Repression by SSN 6-TUP 1 is directed by MIG 1 , a repressor / activator protein ( transcription / yeast / zinc-finger protein / glucose repression )

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). LexATUP1 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 SSN6The 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. §1734 solely to indicate this fact. TUP1 complex to aand haploid-specific genes, respectively (8). Other candidate DNA-binding proteins include MIG1 (14) and ROX1 (15), which may recruit SSN6-TUP1 to glucose-repressible and hypoxic genes, respectively. Here we test this model by examining the role of MIG1 in repression. MIG1 is a Cys2His2 zinc-finger protein related to the mammalian EGR1/Zif268 and WT1 (Wilms tumor) proteins (14). MIG1 binds to the promoters of several glucose-repressible genes, including SUC2, GAL4, and GAL1, and mutation of MIG1 or its binding sites partially relieves glucose repression (14, 16-18). Genetic evidence suggests that MIG1 functions with SSN6-TUP1. migl, ssn6, and tupl mutations all suppress defects caused by loss of SNF1, a protein kinase required for release from glucose repression, and migl and ssn6 show no synergistic effects on repression, suggesting that MIG1 acts in the same pathway as SSN6 (7, 19). For SUC2, however, ssn6 and tupl relieve repression much more effectively than migl, indicating that repression by SSN6-TUP1 also involves MIG1independent mechanisms (19). Thus, genetic evidence supports, but does not prove, the model that MIG1 recruits SSN6-TUP1 to glucose-repressible promoters. To test its function as a transcriptional repressor, we fused MIG1 to the LexA DNA-binding domain so that binding could be directed to target genes containing lexA operators. We show that LexA-MIG1 represses target gene transcription and that repression requires SSN6 and TUP1. These studies also revealed an unexpected transcriptional activation capability for MIG1 when SSN6 and TUP1 are absent. Finally, we provide evidence for differential phosphorylation of MIG1 in response to glucose availability. MATERIALS AND METHODS Strains and Genetic Methods. S. cerevisiae MCY829 (MATa his3 lys2 ura3), MCY1974 (MA Ta ssn6-A9 ade2 his3 lys2 trpl ura3), MCY2437 (MATa tupl-A1::TRPl his3 lys2 ura3 trpl LEU2::GALl-lacZ? gal80? ), and MCY3507 (MA Ta pep4::URA3 his3) are derivatives of S288C. MCY3565 is MCY1974 with tupl-Al::TRPl. ssn6-A9 and tupl-Al::TRPJ are null alleles (5, 7). CTY10.5D (MATa ura3::1exAop-lacZ ade2 trpl leu2 his3 gal4 gal80) was constructed by R. Sternglanz (State University of New York, Stony Brook). MCY3567 (MATa tupl--Al::TRPJ ura3::1exAop-lacZ his3 leu2 ade2 gal4? gal80? ) is a segregant from the cross MCY2437 x CTY10.5D. Standard genetic methods were followed (20). Escherichia coli XL-1 Blue was used as host for plasmids. p-Galactosidase Assays. Freshly transformed colonies were grown to logarithmic phase in SC medium (20) containing the indicated carbon source and lacking appropriate supplements to maintain selection for plasmids. 13-Galactosidase activity was assayed (20) in permeabilized cells or in protein extracts (21). Abbreviations: UAS, upstream activation sequence; GAD, GAL4 activation domain.