Biology subjects

Stillman, D. J.

Publications and source records attributed to Stillman, D. J..

4 recordsLinked to original sources

bioRxiv2021

Genetic analysis argues for a coactivator function for the Saccharomyces cerevisiae Tup1 corepressor

The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating type genes. We report here a tup1(S649F) mutant that displays mating irregularities similar to a tup1 null and an -predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 occupancy changes in mutant vs. wild-type in both a and cells. Increased Tup1(S649F) occupancy tended to occur upstream of upregulated genes, whereas locations with decreased occupancy usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including 2. We also found that large changes in mating type-specific gene expression between a and or between mutant and wild-type were not easily explained by the range of Tup1 occupancy levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 occupancy upstream of the a-specific gene MFA2 and the -specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with identification of additional mating related genes upregulated in the tup1(S649F) strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated.

genetics↗

bioRxiv2019

Independent Recruitment of the Mediator Tail Module to the HO Promoter Suggests Mediator Core Limits Coactivator Recruitment in Saccharomyces cerevisiae

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or UAS regions of genes via interactions with transcription factors, and the Kinase domain facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.

genetics↗

bioRxiv2019

Genetic screens identify multiple negative regulators that additively 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.

genetics↗

bioRxiv2018

FACT activity and histone H3-K56 acetylation promote optimal establishment of chromatin architecture independent of ongoing transcription in Saccharomyces cerevisiae

FACT is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone:DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome structure, chromatin integrity, and transcription output. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT in vitro and in vivo as expected, but H3-K56 and FACT mutations caused surprisingly similar changes in transcription of individual genes. Notably, neither the changes in transcript levels nor the effects on nucleosome occupancy resulting from mutations conformed to the model that FACT is needed to overcome nucleosomal barriers during transcription initiation or elongation. Instead, the results suggest that both FACT and H3-K56ac are involved in establishing chromatin architecture prior to transcription and restoring it afterwards. They contribute to a process that optimizes transcription frequency, especially at conditionally expressed genes, and restores chromatin integrity after transcription, especially at the +1 nucleosome to block antisense transcription, but FACT appears to be less involved than expected in directly promoting transcription.

genomics↗