Fig. 3
Overlapping dinucleosome occupancy is dependent on the nucleosome remodeler BRG1. (a) RT-qPCR results of three independent esiRNA knockdowns targeting EGFP (unexpressed control), the esBAF ATPase BRG1 (encoded by Smarca4) and the ISWI ATPase SNF2H (encoded by Smarca5). qPCR primers amplifying Smarca4 (blue) and Smarca5 (red) were used, and all values were normalized to Gapdh mRNA levels using ∆∆Ct and displayed ± standard deviation. n = 3. (b) Peak assignment to gene-based features for OLDNs called from gel extracted MNase-seq samples. Peaks were called using nucleR from replicate-merged bam files that were bioinformatically filtered to fragment sizes between 230–270 bp (n = 3 per condition, peak width = 250 bp) [41]. (c) Putative OLDNs visualized at RNAPII binding sites, sorted by RNAPII occupancy. RNAPII ChIP-seq from GSE98605 [45]. n = 3 merged replicates per condition, shown as a single heatmap. 43,152/45,518 RNAPII ChIP-seq peaks directly overlapped a called “consensus” OLDN peak (94.8%). Gel-extracted libraries were used for these analyses and size-selected for fragments between 230–270 bp. Data were analyzed for significance using a Friedman test (p < 0.001, q < 0.001). (d) Metaplots of OLDN occupancy visualized over ChIP-seq peaks for BRG1 (top, GSE64825 [48]) and SNF2H (bottom, GSE123670 [49]). n = 3 replicates per condition. Shaded area indicates standard error. Samples were gel-extracted between 200–300 bp and bioinformatically size-selected for fragments between 230–270 bp. (e) Metaplots and heatmaps of OLDN occupancy at TSSs for genes with altered expression in Smarca4 conditional KO (GSE98469 [45]). Genes are clustered by log2 fold change of RNAseq expression as follows: up following knockout (≥ 0.25), unchanged (-0.25 ≤ log2 fold change ≤ 0.25), or down following knockout (≤ -0.25). Data were analyzed for significance using Friedman tests for each cluster (p < 0.001 in all cases). All individual comparisons were significant (p < 0.001, q < 0.001) except C2 (EGFP KD vs. Smarca4 KD, low MNase (p = 0.1505, q = 0.0502)) and (Smarca4 KD vs. Smarca5 KD high MNase (p = 0.1443, q = 0.0482)). (f) Metaplots and heatmaps of OLDN occupancy at TSSs for genes with altered expression in Smarca4 conditional KO (e; GSE98469 [45]), or Snf2h KO (f, GSE112136 [47]). Genes are clustered by log2 fold change of RNA-seq expression as in Fig. 3e. Data were analyzed for significance using Friedman tests for each cluster (p < 0.001 in all cases). All individual comparisons were significant (p < 0.001, q < 0.001) except EGFP KD vs. Smarca4 KD under high MNase digestion (p = 0.5388 (C1), 0. 8128 (C2), 0.0454 (C3), q = 0.1798 (C1), 0.2712 (C2), 0.0152 (C3).