The esBAF and ISWI nucleosome remodeling complexes influence occupancy of overlapping dinucleosomes and fragile nucleosomes in murine embryonic stem cells

Abstract

Abstract Background Nucleosome remodeling factors regulate the occupancy and positioning of nucleosomes genome-wide through ATP-driven DNA translocation. While many nucleosomes are consistently well-positioned, some nucleosomes and alternative nucleosome structures are more sensitive to nuclease digestion or are transitory. Fragile nucleosomes are nucleosome structures that are sensitive to nuclease digestion and may be composed of either six or eight histone proteins, making these either hexasomes or octasomes. Overlapping dinucleosomes are composed of two merged nucleosomes, lacking one H2A:H2B dimer, creating a 14-mer wrapped by ~ 250 bp of DNA. In vitro studies of nucleosome remodeling suggest that the collision of adjacent nucleosomes by sliding stimulates formation of overlapping dinucleosomes. Results To better understand how nucleosome remodeling factors regulate alternative nucleosome structures, we depleted murine embryonic stem cells of the transcripts encoding remodeler ATPases BRG1 or SNF2H, then performed MNase-seq. We used high- and low-MNase digestion to assess the effects of nucleosome remodeling factors on nuclease-sensitive or “fragile” nucleosome occupancy. In parallel we gel-extracted MNase-digested fragments to enrich for overlapping dinucleosomes. We recapitulate prior identification of fragile nucleosomes and overlapping dinucleosomes near transcription start sites, and identify enrichment of these features around gene-distal DNaseI hypersensitive sites, CTCF binding sites, and pluripotency factor binding sites. We find that BRG1 stimulates occupancy of fragile nucleosomes but restricts occupancy of overlapping dinucleosomes. Conclusions Overlapping dinucleosomes and fragile nucleosomes are prevalent within the ES cell genome, occurring at hotspots of gene regulation beyond their characterized existence at promoters. Although neither structure is fully dependent on either nucleosome remodeling factor, both fragile nucleosomes and overlapping dinucleosomes are affected by knockdown of BRG1, suggesting a role for the complex in creating or removing these structures.