The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the −1 and +1 nucleosomes flanking NDRs; however, Ino80C’s function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the −1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.

RSC (Remodels the Structure of Chromatin) is a conserved ATP-dependent chromatin remodeling complex that regulates many biological processes, including transcription by RNA polymerase II (Pol II). We report that RSC contributes in generating accessible nucleosomes in transcribed coding sequences (CDSs). RSC MNase ChIP-seq data revealed that RSC-bound nucleosome fragments were very heterogenous (∼80 bp to 180 bp) compared to a sharper profile displayed by the MNase inputs (140 bp to 160 bp), supporting the idea that RSC promotes accessibility of nucleosomal DNA. Notably, RSC binding to + 1 nucleosomes and CDSs, but not with -1 nucleosomes, strongly correlated with Pol II occupancies, suggesting that RSC enrichment s CDSs is linked to transcription. We also observed that Pol II associates with nucleosomes throughout transcribed CDSs, and similar to RSC, Pol II-protected fragments were highly heterogenous, consistent with the idea that Pol II interacts with remodeled nucleosomes in CDSs. This idea is supported by the observation that the genes harboring high-levels of Pol II in their CDSs were the most strongly affected by ablating RSC function. Additionally, rapid nuclear depletion of Sth1 decreases nucleosome accessibility and results in accumulation of Pol II in highly transcribed CDSs. This is consistent with a slower clearance of elongating Pol II in cells with reduced RSC function, and is distinct from the effect of RSC depletion on PIC assembly. Altogether, our data provide evidence in support of the role of RSC in promoting Pol II elongation, in addition to its role in regulating transcription initiation.

2 SUMMARY RSC (Remodels the Structure of Chromatin) is a conserved ATP-dependent chromatin remodeling complex that regulates many biological processes, including transcription by RNA polymerase II (Pol II). We report that not only RSC binds to nucleosomes in coding sequences (CDSs) but also remodels them to promote transcription. RSC MNase ChIP-seq data revealed that RSC-protected fragments were very heterogenous (~80 bp to 180 bp) compared to the sharper profile displayed by the MNase inputs (140 bp to 160 bp), supporting the idea that RSC activity promotes accessibility of nucleosomal DNA. Importantly, RSC binding to +1 nucleosomes and CDSs, but not with -1 nucleosomes, strongly correlated with Pol II occupancies suggesting that the RSC enrichment in CDSs is important for efficient transcription. This is further supported by a similar heterogenous distribution of Pol II-protected fragments. As such, the genes harboring high-levels of RSC in their CDSs were the most strongly affected by ablating RSC function. Altogether, this study provides a mechanism by which RSC-mediated remodeling aids in RNA Pol II traversal though coding sequence nucleosomes in vivo. 3The nucleosome is the fundamental unit of chromatin and is formed by wrapping ~147 base pairs of DNA around an octamer of histones (2 copies of H3, H4, H2A and H2B). DNA wrapped around a histone octamer is inaccessible for DNA-dependent processes, including transcription by RNA polymerase II (Pol II) (Lorch et al., 1987). This nucleosomal impediment can be relieved by chromatin remodeling complexes that use the energy derived from ATP hydrolysis to slide or evict histones in order to expose the underlying DNA (Clapier and Cairns, 2009;Clapier et al., 2016).One such remodeler is the SWI/SNF family member RSC (remodels the structure of chromatin) complex, the only essential remodeler in budding yeast (Cairns et al., 1996).RSC is an important regulator of chromatin organization around gene promoters. It is implicated in establishing 'nucleosome depleted regions' (NDRs) found upstream of transcription start sites (TSSs) and also in positioning of the NDR-flanking nucleosomes referred to as the -1 and +1 nucleosomes (Nuc) (Krietenstein et al., 2016). The DNA sequence forming the +1_Nuc often harbors TSS, and +1_Nuc can therefore be inhibitory for transcription initiation. RSC has been shown to bind both the -1_Nuc and +1_Nuc, and impairing RSC function is associated with the movement of flanking nucleosomes toward the NDR, which results in filling of the NDRs (Badis et al., 2008;Brahma and Henikoff, 2019;Ganguli et al., 2014;Hartley and Madhani, 2009a;Klein-Brill et al., 2019;Kubik et al., 2015;Rawal et al., 2018). Nucleosomes which invade or assemble within NDRs are also cleared by RSC (Brahma and Henikoff, 2019;Kubik et al., 2015). These nucleosomes were bound by RSC and were termed "fragile", given their greater sensitivity to digestion by micrococcal nuclease (MNase). The presence of such nucleosomes is controversial, partly due to the difficulty in detecting ...

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Chromatin remodelers are essential for the maintenance of chromatin structure and gene regulation. In this study, we examined the role of histone acetyltransferases (HATs) Gcn5 and Esa1 in regulating RSC and histone occupancies and their effects on transcription genome-wide. We identified contrasting roles of HATs in modulating RSC occupancies in promoters and ORFs. In HAT mutants, RSC accumulated in nucleosome depleted regions (NDRs) with fragile nucleosomes (FNs) more than those with stable -1 nucleosomes. Moreover, the accumulation was more significant in the Esa1 mutant than in the Gcn5 mutant. However, RSC NDR accumulation was not observed in cells lacking H3 or H4 tails. Furthermore, we observed marked increases in histone occupancies in NDRs in the HAT mutants genome-wide. Overall, these data suggest that FNs use hypoacetylated tails to recruit RSC to NDRs, and subsequent acetylation of the tails promote histone eviction. In contrast to the promoters, RSC occupancies were significantly reduced in transcribed ORFs in the HAT mutants. Additionally, the HAT mutants showed reduced TBP and Pol II binding at promoters. Thus, our data implicate HATs and RSC in maintaining NDRs, regulating chromatin structure, and promoting transcription.