Fig. 6. EZH2 facilitates self-activation of E2F1 and thus promotes transcription of all CPC members.

A Western blot analysis of the protein level of E2F1 in PCa cell lines upon EZH2 knockdown. B Scatter plot showing the relationship between CDCA8 and E2F1 expressions using data from TCGA, with Spearman correlation coefficient (R) and P value as indicated. C Western blot analysis of the protein levels of all CPC members in PCa cell lines upon E2F1 knockdown. D ChIP-seq profiles showing the E2F1 peaks at the promoter region of each CPC member in LNCaP-abl cells. E ChIP-qPCR assay to monitor the enrichment of E2F1 at the promoter region of CDCA8 in C4–2 cells. Two pairs of primers (F1 and F2) were used to amplify fragments inside CDCA8 promoter region while another pair of primers (NC) targeting nearby region was used as negative control. F ChIP-seq profiles showing the peaks of EZH2, H3K27me3 and E2F1 at the promoter region of E2F1 in PCa cells. G ChIP-qPCR assay to monitor the enrichment of EZH2, H3K27me3 and E2F1 at the promoter region of E2F1 in C4–2 cells. Two pairs of primers (F1 and F2) were used to amplify fragments inside E2F1 promoter region while another pair of primers (NC) targeting nearby region was used as negative control. H ChIP-qPCR assay to monitor the change of E2F1 occupancy at the promoter region of E2F1 upon EZH2 inhibition in C4–2 cells. I RT-qPCR analysis of E2F1 mRNA level in EZH2-deficient C4–2 cells overexpressed with either wild-type (WT) or H689A-mutant EZH2. J Rescue assay showing that ectopic expression of either wild-type (WT) or H689A-mutant EZH2 could restore the downregulation of all CPC members in EZH2-deficient cells, as measured by western blot. K The proposed model of this study. EZH2-mediated CDCA8 upregulation in PCa can be attributed to the combination of let-7b repression and E2F1 self-activation.