Multiple Negative Regulators Restrict Recruitment of the SWI/SNF Chromatin Remodeler to the HO Promoter in Saccharomyces cerevisiae.

Activation of the Saccharomyces cerevisiae HO promoter is highly regulated, requiring the ordered recruitment of activators and coactivators and allowing production of only a few transcripts in mother cells within a short cell cycle window. We conducted genetic screens to identify the negative regulators of HO expression necessary to limit HO transcription. Known repressors of HO (Ash1 and Rpd3) were identified, as well as several additional chromatin-associated factors including the Hda1 histone deacetylase, the Isw2 chromatin remodeler, and the corepressor Tup1 We also identified clusters of HO promoter mutations that suggested roles for the Dot6/Tod6 (PAC site) and Ume6 repression pathways. We used ChIP assays with synchronized cells to validate the involvement of these factors and map the association of Ash1, Dot6, and Ume6 with the HO promoter to a brief window in the cell cycle between binding of the initial activating transcription factor and initiation of transcription. We found that Ash1 and Ume6 each recruit the Rpd3 histone deacetylase to HO, and their effects are additive. In contrast, Rpd3 was not recruited significantly to the PAC site, suggesting this site has a distinct mechanism for repression. Increases in HO expression and SWI/SNF recruitment were all additive upon loss of Ash1, Ume6, and PAC site factors, indicating the convergence of independent pathways for repression. Our results demonstrate that multiple protein complexes are important for limiting the spread of SWI/SNF-mediated nucleosome eviction across the HO promoter, suggesting that regulation requires a delicate balance of activities that promote and repress transcription.

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

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.