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. 2002 Feb;22(3):693-703.
doi: 10.1128/MCB.22.3.693-703.2002.

Histone-dependent association of Tup1-Ssn6 with repressed genes in vivo

Judith K Davie  1 , Robert J Trumbly, Sharon Y R Dent
Affiliations

Histone-dependent association of Tup1-Ssn6 with repressed genes in vivo

Judith K Davie et al. Mol Cell Biol. 2002 Feb.

Abstract

The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces cerevisiae and serves as a model for corepressor functions in many organisms. Tup1-Ssn6 does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA binding factors. Full repression by Tup1-Ssn6 appears to require interactions with both the histone tails and components of the general transcription machinery, although the relative contribution of these two pathways is not clear. Here, we map Tup1 locations on two classes of Tup1-Ssn6-regulated genes in vivo via chromatin immunoprecipitations. Distinct profiles of Tup1 are observed on a cell-specific genes and DNA damage-inducible genes, suggesting that alternate repressive architectures may be created on different classes of repressed genes. In both cases, decreases in acetylation of histone H3 colocalize with Tup1. Strikingly, although loss of the Srb10 mediator protein had no effect on Tup1 localization, both histone tail mutations and histone deacetylase mutations crippled the association of Tup1 with target loci. Together with previous findings that Tup1-Ssn6 physically associates with histone deacetylase activities, these results indicate that the repressor complex alters histone modification states to facilitate interactions with histones and that these interactions are required to maintain a stable repressive state.

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Figures

FIG. 1.
FIG. 1.
Tup1 spreads into the coding region of the a cell-specific genes STE6 and STE2. Chromatin immunoprecipitations (IP) were preformed on cell extracts from a and α HA-TUP1 strains by using an antibody against HA. The location of the PCR primer sets is given in kilobases with the starting ATG as a reference (0.0 kb). These positions are also shown on gene diagrams for STE6 (A) and STE2 (B). All PCR primer sets were designed to generate ∼100-bp products. At STE6 and STE2, the α2 operator spans positions −212 to −182 and positions −228 to −200, respectively. The bar graph represents the ratio between the signals from α cells (repressed) and a cells (derepressed). For the graph, four independent experiments were averaged and the error bars are shown. Quantitative PCR products from one representative experiment are shown.
FIG. 2.
FIG. 2.
Tup1 is present only near the Crt1 binding site at RNR2 and RNR3. Chromatin immunoprecipitations (IP) were performed on cell extracts from wild-type (WT) and crt1 HA-TUP1 strains by using an antibody against HA. The location of the PCR primer sets is given in kilobases with the starting ATG as a reference (0.0 kb). These positions are also indicated on gene diagrams for RNR2 (A) and RNR3 (B). PCR fragments were ∼100 bp in length. The Crt1 binding sites are X-box-related sequences, located at positions −436 and −363 for RNR2 and −383, −321, and −272 for RNR3 (19). The bar graph shows the ratio between the signals from wild-type cells (repressed) and crt1 cells (derepressed). For the graph, four independent experiments were averaged and the error bars are shown. Quantitative PCR products from one representative experiment are shown.
FIG. 3.
FIG. 3.
Ssn6 is required for Tup1 recruitment to target loci. (A) Quantitative PCR products from chromatin immunoprecipitations (IP) performed on cell extracts from wild-type (WT) and ssn6 HA-TUP1 strains by using an antibody against HA. PCR primer sets correspond to −0.2 kb for STE6 (as in Fig. 1A) and −0.4 kb for RNR2 (as in Fig. 2A). (B) Bar graph representing the percentage of input DNA immunoprecipitated from wild-type and ssn6 extracts. The wild-type value was set at 1 for each gene to allow comparison between genes. The standard deviation of the SSN6+/ssn6 ratios was less than 0.01. Note that for both classes of genes, the reductions in immunoprecipitation efficiency were almost identical.
FIG. 4.
FIG. 4.
Srb10 is not required for Tup1 recruitment to target loci. Chromatin immunoprecipitations (IP) were performed on cell extracts from wild-type (WT) and srb10 strains containing HA-TUP1 by using an antibody against HA. Primer sets spanning both STE6 and RNR2 were used. The location of the PCR primer sets is as in Fig. 1A for STE6 and Fig. 2B for RNR2. Quantitative PCR products from one representative experiment are shown for position −0.2 kb of STE6 (A) and position −0.4 kb of RNR2 (C). Data for all primer sets are graphed for STE6 (B) and RNR2 (D). For STE6, the bar graph represents the ratio between the signals from α cells and a cells as well as the ratio between the signals from srb10 α cells and srb10 a cells. For RNR2, the bar graph represents the ratio between the signals from WT cells and crt1 cells as well as the ratio between signals from srb10 cells and crt1 cells. For the graph, three independent experiments were averaged and the error bars are shown.
FIG. 5.
FIG. 5.
Histone mutations severely cripple the association of Tup1 with target loci. (A) Quantitative PCR products from chromatin immunoprecipitations (IP) preformed on cell extracts from wild-type (WT) (MSY590) and H3 Δ1–28 H4 K12QK16Q (MSY 577) strains by using an antibody against Tup1. PCR primer sets correspond to −0.2 kb for STE6 (as in Fig. 1A), −0.3 kb for STE2 (as in Fig. 1B), −0.4 kb for RNR2 (as in Fig. 2A), and −0.4 kb for RNR3 (as in Fig. 2B). (B) Bar graph representing the percentage of input DNA immunoprecipitated from MSY590 and MSY577 extracts. The wild-type value was set at 1 to allow comparison between genes. For the graph, four independent experiments were averaged. The standard deviation of the MSY590/MSY577 ratios was less than 0.01. Note that for each gene, the reductions in immunoprecipitation efficiency were almost identical.
FIG. 6.
FIG. 6.
Decreases in H3 acetylation colocalize with Tup1 at STE6. Chromatin immunoprecipitations (IP) using antibodies against acetylated H3 (Ac H3) (A) and unacetylated H3 (unAc H3) (B) were performed on cell extracts from a, α, and α tup1 strains. The location of the primer sets is as in Fig. 1A for STE6. The data are presented as signal normalized to both PCR amplification of ACT1 from each sample (to control for sample variation) and input DNA levels (to allow comparison of relative acetylation levels along the gene). Three experiments were averaged for the graphs, and error bars are shown. Quantitative PCR products from one representative experiment are shown.
FIG. 7.
FIG. 7.
Decreases in H3 acetylation colocalize with Tup1 at RNR2. Chromatin immunoprecipitations (IP) using antibodies against acetylated H3 (Ac H3) (A) and unacetylated H3 (unAc H3) (B) were performed on cell extracts from wild-type (WT) and tup1 strains. The location of the primer sets is as in Fig. 2A for RNR2. The data are presented as signal normalized to both PCR amplification of ACT1 from each sample (to control for sample variation) and input DNA levels (to allow comparison of relative acetylation levels along the gene). Three experiments were averaged for the graphs, and error bars are shown. Quantitative PCR products from one representative experiment are shown.
FIG. 8.
FIG. 8.
Mutations in histone deacetylases severely cripple the association of Tup1 with target loci. (A) Quantitative PCR products from chromatin immunoprecipitations (IP) performed on cell extracts from wild-type (WT) (DY151) and rpd3 hos1 hos2 (DY4565) strains using an antibody against Tup1. PCR primer sets correspond to −0.2 kb for STE6 (as in Fig. 1A), −0.3 kb for STE2 (as in Fig. 1B), −0.4 kb for RNR2 (as in Fig. 2A), and −0.4 kb for RNR3 (as in Fig. 2B). (B) Bar graph representing the percentage of input DNA immunoprecipitated from DY151 and DY4565 extracts. The wild-type value was set at 1 to allow comparison between genes. For the graph, four independent experiments were averaged. The standard deviation between the DY151/DY4565 ratios was less than 0.01. (C) Quantitative PCR products from chromatin immunoprecipitations performed on cell extracts from both wild-type cells (DY151) and rpd3 hos1 hos2 (DY4565) cells containing a plasmid expressing myc-Crt1 (PMH190), using an antibody against the c-myc epitope. Cell extract from DY151 without PMH190 was also used as a control. PCR primer sets correspond to −0.4 kb for RNR2 (as in Fig. 2A) and −0.4 kb for RNR3 (as in Fig. 2B).
FIG. 9.
FIG. 9.
Reciprocal interactions between Tup1-Ssn6 and histones. Upon recruitment of Tup1-Ssn6 by sequence-specific repressor proteins (black diamond), associated histone deacetylase (HDAC) activities reduce the acetylation (Ac) of histones in neighboring nucleosomes, facilitating interactions with Tup1. These interactions convert the active chromatin (dashed nucleosomes) to a repressive structure (solid nucleosomes). Moreover, Tup1-histone interactions are self-reinforcing in that they stabilize the association of the corepressor complex with the target promoter and help to maintain the histones in an underacetylated state.
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