Genome-wide Regulation of Pol II, FACT, and Spt6 Occupancies by RSC in Saccharomyces cerevisiae

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.RSC（染色质结构重塑复合物，remodels the structure of chromatin）是酿酒酵母（Saccharomyces cerevisiae）中不可或缺的ATP依赖型染色质重塑复合物。其催化亚基Sth1借助ATP酶活性滑动或移除核小体（nucleosome）。已有研究表明，RSC可通过将侧翼核小体从核小体缺失区域（nucleosome-depleted regions，NDRs）处移开，调控NDRs的宽度。当RSC被耗竭时，核小体会侵入NDRs，进而引发转录起始缺陷。本研究探讨了催化失活型Sth1对转录的影响，并将其与通过生长素诱导降解子策略快速、急性耗竭Sth1时观察到的效应进行了对比。研究发现，Sth1的快速耗竭会降低高转录基因中TATA盒结合蛋白（TATA-box binding protein，TBP）与RNA聚合酶II（RNA polymerase II，Pol II）的招募丰度，这与其在启动子处调控染色质结构的功能相符。与之相反，携带催化失活型Sth1的细胞中TBP结合出现显著降低，但令人意外的是，其编码区的Pol II结合丰度却出现了大量积累。在催化失活突变体中耗竭内源Sth1后，我们观察到Pol II结合丰度进一步升高，这表明失活的Sth1参与了此前观测到的编码区Pol II积累现象。尽管Pol II结合丰度升高，但该突变体中组蛋白伴侣FACT和Spt6的开放阅读框（Open Reading Frame，ORF）结合丰度均显著降低。上述结果提示，RSC可能在编码区招募或留存这些组蛋白伴侣中发挥作用。在催化失活突变体中，尽管TBP、FACT及Spt6的结合丰度大幅降低，但Pol II仍出现积累，这可能预示着严重的转录延伸与终止缺陷。这类缺陷与此前研究中观察到的RSC以转录依赖方式被招募至编码区的结果相一致。因此，本研究结果表明，除已明确的转录起始调控功能外，RSC在转录延伸与终止过程中也发挥着一定作用。