Fig. 3: EZH2 inhibition induces bivalent promoters in NEPC.

A Clustering analysis of RNA-seq data (n = 3/condition/model) of PRAD or NEPC models treated with vehicle (Veh; DMSO) or 5 μM of tazemetostat (Taz) for 6 days. Unsupervised k-means clustering was performed on scaled (z-score) FPKM values of genes that were upregulated upon tazemetostat treatment in both models (log₂FC > 0, FDR < 0.05). Optimum number of clusters was determined using sum of squared errors (k = 7). Z-score: red, high; blue, low; turquoise, cluster 3 corresponding to genes tazemetostat-induced in WCM154; teal, cluster 6 corresponding to genes tazemetostat-induced in LNCaP-abl. B H3K27me3/H3K4me3 CUT&RUN from LNCaP-abl and WCM154 was analyzed and plots show profiles of normalized H3K27me3 and H3K4me3 peaks at ±3 kb of gene bodies or TSS of genes upregulated upon EZH2i treatment in LNCaP-abl (turquoise) or WCM154 (teal). C Screenshots from Integrative Genomics Viewer (IGV) of CUT&RUN H3K27me3 or H3K4me3 or IgG levels in LNCaP-abl and WCM154 and the genomic loci of indicated MHC class I genes with vehicle or tazemetostat treatment; gray, IgG; red, H3K27me3; blue, H3K4me3. D FPKM values of HLA-B from the RNA-Seq (Fig. 3A) in vehicle or tazemetostat treated conditions in indicated models. Columns, means of values (n = 3); white, vehicle treatment; red, tazemetostat treatment; bars, SEM values; *p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. E Scatter plot of log2FoldChange of H3K27me3 and H3K4me3 upon tazemetostat treatment as described in (B) on bivalent promoters in WCM154; light blue, bivalent promoters in vehicle condition (n = 1535); red, bivalent promoters losing H3K27me3 and corresponding genes upregulated upon tazemetostat treatment (n = 209; log₂FoldChange > 0; p-value < 0.05); r value corresponding p-value calculated from Pearson’s product-moment correlation analysis; gray line, linear regression; gray band, standard error of regression. F Gene ontology of bivalent promoters losing H3K27me3 and corresponding genes upregulated upon tazemetostat treatment (n = 209, log₂FoldChange > 0; p-value < 0.05;) as described in (E); size, -log₁₀FDR; orange-black gradient, gene ratio. G HOMER analysis was performed setting the promoter region as 1 kb upstream or downstream from TSS of bivalent genes in WCM154 and significantly enriched transcription factor motifs are highlighted.