Fig. 5.

HDAC isoforms 1, 2 and 3 are co-essential for complete ablation of CR TF functionality. a Chemical structures of LW3 and Merck60. b Overlay of MD model of LW3 in HDAC3 (green) and Merck60 in HDAC2 (magenta). c Super-enhancer selective transcriptional assay shows LW3 is selective for SE activity, compared to CMV-driven transcription. Experiments were performed in quadruplicate, and error bars represent the standard deviation. d HDAC inhibition across isoforms HDAC1–9 reveals selectivity profiles for Merck60, LW3, and Entinostat. Experiments were performed in duplicate for each concentration, with symbols representing the mean and the error bars representing the standard deviation. e On-target effect of LW3 observed at the HDAC substrate (acetylated histone 3 lysine residues), seen by western blot after 6 h of treatment at 0, 1, and 10 µM of LW3 (left), and phenocopied by genetic disruption of HDAC3 by CRISPR-cas9 KO at 48 h (right). f Core regulatory TF transcription (GSEA, left) is selectively halted by LW3 and Entinostat but not Merck60 (at 6 h in RH4 cells, compared to DMSO). Combination of Merck60 (HDAC1 + 2i) and LW3 (HDAC3i) mimics the strong effect of triple HDAC1 + 2 + 3 inhibitor Entinostat. Expression changes are shown for top CR TFs, for all three benzamide HDAC inhibitors (right). NS = not significant, **P < 0.008, ***P < 0.0001, determined by GSEA. g HDAC1, HDAC2, and HDAC3 ChIP-seq peaks overlap with one another at sites of CR TF binding, with greater frequency of all three HDACs overlapping in SEs, shown summarized by Venn diagrams. h ATAC-seq shows increased accessibility to the chromatin template at HDAC2-bound sites, at 1 and 6 h of treatment with Entinostat. Sites are divided into HDAC2-only sites (left) and HDAC2 sites co-bound by all five top core regulatory TFs (SOX8, MYCN, PAX3-FOXO1, MYOG, and MYOD) as measured by ChIP-seq (right). i Aberrant chromatin looping interactions gained, and native interactions lost, upon HDAC inhibition with Entinostat for 6 h, assayed by 4C-seq from the MYOD1 promoter (middle) and the first MYOD1 super enhancer (bottom), with ChIP-seq tracks of CTCF, cohesin (RAD21), p300, YY1, HDAC1, HDAC2, and HDAC3