The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription inSaccharomyces cerevisiae

Abstract

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 ...

High-Resolution Genome-Wide Occupancy in Candida spp. Using ChEC-seq

“…Compared to the other ChIP-based techniques, the ChEC procedure relies on total DNA extraction instead of chromatin solubilization and does not require protein-DNA cross-linking or sonication, thus avoiding artifacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon ( 42 , 43 ). So far, ChEC has been exclusively used in the model yeast S. cerevisiae to map chromatin occupancy of general transcriptional regulators ( 44 ), chromatin remodelers ( 27 , 30 , 45 ), and histone modifiers ( 31 , 32 ) in addition to transcription factors ( 46 , 47 ). As many transcriptional regulators and chromatin remodelers are key virulence and drug resistance factors in C. albicans and other fungi ( 6 , 13 , 17 , 48 – 50 ), ChEC-seq represents an attracting tool to unbiasedly decipher transcriptional regulatory networks of fungal fitness.…”

Global and Gene-specific Transcriptional Responses to Acute Stress

“…Whether these changes are controlled by active chromatin remodelling by ATPases such as Isw1, Isw2, RSC or Swi/Snf (76)(77)(78), recruited via stress-related transcription factors or reflect the remodelling activity of Pol2 itself is not clear at present. This dynamic relationship between the onset of transcription and nucleosome positions would not have been evident had nucleosome positions been related to changes in transcript levels, as these are temporally offset early in the acute stress response (68) (this study), or if the system used lacked the necessary temporal resolution.…”

High resolution genome-wide occupancy inCandida spp. using ChEC-seq

“…Compared the other ChIP-based techniques, the ChEC procedure relies on total DNA extraction instead of chromatin solubilization and does not require protein-DNA crosslinking or sonication avoiding thus artefacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon (50,51). So far, ChEC has been exclusively used in the model yeast S. cerevisiae to map chromatin occupancy of general transcriptional regulators (42), chromatin remodelers (27,30,52) and histone modifiers (31,32) in addition to transcription factors (53,54). As many transcriptional regulators and chromatin remodelers are key virulence and drug resistance factors in C. albicans and other fungi (6,13,17,(55)(56)(57), the ChEC-seq represents thus an attracting tool to unbiasedly decipher transcriptional regulatory networks of fungal fitness.…”