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

Abstract

Functional 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.

Figure 1. Klf4 binding is esBAF-dependent

(A) Log₂ fold change of small read (left: Mbd3/EGFP KD, right: Smarca4/EGFP KD) spanning 200 bp directly over binding peaks sorted by either Mbd3 (left) or Brg1 (right) occupancy. (B) Aggregation plot of normalized small reads (≤80 bp) and nucleosome size reads (135–165 bp) of MNase-seq upon EGFP, Mbd3, or Smarca4 KD over Klf4 binding sites. Klf4 binding sites were called from GEO:GSE11431 (Chen et al., 2008). Asterisks (*, **) indicate p-values (<0.05, <0.001) reflecting statistical significance of changes in MNase footprints over the relative to EGFP KD, colored as indicated in key. (C) Histone H3 ChIP-seq in EGFP, Mbd3, or Smarca4 KD ESCs shown as normalized occupancy aggregated over Klf4 binding sites. (D) Klf4 ChIP-seq in EGFP, Mbd3, or Smarca4 KD ESCs shown as normalized Klf4 occupancy aggregated over Klf4 binding sites. (E) Heatmaps of Klf4 occupancy over Klf4 binding sites in EGFP (left) and Smarca4 (right) KD. Occupancy is indicated as log₂(normalized reads). (F) Equal levels of nuclear Klf4 in EGFP and Smarca4 KD ESCs confirmed by Western blotting. GAPDH and Pol II are specificity controls for cytoplasmic and nuclear fractions, respectively.

Figure 2. esBAF and Mbd3/NuRD complexes regulate nucleosome occupancy over factor binding sites

(A,B) Scatterplot of log₂ fold change values of nucleosome size reads versus small reads in Mbd3 (A) or Smarca4 (B) KD ESCs relative to EGFP KD. (C) Aggregation plots of histone H3 ChIP-seq over a subset of factor binding sites of EGFP, Mbd3, and Smarca4 KD ESCs. Asterisks (*, **) indicate p-values as described in Figure 1.

Figure 3. esBAF promotes nucleosome formation at the expense of subnucleosomes

(A,B) Scatterplot of log₂ fold change values of nucleosome size reads versus medium size reads in Mbd3 (A) or Smarca4 (B) KD ESCs relative to control ESCs. (C) Aggregation plots of relative nucleosome (135–165 bp) and subnucleosome (100–130 bp) occupancy of MNase-seq data upon EGFP, Mbd3, or Smarca4 KD over various factor binding sites. Asterisks (*, **) indicate p-values as described in Figure 1.

Figure 4. Brg1 is required for H2A.Z occupancy at a subset of locations

(A–C) Heatmaps of H2A.Z occupancy over indicated regions in EGFP (left) and Smarca4 (right) KD. Binding sites were taken from the following datasets: H2A.Z (GEO:GSE34483), TSSs (mm9), and Pwp1 (GEO:GSE59389). Occupancy is indicated as log₂(normalized reads). (D) Maturation model of subnucleosome-nucleosome transition by esBAF. esBAF is required for converting subnucleosomes into a form suitable for H2A.Z/H2B incorporation by p400 or SRCAP, potentially by organizing wrapping of DNA around the H3/H4 tetramer, promoting accessibility of H3/H4 binding interfaces, or another mechanism. (E) Stabilization model of subnucleosome regulation by esBAF. esBAF prevents disassembly of nucleosomes into subnucleosomes (such as hexasomes or half-nucleosomes) by preventing loss of H2A.Z/H2B or H2A/H2B dimers from the histone octamer. Maturation and stabilization of subnucleosomes could be occurring simultaneously and esBAF could regulate H2A.Z occupancy based on interactions with additional regulatory factors.