Genome-Wide Reconstitution of Chromatin Transactions: RSC Preferentially disrupts H2A.Z-Containing Nucleosomes

Abstract

Running title: RSC Preferentially disrupts H2A.Z Nucleosomes 2 Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mono-nucleosomes harboring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the intrinsic stability of individual nucleosomes, and -as proof of principlethe nucleosome preference of the chromatin remodeling complex, RSC. Our data indicate that the natural stability of nucleosomes differs greatly, with nucleosomes on tRNA genes and on promoters of protein-coding genes standing out as intrinsically unstable. Interestingly, RSC shows a distinct preference for nucleosomes derived from regions with a high density of histone variant H2A.Z, and this preference is indeed markedly diminished using nucleosomes from cells lacking H2A.Z. Importantly, the preference for H2A.Z remodeling/nucleosome ejection can also be reconstituted with recombinant nucleosome arrays. Together, our data indicate that, despite being separated from their genomic context, individual nucleosomes can retain their original identity as promoteror TSS-nucleosomes. Besides shedding new light on nucleosome stability and the chromatin remodeler RSC, the simple experimental system outlined here should be generally applicable to the study of chromatin transactions.

Genome-wide chromatin accessibility is restricted by ANP32E

“…We propose that increased nucleosome remodeling at H2A.Z sites leads to increased nucleosome repositioning, which uncovers TF-binding motifs and promotes increased gene expression. This model is in agreement with studies of chromatin remodelers in yeast and plants, which demonstrate that ISWI 15 , RSC 14 , and BRM 16 prefer to remodel H2A.Z-containing nucleosomes. Although this mechanism has not yet been demonstrated in animals, SMARCA4 and H2A.Z do physically interact in mESCs 5 .…”

“…ANP32E may restrict chromatin accessibility through changes in nucleosome turnover, wrapping, or remodeling, among other possibilities. Studies of nucleosome remodelers in yeast indicate that SWI/SNF and ISWI family nucleosome remodelers preferentially reposition H2A.Z-containing nucleosomes 14 , 15 . If conserved in mammals, this is a potential pathway by which ANP32E might function to control chromatin patterns.…”

“…These include differences in the wrapping of DNA, differences in nucleosome turnover rates, and differences in chromatin stability at H2A.Z nucleosomes 12,13 . Additionally, increased nucleosome remodeling at H2A.Z sites, which has been observed in yeast and plants 5,[14][15][16] , might allow for better access to DNA at H2A.Z nucleosomes compared with canonical nucleosome sites. Notably, it has not yet been established whether these characteristics of H2A.Z are important for mammalian TF binding.…”

Transcription Promotes the Interaction of the FAcilitates Chromatin Transactions (FACT) Complex with Nucleosomes inSaccharomyces cerevisiae

“…First, the timing of RNAPII and FACT recruitment to induced genes during heat shock differs (Vinayachandran et al 2018). Second, FACT is also found at regions transcribed by RNAPI and III (Birch et al 2009;Tessarz et al 2014;Cakiroglu et al 2018). Finally, histone mutants cause delocalization of FACT independently of RNAPII and other elongation factors (Duina et al 2007;Lloyd et al 2009;Nguyen et al 2013;Pathak et al 2018).…”

Chromatin regulation and dynamics in stem cells

“…In addition to INO80, ISWI complexes display enhanced activity at H2A.Zcontaining nucleosomes; H2A.Z may therefore regulate ISWI complexes, serving as a stimulatory cue for nucleosome remodeling activity (Goldman et al, 2010). In eukaryotes, SWI/SNF family remodelers (RSC and esBAF) act on H2A.Z-containing nucleosomes (Cakiroglu et al, 2019;Hainer & Fazzio, 2015).…”