2.3. Chromatin immunoprecipitation and quantitative PCR

Chromatin immunoprecipitation was performed as described previously [8]. In brief, 1 × 10⁷ cells were crosslinked with 1% formaldehyde, lysed by lysis buffer, and chromatin in the lysate was fragmented into 100–500 bps by sonication. Protein-DNA complexes were immunoprecipitated (IP) by 2 μg antibodies coupled with Dynal magnetic beads (Invitrogen). ChIP-grade antibodies used were anti-AR (Millipore; 06-680), anti-EZH2 (Cell signaling; #4905), anti-H3K27me3 (Diagenode; C15200181-50) and ant-IgG (Abcam; ab2410). The IP or input DNA was subjected to elution, reverse crosslink and purification. Purified IP and input DNA were analyzed by PCR quantification using SYBR® Premix Ex Taq™ Kit (TaKaRa). The primer sequences for individual genes were listed in the supplementary information. Promoter enrichment of EZH2, AXIN2, NKD1, PPP2R2B, PRICKLE1 and SFRP5 conjugated with respective proteins was determined and shown as the percentage of input DNA. IgG antibody was used as a negative control.