Pou4f1

Protein was extracted from the cells with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Cat# 78 440, Thermo Fisher Scientific). The protein concentration was measured by Pierce BCA Protein Assay kit (Cat# 23 225, Thermo Fisher Scientific). Quantified protein lysates were resolved on SDS–PAGE gels, then transferred to polyvinylidene difluoride membranes. The primary antibodies against POU4F1 (Cat# ab81213 or Cat# ab245230, abcam), ERα (Cat# 8644, Cell Signaling Technology), Phospho‐Rb (Cat# 9308, Cell Signaling Technology), E2F2 (Cat# ab138515, abcam), CCND1 (Cat# 2978, Cell Signaling Technology), CDK2 (Cat# ab32147, abcam), Phospho‐CDK2 (Thr160)(Cat# 2561, Cell Signaling Technology), EZH2 (Cat# 5246, Cell Signaling Technology), PR (Cat# 8757, Cell Signaling Technology), H3K27me3 (Cat# 9733, Cell Signaling Technology), Phospho‐EZH2 (Thr416)(AF3585, Affinity Biosciences), GAPDH (Cat# 5174, Cell Signaling Technology) were used. HRP‐conjugated secondary anti‐rabbit or mouse antibody (Cat# 7074 or Cat# 7076, Cell Signaling Technology) was used and the antigen–antibody reaction was visualized using an enhanced chemiluminescence assay (Cat# 34 577, Thermo Fisher Scientific).

ChIP‐qPCR was performed as previously described.^[48 (link)
^] Briefly, cells were washed with PBS, crosslinked with 1% formaldehyde for 10 min at RT and neutralized by glycine (1.25 M), followed by lysed and sonicated in Bioruptor Sonicator (Diagenode, USA). The resulting whole cell extract was incubated with H3K27me3 (Cat# 9733, Cell Signaling Technology), H3K4me3 (Cat# ab8580, abcam), EZH2 (Cat# 5246, Cell Signaling Technology), POU4F1 (Cat# ab245230, abcam) and normal rabbit IgG (Cat# 2729, Cell Signaling Technology) overnight at 4 °C and then Protein G magnetic beads (Invitrogen) was added for additional 4 h. The bound complexes were eluted from the beads and inverse‐crosslinked at 65 °C overnight. The enriched DNA was purified by treatment with Rnase A, proteinase K and HiPure DNA Clean Up Kit (Cat# D2141, Magen Biotechnology) and then subjected to qPCR analysis. The primers were listed in Table S6 (Supporting Information).