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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 89, 365±371, May 2, 1997, Copyright 1997 by Cell Press Repression by Ume6 Involves Recruitment of a Complex Containing Sin3 Corepressor and Rpd3 Histone Deacetylase to Target Promoters David Kadosh and Kevin Struhl* initially inferred to be a corepressor from the observation Department of Biological Chemistry that a LexA±Sin3 fusion protein can repress transcription and Molecular Pharmacology when brought to a heterologous promoter (Wang and Harvard Medical School Stillman, 1993). Furthermore, a Sin3-related protein from Boston, Massachusetts 02115 mouse functions as a corepressor for repression by Mad and Mxi1, proteins that bind DNA as heterodimers with Max (Ayer et al., 1995; Schreiber-Agus et al., 1995). How- Summary ever, yeast DNA-binding proteins that specifically re- quire Sin3 and Rpd3 for repression have yet to be iden- Sin3 and Rpd3 negatively regulate a diverse set of tified. yeast genes. A mouse Sin3-related protein is a tran- Rpd3 is 60% identical in sequence to a human histone scriptional corepressor, and a human Rpd3 homolog deacetylase (Taunton et al., 1996), and it is likely to is a histone deacetylase. Here, we show that Sin3 and possess this activity because rpd3 mutant strains show Rpd3 are specifically required for transcriptional re- increased acetylation of lysines 5 and 12 of histone H4 pression by Ume6, a DNA-binding protein that regu- (Rundlett et al., 1996). Rpd3 is also homologous to a lates genesinvolved in meiosis. A short region of Ume6 mouse protein that functions as a corepressor for the is sufficient to repress transcription, and this repres- DNA-binding protein YY1 (Yang et al., 1996a) and a Dro- sion domain mediates a two-hybrid and physical inter- sophila protein that affects position-effect variegation action with Sin3. Coimmunoprecipitation and two- (De Rubertis et al., 1996). As deacetylated histones are hybrid experiments indicate that Sin3 and Rpd3 are generally associated with transcriptionally inactive re- associated in a complex distinct from TFIID and Pol II gions (Roth and Allis, 1996; Wolffe and Pruss, 1996), holoenzyme. Rpd3 is specifically required for repres- the histone deacetylase activity of Rpd3 is likely to be sion by Sin3, and artificial recruitment of Rpd3 results relevant for its repression function. However, the molec- in repression. These results suggest that repression ular relationshipbetween Sin3 and Rpd3 and the mecha- by Ume6 involves recruitment of a Sin3±Rpd3 complex nism by which Rpd3 mediates gene-specific repression and targeted histone deacetylation. are unknown. Here, we identify a yeast DNA-binding protein, Ume6, Introduction that directs transcriptional repression in a manner de- pendent upon Sin3 and Rpd3. We show that a short Understanding of the relationship between chromatin region of Ume6 is sufficient to recruit the Sin3 corepres- structure and gene expression in eukaryotic organisms sor to promoters, that Sin3 and Rpd3 are physically has been dramatically enhanced by the identification of associated, that Rpd3 is specifically required for repres- nucleosome-modifying activities such as the Swi/Snf sion by the Sin3 corepressor, and that artificial targeting and related complexes (Peterson, 1996), histone ace- of Rpd3 to promoters inhibits transcription. These ob- tylases Gcn5 (Brownell et al., 1996), P/CAF (Yang et servations strongly suggest that repression by Ume6 al., 1996b), p300/CBP (Bannister and Kouzarides, 1996; occurs by targeted histone deacetylation. Ogryzko et al., 1996), TAF130/250 (Mizzen et al., 1996), and Rpd3 histone deacetylase (Taunton et al., 1996). Results Yeast cells that lack Swi/Snf, Gcn5, or Rpd3 are viable but have selective effects on gene expression. An attrac- URS1 Elements Are Sufficient to Direct tive hypothesis is that such selective transcriptional ef- Sin3- and Rpd3-Dependent Repression fects are due to the targeting of chromatin-modifying IME2, a key meiosis regulator, and INO1, a gene involved activities to specific promoters. However, it is also pos- in phospholipid biosynthesis, are negatively regulated sible that these nucleosome-modifying activities have by Sin3 (Bowdish and Mitchell, 1993; Slekar and Henry, untargeted, genome-wide effects that selectively affect 1995), and their promoter regions both contain URS1, a limited set of promoters with particular configurations a GC-rich upstream repression sequence found at a of promoter elements (location and quality) and inherent number of yeast promoters. To determine whether URS1 chromatin structures (nucleosome positioning, stability, is sufficient to direct repression in a Sin3- and Rpd3- or density). dependent manner, we inserted fragments containing In this paper, we define a novel form of transcriptional URS1 elements from the INO1 and IME2 promoters up- repression that involves the yeast Sin3 and Rpd3 pro- stream of the intact CYC1 promoter and LacZ structural teins. Sin3 and Rpd3 negatively regulate a diverse set gene, and analyzed transcription in wild-type, sin3, and of genes including those involved in meiosis, cell-type rpd3 strains (Figure 1A). In both cases, the URS1 frag- specificity, potassium transport, phosphate metabo- ments direct significant levels of repression (9- to 13- lism, methionine biosynthesis, and phospholipid metab- fold) in a wild-type strain, whereas repression is nearly olism (Vidal and Gaber, 1991; McKenzie et al., 1993; abolished in a sin3 deletion strain and is significantly Jackson and Lopes, 1996; Stillman et al., 1994). Sin3 was reduced in a rpd3 deletion strain. Thus, URS1 elements are sufficient to direct Sin3- and Rpd3-dependent re- *To whom correspondence should be addressed. pression from a heterologous promoter. Cell 366 Table 1. Localization of the Ume6 Repression Domain Promoter Fold LexA Hybrid Protein 4 LexAop 2LexAop Repression Ume6 (1±836) 7 49 7.0 Ume6 (118±836) 9 52 5.8 Ume6 (160±836) 7 49 7.0 Ume6 (508±836) 7 59 8.4 Ume6 (653±836) 72 65 0.9 Ume6 (508±802) 54 79 1.5 Ume6 (508±744) 41 82 2.0 Ume6 (508±651) 50 78 1.6 Ume6 (508±594) 11 73 6.6 Ume6 (1±509, 596±836) 31 56 1.8 LexA 54 69 1.3 b-galactosidase activities of wild-type strains containing the indi- cated promoters and expressing the indicated proteins (1±836 aa 5 full length). Fold repression represents the ratio of b-galactosidase activities in strains containing plasmids that either lack or contain four LexA operators upstream of the CYC1 promoter fused to the LacZ structural gene. function as a meiosis-specific transcriptional activator. These results suggest that recruitment of Ume6 to the promoter is sufficient to direct repression in a Sin3- and Rpd3-dependent manner. Figure 1. Repression by URS1 Elements and LexA±Ume6 Requires Sin3 and Rpd3 (A) b-galactosidase activities of wild-type and the indicated deletion A Short Domain of Ume6 That Mediates strains harboring CYC1 promoter derivatives that do (arrows) or do Transcriptional Repression not contain one copy of the INO1 oligonucleotide (middle) or two The repression function of Ume6 was localized by a copies of the IME2 oligonucleotide (right). deletion analysis of LexA±Ume6 (Table 1). Deletions that (B) b-galactosidase activities of wild-type or the indicated deletion removed as many as 507 N-terminal residues behave strains expressing LexA±Ume6, LexA±Ume6 , or LexA. Fold- 508±594 indistinguishably from the full-length protein, whereas repression represents the ratio of b-galactosidase activities in strains containing plasmids that either lack (shaded bars) or contain removal of 652 N-terminal residues abolishes repres- (open bars) 4 LexA operators upstream of the CYC1 promoter. sion. Three C-terminal deletions of LexA±Ume6508±836 confer little or no repression, but the most extensively deleted derivative (LexA±Ume6508±594) directs repression Ume6, a DNA-Binding Protein, Directs Repression at wild-type levels. Conversely, a LexA±Ume6 derivative Mediated by Sin3 and Rpd3 lacking residues 510±595 is severely compromised for The above results indicate that a URS1-binding pro- repression. Thus, an 87±amino acid region (residues tein(s) can repress transcription in a manner dependent 508±594) is necessary and sufficient to mediate the re- on Sin3 and Rpd3. Ume6 was a good candidate for pression function of Ume6; hence, it defines a minimal such a DNA-binding protein, because it is required for repression domain. This minimal repression domain repression via URS1 in the IME2, CAR1, and INO1 pro- shows no significant similarity to the repression domain moters (Park et al., 1992; Bowdish and Mitchell, 1993; of Mad. As expected, repression mediated by the mini- Jackson and Lopes, 1996), and it binds the URS1 site mal Ume6 domain is nearly abolished in a sin3 deletion in the promoter of SPO13, another gene subject to Sin3 strain and is significantly reduced in an rpd3 deletion control (Strich et al., 1994). In accord with these results, strain (Figure 1B), even though the protein is stably ex- repression directed by the isolated URS1 elements is pressed in these strains (data not shown). Thus, Sin3 completely abolished in a ume6 deletion strain (Fig- and Rpd3 histone deacetylase are required for the func- ure 1A). tion of the Ume6 repression domain. We therefore tested whethera LexA±Ume6 fusion pro- tein could repress transcription when artificially re- Interaction between Sin3 and Ume6 cruited to a heterologous promoter (Figure 1B). Indeed, The functional relationship between Ume6 and Sin3 sug- LexA±Ume6 represses transcription by a factor of 6, and gested that these proteins might interact with one this repression is nearly abolished in a sin3 deletion another. In support of this hypothesis, we detect a strain and is significantly reduced in an rpd3 deletion two-hybrid interaction between Sin3±VP16 and either background. Although LexA±Ume6 can function as a LexA±Ume6 or (more strongly) LexA±Ume6508±594 (mini- transcriptional activator in cells undergoing meiosis mal repression domain)(Figure 2).

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