Fig. 3

An AR-binding site (P10) ~ 25 Kb downstream of the MYC gene was androgen-induced for interaction with MYC promoter and epigenetically repressed upon androgen treatment. A Schematic showing of the MYC-P10 locus in alignment with VCaP AR ChIP-Seq tracks, the 3C assay strategy based on PstI digestion (the targeting bait (anchor), relevant PstI sites, control sites and PCR primers). B Two-round PCR (nest-PCR) assay was performed to amplify the 3C signals of MYC-Pro-P10 hybrids, shown here was the round-2 PCR result. Highlighted DNA bands (B1–B7) were excised and purified for DNA-Seq, confirming androgen-induced MYC-Pro-P10 interaction in the B6 and B7 bands that are derived from two P10-proximal PstI sites. PC-F/R: positive loading control primers that target the MYC-Pro locus without PstI site in the amplicon. NC: negative control primer that targets a distal Chr8 site with proximal PstI site but no AR binding. C RT-qPCR analysis of the 3C signal between MYC-Pro and P10, with GAPDH (no PstI site in its amplicon) as reference. Data are mean values ± SD for two biologically independent samples. D 3C-ddPCR analysis of MYC-Pro-P10 interaction. The copy number reference is an RPPH1 probe that does not contain PstI site in the amplicon. E–G VCaP cells in androgen-depleted medium were treated with 10 nM of DHT for 4 h and then subjected to ChIP-qPCR analysis. Occupancy at the MYC-Pro and P10 sites are assessed by RT-qPCR that was normalized to input. Data are mean values ± SD for three biologically independent samples. H Global calculation of genomic BRD4 and H3K27ac chromatin occupancy peak counts based on the GSE55062 datasets. See Additional file 1: Fig. S1–S3 for additional information