Supplementary Figure 3: HDAC1/2/3 epigenomics and transcriptional contributions. | Nature Genetics

Supplementary Figure 3: HDAC1/2/3 epigenomics and transcriptional contributions.

From: Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma

Supplementary Figure 3

a, ChIP-seq of HDAC1 (left), HDAC2 (center) and HDAC3 (right) along the body of genes, categorized as Repressed, Low Expression, Mid Expression, High Expression, or Core Regulatory TF target genes. Shading shows the SEM. b, Peak overlap measurement (top) among HDAC isoforms 1, 2 and 3 (and % overlap with CR TFs) across the RH4 epigenome (top) and visualized via ChIP-seq heatmap (bottom). c, HDAC inhibition with Entinostat (at 1, 6 and 24 hours in RH4 cells) reduced SOX8, MYOD1, MYOG and MYCN. Data shown are from a single experiment, and is representative of similar results obtained in RH41 cells with similar HDAC inhibitors (data not shown). d, Gene set enrichment analysis (GSEA) of time course RNA-seq from Entinostat treatment (1, 6 and 24 hours). P-values for gene sets are indicated by size of the circles, while color indicates both sign and extend of the normalized enrichment score (NES). e, GSEA plots of CR TF transcription after sgRNA disruption of individual HDACs. Data are for 72 hours of CRISPR editing, compared to 6 hours of Entinostat. 2 sgRNAs per HDAC enzyme where delivered to guide CRISPR–cas9 machinery in RH4 cells, targeting the enzymatic active site responsible for deacetylation. Expression changes of HDACs is shown (right). f, Formulas for calculating mRNA decay rates and half-lives from integrated analysis of nascent RNA and total RNA-seq. g, Calculated relative transcript half-lives shown as box (1st quartile, median and 3rd quartile) and whisker (1.5 * interquartile range) plots, overlapping distribution shown as violin plots, for housekeeping and core regulatory TF transcripts. P-value was calculated using a two sided student’s T-test with Welch’s correction (unpaired). h, Hypothetical graph of mRNA decay assuming halted transcription, 100 molecules at time zero, and indicated half-lives. i, Gene set of apoptotic genes activated by HDACi and measured at the single cell level. The sample sizes per condition that were aggregated at the gene level were DMSO (n = 2,925 cells), Entinostat 1 hr (n = 3,805 cells) or 6 hrs (n = 3,240 cells).

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