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Nucleosome position mapping by micrococcal nuclease analysis of S. cerevisiae anchor-away Sth1, Swi2 and/of TBP cells in raffinose and galactose-rich media in rapamycin or DMSO

ABSTRACT: We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease on yeast strains that were grown in raffinose or galactose containing media (synthetic complete). Strains contained FRB-tags to Sth1, Swi2, Sth1 & Swi2 or Sth1 and TBP, where TBP was tagged on one of two alleles in a diploid strain. Cells were treated for 60 min either with 7.5 uM rapamycin or the same volume of DMSO.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE190737 | GEO | 2023/01/11

REPOSITORIES: GEO

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Project description:RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. The catalytic subunit of RSC, Sth1 uses its ATPase activity to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such the nucleosomes encroach NDRs when RSC is depleted and leads to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to the effects observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 exhibited a severe reduction in TBP binding, but surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. After depleting endogenous Sth1 in the catalytic dead mutant, we observed a further increase in Pol II occupancies, suggesting that the inactive Sth1 contributed to the observed accumulation of Pol II in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could be indicative of severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.

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Nucleosome position mapping by micrococcal nuclease analysis of S. cerevisiae anchor-away Sth1, Swi2 and/of TBP cells in raffinose and galactose-rich media in rapamycin or DMSO

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