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

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.

The Paf1 Complex: A Keystone of Nuclear Regulation Operating at the Interface of Transcription and Chromatin.

The regulation of transcription by RNA polymerase II is closely intertwined with the regulation of chromatin structure. A host of proteins required for the disassembly, reassembly, and modification of nucleosomes interacts with Pol II to aid its movement and counteract its disruptive effects on chromatin. The highly conserved Polymerase Associated Factor 1 Complex, Paf1C, travels with Pol II and exerts control over transcription elongation and chromatin structure, while broadly impacting the transcriptome in both single cell and multicellular eukaryotes. Recent studies have yielded exciting new insights into the mechanisms by which Paf1C regulates transcription elongation, epigenetic modifications, and post-transcriptional steps in eukaryotic gene expression. Importantly, these functional studies are now supported by an extensive foundation of high-resolution structural information, providing intimate views of Paf1C and its integration into the larger Pol II elongation complex. As a global regulatory factor operating at the interface between chromatin and transcription, the impact of Paf1C is broad and its influence reverberates into other domains of nuclear regulation, including genome stability, telomere maintenance, and DNA replication.