Supplementary Figure 7: Chromatin features and accessibility changes at SEs upon HDAC treatment. | Nature Genetics

Supplementary Figure 7: Chromatin features and accessibility changes at SEs upon HDAC treatment.

From: Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma

Supplementary Figure 7

a, Time-course ChIP-Rx of H3K27ac during Entinostat treatment, at core regulatory TFs (left) compared to all other TFs (right), both along the gene body (top) and the transcriptional end site (TES, bottom). b, Quantification of H3K27ac density along the promoter, transcriptional start site region (TSSR), the gene body, and the transcriptional end site region (TESR), shown for all Pol2 bound coding genes (n = 28,974), all housekeeping genes (n = 5,034), all super enhancer associated genes (n = 1,284), and SE associated TF genes (n = 114). Adjacent box plots are shown for DMSO (6 hours) and Entinostat (1 hour and 6 hours), where the boxes show the 1st quartile, median and 3rd quartile, and the whiskers show the 1.5 * interquartile range. c, Genic spread ratio of H3K27ac (formula above) shown for various gene sets compared to core regulatory TFs, split into gene size categories: less than 10 kb, 10 to 100 kb, and genes greater than 100 kb. Box plots are used to display the median (center thick line) surrounded by the 1st and 3rd quartiles, with whiskers showing 1.5 * the interquartile range. P-values were calculated using a two-sided t-test with no adjustments for multiple comparisons. d, Cumulative distribution of Traveling Ratios for RNA Polymerase 2 (Pol2) showing increased elongation at CR TF genes compared to all genes (left, p < 0.0001), a general increase in pausing induced by Entinostat treatment (center, p < 2.2x10-16), and no statistically significant change in pausing at CR TF genes under DMSO or Entinostat treatment (right, p = 0.5). Traveling Ratio distribution differences were evaluated by Welch’s two-tailed t-test. e, RNA-Pol2 is heavily reduced at SEs by HDAC inhibition. Scatter plots of Pol2 ChIP-Rx signal, log2-fold change vs ChIP-Rx density in RRPM (left) and summarized by box-plots (right; boxes show 1st and 3rd quartiles with center as the median value, while whiskers show 1.5 * the interquartile range; the p-values were calculated by a two-sided unpaired student’s t-test with Welch’s correction). Pol2 sites outside of SEs were (n = 16,268) and Pol2 sites within SEs were (n = 472). Peaks were stitched together when multiple Pol2 peaks were present in a SE. f, Super resolution imaging (iSIM methodology, see Online Methods for details) of RNA-Pol2-GFP clusters in live RH4 cells. Fluorescence images are pseudo-colored green. Individual Pol2 puncta ranked by density (area times brightness) plots reveal asymmetric distribution of Pol2 in control conditions (DMSO) which are reduced by 6 hours of HDACi (Entinostat) treatment. The experiment was repeated twice with similar results.

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