Fig. 4: EZH2i in NEPC models induce NE-lineage genes bearing bivalent promoters.

A Screenshots from Integrative Genomics Viewer (IGV) of CUT&RUN H3K27me3 or H3K4me3 or IgG levels in WCM154 treated with vehicle or tazemetostat as described in Fig. 1A at the genomic loci of ASCL1 with vehicle or tazemetostat treatment; gray, IgG; red, H3K27me3; blue, H3K4me3. B WCM154 was treated as mentioned in Fig. 1A and samples were processed for qRT-PCR. Target gene (ASCL1) mRNA levels were normalized to GAPDH expression and are represented as relative expression using one of the values obtained from vehicle-treated conditions as 1. Columns, means of values (n = 3); white, vehicle; red, tazemetostat; bars, SEM values; *p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. C Volcano plot of differentially expressed genes in WCM154 RNA-Seq data as described in Fig. 3A (n = 3/condition); blue, significantly downregulated genes upon tazemetostat treatment (log₂FoldChange < 0; p-value < 0.05); red, significantly upregulated genes upon tazemetostat treatment (log₂FoldChange < 0; p-value < 0.05); burgundy, NE-lineage transcription factors upregulated upon tazemetostat treatment. D Western blot analysis for indicated proteins showing efficiency of knockout of EZH2 in WCM154 using two independent sgRNAs targeting EZH2. Blots were reprobed for β-actin as loading control. Representative western blot from three independent experiments is shown. E WCM154 control or EZH2 knockout lines were processed for qRT-PCR. Target gene (ASCL1) mRNA levels were normalized to GAPDH expression and are represented as a relative expression using one of the values obtained from WCM154-sgINC conditions as 1. Columns, means of values (n = 3); white, WCM154-sgINC; orange, WCM154-sgEZH2 (#1 and #2); bars, SEM values; *p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. F Volcano plot of differentially expressed genes in WCM154 RNA-Seq data as described in (D; n = 3/condition); blue, significantly downregulated genes upon EZH2 knockout treatment (log₂FoldChange < 0; p-value < 0.05); red, significantly upregulated genes upon EZH2 knockout treatment (log₂FoldChange < 0; p-value < 0.05); burgundy, NE-lineage transcription factors upregulated upon EZH2 knockout. G Screenshots from Integrative Genomics Viewer (IGV) of CUT&RUN H3K27me3 or H3K4me3 or IgG levels in WCM154-sgINC or sgEZH2 #1 as described in (D) at the genomic loci of ASCL1 or LHX2; gray, IgG; red, H3K27me3; blue, H3K4me3. H Schematic of the WCM154 knockout of endogenous EZH2 and rescue using a recombinant dTAG-fused EZH2 and treatment with degrader compound (dTAGv-1) for 9 days. H3K27me3 levels are lost after EZH2 knockout and rescued (partially) after re-introduction of recombinant dTAG-EZH2 and again lowered upon 9 days of dTAGv-1 mediated degradation of EZH2. Figure 4/panel H Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. I Western blot analysis for indicated proteins showing the efficiency of knockout of EZH2 and rescue using recombinant dTAG-EZH2 in WCM154. Impact of dTAGv-1 treatment at 8 h and 9 days is shown on levels of EZH2 and H3K27me3. Blots were reprobed for β-actin as loading control. Representative western blot from two independent experiments is shown. J WCM154 models with knockout of EZH2 (sgEZH2 #1) and rescue using recombinant dTAG-EZH2 were processed for RNA-Seq (n = 3/condition). Volcano plot of differentially expressed genes; blue, significantly downregulated genes upon EZH2 knockout treatment (log₂FoldChange < 0; p-value < 0.05); red, significantly upregulated genes upon EZH2 knockout treatment (log₂FoldChange < 0; p-value < 0.05); orange, EZH2 levels shown as control; burgundy, NE-lineage transcription factors upregulated upon rescue of EZH2 expression. K WCM154 models with recombinant dTAG-EZH2 treated with vehicle or dTAGv-1 for 9 days were processed for RNA-Seq (n = 3/condition). Volcano plot of differentially expressed genes; blue, significantly downregulated genes upon dTAGv-1 treatment (log₂FoldChange < 0; p-value < 0.05); red, significantly upregulated genes upon dTAGv-1 treatment (log₂FoldChange < 0; p-value < 0.05); burgundy, NE-lineage transcription factors upregulated upon rescue of EZH2 expression.