In the face of such functional redundancy, complete loss of Cyc8-Tup1 function would require inactivating all of these independent targets or pathways. Third, Cyc8-Tup1 repression might involve a novel function that has yet to be revealed through mutational analysis.

Here, we provide evidence that various forms of functional redundancy do not account for the modest effects of the mutations on Cyc8-Tup1 repression. Specifically, we demonstrate that multiple mutations within Srbs8–11, between Srbs8–11 and Sin4, and between Srbs8–11 and histone tails affect Cyc8-Tup1 repression to the same modest extent as the individual mutations. We were unable to examine the effects of more complex combinations of mutations, because the resulting strains were extremely sick or nonviable. Nevertheless, the failure to observe any additional defect in the multiply mutated strains tested here is noteworthy, because eliminating redundant functions should lead to increased loss of Cyc8-Tup1 repression even if some redundant functions remain. In this regard, the CTD kinases Srb10 and Ctk1 can independently affect Cyc8-Tup1 repression, although the double mutant strain still retains considerable Cyc8-Tup1 function (KUCHIN and CARLSON 1998 ). Taken together, our results argue that components of Pol II holoenzyme make only a minor contribution to Cyc8-Tup1 repression of natural promoters.

Our suggestion that Pol II holoenzyme plays a minor role in Cyc8-Tup1 repression is consistent with the observation that Cyc8-Tup1 blocks the association of TATA-binding protein (TBP) with natural promoters in vivo (KURAS and STRUHL 1999 ). As numerous biochemical experiments indicate that TBP is required for the association of the remainder of the Pol II machinery, this observation suggests that Cyc8-Tup1 repression occurs under conditions where the entire Pol II machinery is not associated with promoters. Thus, we disfavor models in which Cyc8-Tup1 interacts with holoenzyme components associated at the promoter and blocks transcriptional activity at a later step such as phosphorylation of the C-terminal tail of Pol II. Indeed, blocking phosphorylation of the Pol II tail by inactivating the Kin28 kinase subunit of TFIIH does not affect TBP occupancy, even though it eliminates transcription (KURAS and STRUHL 1999 ). More generally, it is difficult to explain how direct interaction of Cyc8-Tup1 with targets in the Pol II machinery makes a major contribution to repression given that Cyc8-Tup1 blocks association of the machinery with promoters.

If the Pol II holoenzyme plays a minor role in repression of natural promoters by Cyc8-Tup1, what is the predominant mechanism? One possibility is that Cyc8-Tup1 functions predominantly through a chromatin mechanism and that the current experiments have adequately addressed the issue of redundancy. In this regard, if interactions of Cyc8-Tup1 with histone tails are crucial (EDMONDSON et al. 1996 ), it would be difficult to completely remove all potential "targets" without killing the cell. Alternatively, repression of natural promoters might involve a distinct function of Cyc8-Tup1 that has not been revealed by mutations. For example, a great deal of Cyc8-Tup1 repression of natural promoters might be due simply to steric hindrance. Binding of LexA to operators located between the enhancer and TATA elements can inhibit transcription by a factor of 5–10 (BRENT and PTASHNE 1984 ), and such a blocking effect is likely to be more significant if a large complex such as Cyc8-Tup1 is located at a similar position. The complex, consisting of one molecule of Cyc8 and three to four molecules of Tup1 (VARANASI et al. 1996 ; REDD et al. 1997 ), is ~600 kD, and multiple complexes are likely to be recruited to promoters given the multimeric nature of the DNA-binding proteins.

The effects of the srb mutations vary among the different promoters regulated by Cyc8-Tup1. As shown here and elsewhere (KUCHIN and CARLSON 1998 ), srb mutant strains affect repression of artificial promoters by LexA-Cyc8 and LexA-Tup1 more significantly than repression of natural promoters. Steric interference by Cyc8-Tup1 might account for this observation, particularly because the LexA-binding sites in the artificial promoters are located ~100 bp upstream of the enhancer regions, a position that should minimize potential steric effects. However, we cannot exclude the possibility that LexA-Cyc8 and LexA-Tup1 are partially compromised for repression function such that the effects of the srb mutations are exaggerated. Our results also indicate that repression of SUC2 is more sensitive to the various mutations tested than repression of ANB1, MFA2, and RNR2. Although we do not understand the basis for this observation, the apparent specificity for SUC2 might be related to Sfl1, a protein that binds the SUC2 promoter and associates with the Srbs and other holoenzyme components (SONG and CARLSON 1998 ).

Although the functions of Srbs8–11 appear indistinguishable with respect to repression by Cyc8-Tup1, our results indicate that these proteins are not functionally equivalent in other respects. For example, loss of both Srb8 and Srb10 causes a failure to grow at high temperature and on galactose medium, and these effects are reversed by loss of Srb11. In addition, loss of Srb11 causes a ts phenotype that is reversed by loss of either Srb8 or Srb10. These observations suggest that Srb11 cyclin can negatively regulate the activity of Srb10, its associated kinase, and that Srb8 can both positively regulate the function of Srb10 and also contribute functions independent of the kinase. While we do not understand the molecular or biological bases for the distinct phenotypes and suppression properties conferred by the various srb mutations, it is clear that the Srb8–11 module is more complicated than previously expected. One explanation for these genetic results is that Srbs8–11 might be present in distinct versions of Pol II holoenzyme and/or other complexes lacking Pol II. In this regard, the mammalian NAT and SMCC complexes contain homologues of Srb8 and Srb10 but not the other Srbs associated with mediator, and these complexes can negatively regulate transcription in vitro (SUN et al. 1998 ; GU et al. 1999 ). It is becoming increasingly clear that eukaryotic transcriptional regulatory proteins are often present in multiple complexes, and it is almost certain that this contributes to the complex patterns of gene expression.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Beckman Center B-215, Howard Hughes Medical Institute, Stanford University Medical School, Stanford, CA 94305.

	ACKNOWLEDGMENTS

We thank David Chao, Christoph Hengartner, and Rick Young for providing the initial srb mutant strains and DNAs as well as their assistance during the initial stages of this work, Michael Grunstein for providing the histone strains, and Fred Winston and Steve Buratowski for their advice during the project. We also thank Susanna Chou, Lisete Fernandes, Ada Garcia, and Irene Wu for their discussions and critical reading of the manuscript. This work was supported by a predoctoral fellowship to M.L. from the Howard Hughes Medical Institute and by a research grant to K.S. from the National Institutes of Health (GM 53720).

Manuscript received February 28, 2000; Accepted for publication April 10, 2000.

	LITERATURE CITED

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