FIGURE 6.

HSF1 regulated target genes expression via PRMT5‐WDR5 axis‐mediated histone methylation. (A‐D) Chromatin immunoprecipitation (ChIP) ‐qPCR analysis of (A) H3R2me1, (B) H3R2me2s, (C) WDR5, and (D) H3K4me3 enrichment on the promoters of LEF1, MMP9, CCL20 and E2F2 in UM‐UC‐3 and T24 cells. (E) Quantification of LEF1, MMP9, CCL20 and E2F2 mRNA expression by qPCR after WDR5 knockdown in UM‐UC‐3 and T24 cells. (F) Western blotting assay of the protein levels of LEF1, MMP9, and E2F2, and (G) ELISA measuring CCL20 levels in WDR5‐silenced UM‐UC‐3 and T24 cells. (H‐K) ChIP‐qPCR analysis of HSF1, PRMT5, WDR5, H3R2me1, H3R2me2s, H3K4me3 and RNA polymerase‐II (Pol‐II) status at the (H) LEF1, (I) MMP9, (J) CCL20 and (K) E2F2 promoters in HSF1‐silenced UM‐UC‐3 cells. (L‐O) ChIP‐qPCR analysis of PRMT5, HSF1, WDR5, H3R2me1, H3R2me2s, H3K4me3 and Pol‐II enrichment at candidate HSF1 target genes promoters in PRMT5‐silenced UM‐UC‐3 cells. The error bars represent the standard deviations of three independent experiments. * P < 0.05 and ** P < 0.01. Abbreviations: ChIP, chromatin immunoprecipitation; qPCR, quantitative real‐time polymerase chain reaction; Pol‐II, RNA polymerase‐II; ns, not statistically significant