SWI/SNF binding to the HO promoter requires histone acetylation and stimulates TATA-binding protein recruitment

Abstract

We use chromatin immunoprecipitation assays to show that the Gcn5 histone acetyltransferase in SAGA is required for SWI/SNF association with the HO promoter and that binding of SWI/SNF and SAGA are interdependent. Previous results showed that SWI/SNF binding to HO was Gcn5 independent, but that work used a strain with a mutation in the Ash1 daughter-specific repressor of HO expression. Here, we show that Ash1 functions as a repressor that inhibits SWI/SNF binding and that Gcn5 is required to overcome Ash1 repression in mother cells to allow HO transcription. Thus, Gcn5 facilitates SWI/SNF binding by antagonizing Ash1. Similarly, a mutation in SIN3, like an ash1 mutation, allows both HO expression and SWI/SNF binding in the absence of Gcn5. Although Ash1 has recently been identified in a Sin3-Rpd3 complex, our genetic analysis shows that Ash1 and Sin3 have distinct functions in regulating HO. Analysis of mutant strains shows that SWI/SNF binding and HO expression are correlated and regulated by histone acetylation. The defect in HO expression caused by a mutant SWI/SNF with a Swi2(E834K) substitution can be partially suppressed by ash1 or spt3 mutation or by a gain-of-function V71E substitution in the TATA-binding protein (TBP). Spt3 inhibits TBP binding at HO, and genetic analysis suggests that Spt3 and TBP(V71E) act in the same pathway, distinct from that of Ash1. We have detected SWI/SNF binding at the HO TATA region, and our results suggest that SWI/SNF, either directly or indirectly, facilitates TBP binding at HO.

FIG. 1.

SWI/SNF binding to HO is restored in the swi5 sin3 mutant. (A) Map of the HO promoter showing positions of URS1, URS2, and TATA. (B) HO is expressed in the swi5 sin3 mutant. RNAs were prepared from strains DY150, DY408, DY5270, DY3498, DY984, DY986, DY2870, and DY3499 and used for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (C) ChIP was performed with an untagged strain (DY150) and with Swi2-Myc strains that were wild type (DY6151), swi5 (DY9395), or swi5 sin3 (DY9391). SWI/SNF binding to either HO URS1 or URS2 was measured by real-time PCR, and the units are arbitrary after normalization to a YDL224c internal control. The error bars show the standard deviations of the ChIP PCRs performed in triplicate. (D) SWI/SNF binding to HO is restored in a gcn5 sin3 strain. ChIP was performed with an untagged strain (DY150) and with Swi2-Myc strains that were wild type (DY6151), gcn5 (DY8738), sin3 (DY9923), or gcn5 sin3 (DY9927). SWI/SNF binding to either HO URS1 or URS2 was measured by real-time PCR, and the units are arbitrary after normalization to a YDL224c internal control. The error bars show the standard deviations of the ChIP PCRs performed in triplicate.

FIG. 2.

Ash1 overcomes the gcn5 requirement for SWI/SNF binding and HO expression. (A) SWI/SNF binding to HO URS1 and URS2 is restored in a gcn5 ash1 mutant. ChIP was performed with an untagged strain (DY150) and with Swi2-Myc strains that were wild type (DY6151), gcn5 (DY8738), ash1 (DY7403), or gcn5 ash1 (DY8736). SWI/SNF binding to HO URS1 and URS2 was measured by real-time PCR, and the units are arbitrary after normalization to a YDL224c internal control. The error bars show the standard deviations of the ChIP PCRs performed in triplicate. (B) SWI/SNF binding to HO URS1 and URS2 during the cell cycle. Swi2-Myc-tagged strains DY6151 (wild type), DY8738 (gcn5), DY7403 (ash1), and DY8736 (gcn5 ash1) were synchronized by α-factor arrest and release, and samples were taken at various times for ChIP. SWI/SNF binding to HO URS1 and URS2 was measured by real-time PCR, and the units are arbitrary after normalization to a YDL224c internal control. The error bars show the standard deviations of the ChIP PCRs performed in triplicate. (C) RNAs were prepared from strains DY150 (wild type), DY5265 (gcn5), DY4394 (ash1), and DY5268 (gcn5 ash1) and used for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels.

FIG. 3.

HO expression in mothers and daughters in a gcn5 ash1 strain. Strains were arrested with α-factor to synchronize in the cell cycle, and samples were collected between 2 and 2.5 hours following release. Fluorescent images (left) and corresponding DIC images (right) are shown. The following strains were used: (A) DY9862 (HO::GFP-NLS-PEST), (B) DY9864 (HO::GFP-NLS-PEST ash1), (C) DY9865 (HO::GFP-NLS-PEST gcn5), and (D) DY9868 (HO::GFP-NLS-PEST gcn5 ash1). The α-factor arrest results in changed cell shape, marking mother cells. This shape change is not as visible in the gcn5 ash1 strain, possibly because the strain arrested poorly with α-factor.

FIG. 4.

Gcn5 catalytic activity is necessary for sustained SWI/SNF binding. (A) sin3 and ash1 are additive in suppressing gcn5. RNAs were prepared from strains DY150 (wild type), DY984 (sin3), DY4394 (ash1), DY6394 (sin3 ash1), DY5265 (gcn5), DY5297 (gcn5 sin3), DY7387 (gcn5 ash1), and DY7385 (gcn5 sin3 ash1) and used for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (B) Gcn5 catalytic activity is necessary for HO expression. RNAs were prepared from strains DY150 (no tag), DY6151 (Swi2-Myc), DY8738 (Swi2-Myc gcn5Δ), and DY9754 (Swi2-Myc gcn5-E173Q) and used for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (C) Gcn5 catalytic activity is necessary for sustained SWI/SNF binding. ChIP was performed with an untagged strain (DY150) and with Swi2-Myc strains that were wild type (DY6151), gcn5Δ (DY8738), and gcn5-E173Q (DY9754). SWI/SNF binding to either HO URS1 or URS2 was measured by real-time PCR, and the units are arbitrary after normalization to a YDL224c internal control. The error bars show the standard deviations of the ChIP PCRs performed in triplicate.

FIG. 5.

SWI/SNF binds to the HO TATA. (A) Strain DY6151 (Swi2-Myc) was synchronized by α-factor arrest and release, and samples were taken at various times for ChIP and mRNA analysis. SWI/SNF binding to the URS1, URS2, or TATA region of the HO promoter was measured by real-time PCR, and the units are arbitrary after normalization to a YDL224c internal control. The error bars show the standard deviations of the ChIP PCRs performed in triplicate. HO mRNA was measured by S1 nuclease protection, quantitated by phosphorimager, and normalized to CMD1 levels (loading control). (B) Map showing relative positions of PCR primers used. Strains DY6151 (Swi2-Myc) and DY150 (untagged) were synchronized by α-factor arrest and release, and samples were taken at 90, 100, and 110 min after release for ChIP. The data shown are for the 100-min time point, but similar results were obtained with the other samples. The samples labeled HindIII were digested with HindIII after the immunoprecipitation step.

FIG. 6.

Genetic suppression shows that SWI/SNF acts at the HO TATA. (A) RNAs were prepared from strains DY150 (wild type), DY9726 (swi2-E834K), DY9846 (sin3), and DY10145 (swi2-E834K sin3) and used for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (B) RNAs were prepared from strains DY150 (wild type), DY4394 (ash1), DY6806 (spt3), DY7131 (ash1 spt3), DY9726 (swi2-E834K), DY9711 (swi2-E834K ash1), DY9709 (swi2-E834K spt3), and DY9715 (swi2-E834K ash1 spt3) and used for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (C) YCp-TRP1 plasmids with either wild-type TBP or the indicated TBP mutants were transformed into DY7242 [SWI2 spt15Δ YCp-URA3-TBP(wild type)] or DY10009 [swi2-E834K spt15Δ YCp-URA3-TBP(wild type)] and then grown on 5-FOA medium to eliminate the YCp-URA3-TBP(wild type) plasmid. Cells with the indicated TBP plasmid as the sole source of TBP in the cell were then grown in selective medium, and the RNAs were isolated for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (D) Strains DY150 (wild type) and DY9726 (swi2-E834K) were each transformed with two plasmids, one with a TRP1 marker and one with a URA3 marker, as follows: lanes 1 and 7, pRS314 vector and pRS316 vector; lanes 2 and 8, YCp-TBP(wild type) and pRS316 vector; lanes 3 and 9, YCp-TBP(V71E) and pRS316 vector; lanes 4 and 10, YCp-TBP(N159K) and pRS316 vector; lanes 5 and 11, YEp-TBP(wild type) and pRS316 vector; and lanes 6 and 12, pRS314 vector and YCp-SWI2. The cells were grown on selective medium to maintain both plasmids, and RNAs were isolated for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (E) Strains DY150 (wild type), DY4394 (ash1), DY6806 (spt3), DY9726 (swi2-E834K), DY9711 (swi2-E834K ash1), and DY9709 (swi2-E834K spt3) were each transformed with either YCp-TRP1-TBP(wild type) or YCp-TRP1-TBP(V71E). The cells were grown on selective medium to maintain the plasmid, and RNAs were isolated for S1 nuclease protection assays to measure HO and CMD1 (internal control) RNA levels. (F) Ribbon diagram of the TBP structure (49, 50), with the V71, N159, and G174 residues highlighted.

FIG. 7.

Proposed model of HO regulation. Green arrows represent stimulation or recruitment, red bars represent inhibition, and gray arrows represent hypothetical interactions. (A) Original model for factor recruitment to HO based on Cosma et al. (25). In mother cells, Swi5 recruits SWI/SNF, which recruits SAGA (with Gcn5), and Gcn5 is required for SBF binding. Ash1 inhibits SWI/SNF binding in daughter cells. (B) Revised model for factor recruitment to HO. Swi5 recruits SWI/SNF and possibly SAGA to HO, and SWI/SNF and SAGA are mutually required for stable binding to the promoter. Swi5 and SWI/SNF are both required for Mediator binding to the URS1 region of the promoter. Ash1 and Sin3/Rpd3 inhibit SWI/SNF binding to HO; the dashed line between Ash1 and Sin3/Rpd3 indicates that, despite the fact that they can be found in the same complex, ash1 and sin3 mutations have additive effects on the regulation of HO. In the URS2 region, Gcn5 is required for SBF binding and SBF is required for Mediator binding. SAGA and Mediator have been shown to stimulate each other's binding at some promoters, and a hypothetical interaction is indicated. (C) Regulation of TBP binding at HO. SWI/SNF and Gcn5 both stimulate TBP binding to HO, and Spt3 and Ash1-Sin3/Rpd3 inhibit TBP binding. A molecular role for Mediator in stimulating HO transcription has not been clearly defined, and it may also stimulate TBP binding.