Fig. 7: Convergence of dual AR regulatory functions on PCAT1 SEs and MYC regulation.

a Annotation of AR and co-factor binding at PCAT1-SE in clinical PCa. Binding profiles annotated: AR, H3K27ac, FOXA1, and HOXB13 (GSE130408). For each the profiling was based on average of normalized signals across the cases as indicated. Bottom track is an alignment of VCaP MYC ChIP-seq generated in this study. N: normal; T: primary PCa; M: metastatic CRPC. b Combined AR HiChIP, H3K27ac ChIP-seq and AR ChIP-seq annotation showing confident AR-mediated interactions anchored at MYC promoter. c ChIP-qPCR of H3K27ac, BRD4, and MYC at MYC binding site in PCAT1 SE with BRD4 antagonist JQ1 (500 nM) or DHT (10 nM) treatment for 2 h in VCaP cells in androgen-depleted medium. d VCaP cells in androgen-depleted medium treated with DHT and/or JQ1. Total RNA was subjected to qRT-PCR analysis of MYC with GAPDH as internal control. e, f LNCaP-AR stable pools were generated to overexpress Flag-AR versus mutants: M1 (R598A-N599A: mutation at the DBD zinc finger 2 (ZF2) D-box that mediates AR dimerization); M2 (C619Y: mutation at AR DBD between ZF2 and hinge domain, defective in DNA binding but not nuclear localization; M3 (C562A-C595A: mutations in AR DBD at two zinc finger cysteine residues that are in ZF1 and ZF2, respectively); and M4 (K630A-K632A-K633A: mutation in the NLS (nuclear localization signal) at the hinge region, defective in both nuclear localization and transactivation. Ctrl: parental LNCaP. The Western blotting experiment was repeated independently three times with similar results. g Parental LNCaP (ctrl, −), LNCaP-AR WT, and mutants stable pools were cultured in androgen-depleted CDS medium for 2 days, and then subjected to androgen treatment (10 nM of DHT) as indicated. Total RNA was submitted to RT-PCR analysis of MYC (GAPDH as internal control) and the ratio was normalized to the vehicle-treated control, which was set at 1. For c, d, and g, Data are presented as mean values ± SD. For each test, n = 3 biologically independent samples. P values were determined by two-sided Student’s t test. N.S., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Source data are provided in Histogram and Immunoblot Source Data files.