Ssn6-Tup1 interacts with class I histone deacetylases required for repression

Abstract

Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions.

Figure 1

Ssn6–Tup1-mediated repression is abolished in rpd3 hos1 hos2 cells. (A) Endogenous MFA2 RNA levels in the indicated wild-type (WT) or mutant a and α strains were assayed by S1 nuclease protection. A representative gel and averages of MFA2 RNA levels normalized to ACT1 RNA levels from three independent experiments are shown. Fold derepression values reflect the normalized MFA2 signals relative to those observed in wild-type α cells. (B) Endogenous SUC2 mRNA levels were assayed by S1 nuclease protection and normalized to ACT1 RNA levels as in (A). Fold derepression values reflect the amount of SUC2 signal relative to that observed in wild-type cells under fully repressing conditions (lane 2). Values shown are averaged from three independent experiments.

Figure 2

Acetylation of histones H3 and H4 is increased at Ssn6–Tup1 regulated promoters in rpd3 hos1 hos2 cells. Chromatin immunoprecipitations using antibodies specific for H3, AcH3 9,18, or AcH4 (as indicated) were carried out with chromatin extracted from wild-type or rpd3 hos1 hos2 cells. Immunoprecipitated DNA was applied to slot blots and probed for (A) MFA2- (B) STE6-, or (C) SUC2-specific promoter sequences. Signals were quantitated by PhosphorImage analysis and normalized to the amounts of promoter sequences detected in the chromatin input. The ratio of these normalized values is presented in the right-hand column.

Figure 3

Bulk histone acetylation in wild-type or HDAC mutant cells. Yeast histones isolated from wild-type or various HDAC mutant strains (as indicated) were resolved by acid-urea gel electrophoresis. Immunoblots were performed using antibodies specific for acetylated isoforms of H3 (A) or H4 (B). Two separate blots are shown in each panel. Lanes 1–5 in A and B are from a single immunoblot of a sister to the gel shown in C, probed with the different antibodies. Equal amounts of protein were loaded into each lane, as confirmed by Coomassie blue staining (C).

Figure 4

Ssn6–Tup1 interacts with HDACs in vivo and in vitro. (A) Anti-HA immunoprecipitations were performed on cell extracts from yeast strains carrying the indicated expression plasmids and the presence of tagged fusion proteins determined by Western blot. Shown are cell extracts and immunoprecipitated fractions. The upper panel was probed with the anti-HA antibody and the lower panel with the anti-LexA antibody. LexA–Ssn6 coimmunoprecipitates with HA-Gal4–Rpd3 and HA-Gal4–Hos2. In the far right panel, the experiment was repeated in the presence of ethidium bromide (50 μg/mL) to demonstrate that the interactions observed are not mediated by DNA. (B) Purified recombinant GST–Ssn6 (amino acids 1–398) or GST alone was incubated with extracts from yeast expressing HA-tagged Hos2 from the native HOS2 locus. The top panel shows an anti-HA antibody immunoblot. HA-Hos2 interacts with GST–Ssn6, but not with GST alone. The lower panel shows a Coomassie blue stain of extract (30% of input) and the recombinant proteins. (C) Extracts from yeast expressing HA-tagged Rpd3 from the native RPD3 locus were immunoprecipitated with anti-Tup1 antibodies. The upper panel is an anti-HA immunoblot showing that HARpd3 coimmunoprecipitates with anti-Tup1 antisera (Tup1) but not with preimmune sera (PRE). The middle and lower panels show parallel blots probed with anti-Tup1 (middle) or anti-Ssn6 (lower) antisera.