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

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.

Ash1 and Tup1 Dependent Repression of theSaccharomyces cerevisiae HOpromoter Requires Activator-Dependent Nucleosome Eviction

“…The ordered recruitment of transcription factors and coactivators required for HO activation has previously been examined extensively by ChIP analysis in cells with a GAL::CDC20 allele that can be arrested at G2/M and then released to allow synchronous progression through the cell cycle [7,21,48]. Three repressive DNA-binding factors, Ash1, Dot6, and Ume6, bind to the promoter after initial association of the Swi5 transcription factor but before HO expression [27]. We examined Tup1 binding using GAL::CDC20 synchronization and found that, as expected, Tup1 associated with the HO promoter at the same time as Ash1, 25 min after the cells were released from the G2/M arrest ( Fig 1D).…”

“…Null alleles of tup1 show delayed progression of cells through G1 and therefore are not useful for monitoring the effect on HO expression in late G1 [27]. For these analyses, we therefore used the tup1(H575Y) hypomorph that does not demonstrate a cell cycle delay.…”

“…The second protein, Ume6, was originally identified as a meiotic regulator, and represses transcription of many genes [35]. It binds to a single site within the HO promoter in a nucleosomal linker between URS1 and URS2 [27].…”

“…Other negative regulators identified in genetic screens for inappropriate transcriptional activation [27] may antagonize the SWI/SNF complex at the HO promoter. The Isw2 ATP-dependent chromatin remodeler promotes the movement of nucleosomes into NDRs and could play a role in opposing the nucleosomal eviction caused by SWI/SNF [36].…”

“…Ume6 is known to recruit both Rpd3 and Isw2 to promoters and could be doing so at HO [37,38]. The Tup1 corepressor protein was also identified as a negative regulator of HO expression activation [27]. Tup1, usually found in complex with Cyc8 in a 4:1 ratio, is recruited to many promoters in yeast by a variety of sequence-specific DNA-binding proteins, and has been suggested to reduce expression by masking the activation domain of its recruiting protein, inhibiting its interaction with SWI/SNF [39][40][41][42].…”