Inhibitor of DNA binding/differentiation protein 4 (ID4) is dominant negative 
helix loop helix transcriptional regulator is epigenetically silenced due to 
promoter hyper-methylation in many cancers including prostate. However, the 
underlying mechanism involved in epigenetic silencing of ID4 is not known. Here, 
we demonstrate that ID4 promoter methylation is initiated by EZH2 dependent 
tri-methylation of histone 3 at lysine 27 (H3K27me3). ID4 expressing (LNCaP) and 
non-expressing (DU145 and C81) prostate cancer cell lines were used to 
investigate EZH2, H3K27me3 and DNMT1 enrichment on ID4 promoter by Chromatin 
immuno-precipitation (ChIP). Enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and 
C81 cell lines compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in 
DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, 
H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent 
manner. ChIP data on prostate cancer tissue specimens and cell lines suggested 
EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data 
indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer 
through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased 
EZH2 but decreased ID4 expression in prostate cancer strongly supports this 
model.