Figure 4: KDM5 histone lysine demethylases are inhibited by (R)-2HG in cells.

(A) Immunoblot analysis of trimethyl-H3K4 (H3K4me3) levels in isogenic TF-1 cell lines expressing empty vector (Empty), wild-type IDH1 (IDH1^WT), or R132H-mutant IDH1 (IDH1^R132H); Cas9 and three non-targeting sgRNAs (sg3xNT) or Cas9 and sgRNAs targeting KDM5A, KDM5C and KDM5D (sgACD); a non-targeting shRNA (shNT) or one of two shRNAs targeting TET2 mRNAs (shTET2-1 and shTET2-2); and TF-1 cells treated with vehicle-control (DMSO) or 50 nM KDM5c70. (B) Volcano plot illustrating the relative enrichment of trimethyl-H3K4 in IDH1^R132H-expressing TF-1 cells when compared to IDH1^WT-expressing TF-1 cells. (C) Tornado plots of the trimethyl-H3K4 ChIP-seq signals at the sites showing differential enrichment between IDH1^WT and IDH1^R132H identified in (B) in the TF-1 cells described in (A). (D) Tornado plots of the trimethyl-H3K4 ChIP-seq signals at sites showing differential enrichment between IDH1^WT and IDH1^R132H identified in (B) in TF-1 cells treated with the indicated concentrations of TFMB-(R)-2-HG [(R)-2HG] or DMSO-control (0 μM (R)-2HG). In all cases, cells were passaged for 21 days after lentiviral infection or 21 days in the presence of DMSO or KDM5c70 prior to harvesting for methyl-histone analysis. (E) GSEA of the transcripts detectable by RNA-seq in TF-1 cells that are associated with loci enriched for trimethyl-H3K4 in both IDH1^R132H TF-1 cells and sgACD TF-1 cells when compared to Empty TF-1 cells and sg3xNT TF-1 cells (n=169 genes). Shown in (A) are representative results from at least two independent experiments. The ChIP-seq and RNA-seq experiments in (B-E) were done in duplicate, and the average signals from the two replicates is shown. All the experiments shown in (A-E) were done on cells cultured in the presence of GM-CSF. NES: normalized enrichment score; FDR: false discovery rate.