Regulation of Nucleosome Architecture and Factor Binding Revealed by Nuclease Footprinting of the ESC Genome

• Alterations in MNase footprints reveal functional interactions among gene regulators
• esBAF and Mbd3/NuRD regulate the occupancy of multiple regulatory proteins in ESCs
• esBAF is required for Klf4 binding in ESCs
• esBAF functions in subnucleosome maturation through regulation of H2A.Z occupancy

SummaryFunctional interactions between gene regulatory factors and chromatin architecture have been difficult to directly assess. Here, we use micrococcal nuclease (MNase) footprinting to probe the functions of two chromatin-remodeling complexes. By simultaneously quantifying alterations in small MNase footprints over the binding sites of 30 regulatory factors in mouse embryonic stem cells (ESCs), we provide evidence that esBAF and Mbd3/NuRD modulate the binding of several regulatory proteins. In addition, we find that nucleosome occupancy is reduced at specific loci in favor of subnucleosomes upon depletion of esBAF, including sites of histone H2A.Z localization. Consistent with these data, we demonstrate that esBAF is required for normal H2A.Z localization in ESCs, suggesting esBAF either stabilizes H2A.Z-containing nucleosomes or promotes subnucleosome to nucleosome conversion by facilitating H2A.Z deposition. Therefore, integrative examination of MNase footprints reveals insights into nucleosome dynamics and functional interactions between chromatin structure and key gene-regulatory factors.

Graphical AbstractFigure optionsDownload as PowerPoint slide