The SSN6-TUP1 protein complex represses transcription of diversely regulated genes in the yeast Saccharomyces cerevisiae. Here we present evidence that MIG1, a zinc-finger protein in the EGR1/Zif268 family, recruits SSN6-TUP1 to glucose-repressed promoters. DNA-bound LexA-MIG1 represses transcription of a target gene in glucose-grown cells, and repression requires SSN6 and TUP1. We also show that MIGI and SSN6 fusion proteins interact in the two-hybrid system. Unexpectedly, we found that LexA-MIG1 activates transcription strongly in an ssn6 mutant and weakly in a tupi mutant. Finally, LexA-MIG1 does not repress transcription in glucose-deprived cells, and MIG1 is differentially phosphorylated in response to glucose availability. We suggest a role for phosphorylation in regulating repression.Transcriptional repression is an important regulatory mechanism in eukaryotes. Repressors have been shown to inhibit transcription by interfering with various steps in the transcriptional prdcess. Some repressors block the function of specific activators, whereas others interfere with the transcriptional machinery (for review, see ref. 1).In the yeast Saccharomyces cerevisiae, the SSN6 (CYC8)-TUP1 protein complex represses transcription of genes regulated by glucose, cell type, oxygen, DNA damage, and other signals. Mutations in SSN6 and TUP1 relieve repression of these genes and also display diverse phenotypes such as clumpiness, temperature-sensitive growth, and defects in sporulation and plasmid maintenance (for reviews, see refs. 2 and 3). These shared phenotypes suggested that SSN6 and TUP1 function together, and biochemical studies showed that the two proteins are associated in a complex (4), herein called SSN6-TUP1. SSN6 and TUP1 contain essential tetratricopeptide (TPR) and f3-transducin (WD40) repeats, respectively (5-7).When bound to DNA, LexA-SSN6 represses transcription of a target gene in a TUPl-dependent manner (8). LexA-TUP1 similarly represses target gene expression but does not require SSN6 (9) and may, therefore, directly mediate repression by the complex. The mechanism of repression is not yet understood. SSN6-TUP1 represses transcription by RNA polymerases I and II, but not RNA polymerase III, suggesting that SSN6-TUP1 interacts with a component common to the RNA polymerase I and II transcription complexes (10). Studies of repression in vitro also point to interaction with the general transcriptional machinery (11). Other evidence suggests that repression by SSN6-TUP1 involves positioned nucleosomes that occlude promoter sequences (12, 13).Repression by SSN6-TUP1 is directed to distinctly regulated genes, yet neither SSN6 nor TUP1 appears to bind DNA (5, 7). It has been proposed that specific DNA-binding proteins recruit the SSN6-TUP1 complex to different promoters (8).Evidence suggests that a2-MCM1 and al-a2 target the SSN6-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1...

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucoserepressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.In Saccharomyces cerevisiae the Ssn6 (Cyc8)-Tup1 complex represses transcription of genes regulated by glucose, cell type, oxygen, DNA damage, and other signals (27,34,45,46,48,52,54,56,57,61). Ssn6-Tup1 is recruited to these promoters by specific DNA-binding proteins, including ␣2-Mcm1, a1-␣2, Mig1, Mig2, Rox1, and Rgt1 (1,27,31,39,47,51), and mediates repression by interacting with chromatin (43) and/or the general transcriptional machinery (20,21,41). In this work we have focused on the role of Mig1 in regulating repression by Ssn6-Tup1 in response to the glucose signal.Mig1 is a Cys 2 -His 2 zinc finger protein (36) that binds to the promoters of SUC, GAL, MAL, and other glucose-repressible genes; mutation of Mig1 or its binding sites partially relieves glucose repression (15,17,22,25,35,36,44,53,55). A LexAMig1 fusion protein represses transcription of a CYC1-lacZ reporter containing lexA operators. Such repression requires Ssn6-Tup1 and occurs only in glucose-grown cells (47, 51). Mig1 is differentially phosphorylated in response to glucose availability (11, 47), and the localization of Mig1 to the nucleus requires glucose (11). In contrast, no difference in modification was detected for Ssn6 or Tup1 (46, 57), and Ssn6 resides in the nucleus regardless of glucose availability (46). These findings strongly suggest that the recruitment of Ssn6-Tup1 to a promoter by Mig1 is regulated by glucose. However, it remains possible that other mechanisms also contribute to regulation of repression by the Mig1-Ssn6-Tup1 complex. Here we present evidence that LexA-Mig1 confers glucose-regulated repression to a promoter that is not otherwise glucose repressed, thereby excluding any requirement for other promoter-bound glucoseregulated factors. We also show that repression by LexA-Ssn6 is not glucose regulated, indica...

The SNF2, SNF5, and SNF6 genes of Sac- (1)(2)(3)(4). Their similar mutant phenotypes suggest that SNF2, SNF5, and SNF6 have related functions. Further genetic evidence for this view is that mutations in these genes show similar interactions with the suppressor mutations ssn6 and spt6 (1,3,6). Previous evidence suggests that SNF5 encodes a transcriptional activator (4). The predicted SNF5 protein has extremely glutamine-and proline-rich regions and an acidic region, which are characteristic of activators (7-10), and the SNF5 product is located in the nucleus (4). Using the method of Brent and Ptashne (11), we showed that a LexA-SNF5 fusion protein, when bound to a lexA operator via the LexA DNA-binding domain, activates expression of a nearby promoter (4). Fusions of LexA to various authentic transcriptional activators function in this assay, whereas fusions to the MATa2 product or bacteriophage 434 repressor do not (10)(11)(12)(13).What is the functional relationship among the SNF2, SNF5, and SNF6 proteins? One model is that they function sequentially in a cascade of events. Another is that the three proteins are physically associated and function together as a unit. A third possibility is that two of the proteins function together, and the third protein either affects or is affected by the other two. Previous studies showed that none of these SNF genes affects expression of the others, nor does increased dosage of one compensate for a mutation in another (2, 3).We examine here the functional relationship of the SNF2, SNF5, and SNF6 proteins in transcriptional activation. We tested activation by DNA-bound LexA-SNF5 fusion protein in snf2 and snff6 mutants, thereby showing that LexA-SNF5 function requires the SNF2 and SNF6 proteins. We report here the sequence of the SNF2 genet and the nuclear localization of its product. A LexA-SNF2 fusion protein was constructed and found to activate gene expression, dependent on SNF5 and SNF6. Finally, we examined the effects of an spt6 mutation, a suppressor of snf2, snfS, and snf6, on transcriptional activation.

The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose-and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MFal and BARI at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNFS-0-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator. Many transcription factors in Saccharomyces cerevisiaehave been identified by a combination of genetic and biochemical approaches. Some affect a narrow subset of genes, and others have a very broad range of action; for example, GAL4 activates transcription of genes controlled by a specific regulatory mechanism, whereas RAP1/GRF1/TUF affects transcription of a broad variety of genes (for a review, see reference 53).The SNF5 gene of S. cerevisiae was originally identified as a gene required for expression of SUC2 (encoding invertase) and other glucose-repressible genes (38). The snf ¶ mutants showed growth defects on raffinose, galactose, and glycerol, and homozygous diploids failed to sporulate. The defect in SUC2 expression lies at the RNA level (1). Further studies revealed that SNF5 is required not only for expression of glucose-repressible genes but also for derepression of acid phosphatase in response to phosphate starvation (1). Mutations in SNF5 also cause increased expression of protease B in stationary-phase cells (34). This evidence argues against a role for SNF5 in a specialized signal transduction pathway and suggests rather that SNF5 has a more general function in transcription.Genetic evidence also supports the notion that SNF5 functions in the transcriptional process. Mutations in SPT61 SSN20, an essential gene that appears to affect transcription (9,12,39,54,61), restore regulated invertase expression in snjS mutants (40). The spt6lssn20 suppressors restore derepression of SUC2 mRNA in mutants lacking both cis-and trans-acting elements that are normally required (39). The suppression of snjS defects by spt6 mutations is consistent with an involvement of SNF5 in transcription.SNF5 was one of six SNF genes identified in a search for mutants (38). Genetic evidence suggests that SNF5 is a member of a group of three functionally related genes. The SNF2, SNF5, and SNF6 genes share similarly pleiotropic mutant phenotypes and show similar patterns of genetic interactions with two extragenic suppressors, spt6lssn20 and * Corresponding author.ssn6 (14,38,40). The properties o...

Concomitant administration of boceprevir with PI/r resulted in reduced exposures of PI and boceprevir. These drug-drug interactions may reduce the effectiveness of PI/r and/or boceprevir when coadministered.