Fig. 5

H2A.Z deposition is strongly tied with greater RNAP2 pausing. a Correlation of TSS chromatin features with PI in four cell types. Correlation was modeled with a multi-dimensional linear regression. H2A.Z had a strong positive coefficient across all four tested cell types, suggesting that H2A.Z occupancy correlated with increased PI. b H2A.Z mean tag density around the TSS in GM12878 and K562 by increasing PI quintile. H2A.Z density increased with increasing PI. c, d Relationship of H2A.Z or H3.3 TSSR density to RNAP2 TSSR and gene body density. H2A.Z correlated with PI (Spearman r = 0.55) whereas H3.3 did not (Spearman r = 0.13). Genes with the same PI values align on the diagonal; one example line with PI = 1 is shown. e–g H2A.Z knockdown in MCF7 cells globally increased RNAP2 pausing. MCF7 cells were treated with H2A.Z or control siRNA. RNAP2 pausing was then determined from RNAP2 ChIP-seq. H2A.Z knockdown in MCF7 cells globally increased RNAP2 pausing (e; ***p <0.001; Mann–Whitney U test). We counted the number of genes with absolute fold-change in RNAP2 density >2 in TSSR or gene body (f). Predominantly, genes increased TSSR RNAP2 density or decreased either TSSR or gene body density, but rarely both. Genes with exclusively ≥2-fold decrease in gene body RNAP2 density had a lower PI pre-knockdown than all genes or genes with exclusively ≥2-fold increase in their TSS RNAP2 density, further suggesting that H2A.Z differentially affects genes based on their PI. (g; ***p <0.001; Mann–Whitney U test.) (h). Antagonizing pause-release predominantly increased H2A.Z enrichment at promoters (all 500 NM FP vs. no FP, ***p <0.001; t-test). MCF7 cells were treated with FP to block pause-release. H2A.Z occupancy at 11 promoters was measured by ChIP-qPCR and normalized to histone H3 occupancy