Abstract
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.