Genome wide VDR binding sites in RWPE1 human prostate epithelial cells

Signaling through the vitamin D receptor (VDR) has been proposed to suppress the development of epithelial cancers, including prostate cancer. We conducted ChIP-seq to identify the VDR binding sites in the genome of the prostate epithelial cell line, RWPE1. This analysis reveals a large number of VDR binding sites in both control cells and in cells treated with 10 nM 1,25 dihydroxyvitamin D3 for 3 hours. These peaks are associated with genes controlling a wide variety of cellular functions. Overall design: 80% cultures of RWPE1 cells were treated with 10 nM 1,25 dihydroxyvitamin D3 (1,25(OH)2D) or ethanol vehicle for 3 h. Chromatin immunoprecipitation (ChIP) was conducted using either normal rabbit IgG polyclonal antibody as a control (12-370, Millipore Sigma, Burlington, MA) or anti-VDR polyclonal antibody (C-20, sc-1008, Santa Cruz Biotechnology, Inc., Dallas, TX). Prior to sending samples for next generation sequencing, we conducted ChIP-PCR to detect ligand-inducible VDR binding to the -252 to -51 bp region of the CYP24A1 gene promoter and lack of VDR binding to the GAPDH gene promoter (42). Three biological replicates that met this criterion were pooled by treatment to generate three samples (control treated-VDR immunoprecipitation, 1,25(OH)2D treated-VDR immunoprecipitation, pooled control and 1,25(OH)2D treated-IgG immunoprecipitation). The ChIP procedure was repeated on these pooled samples and the resultant immunoprecipitations were sent to the Michael Smith Genome Science Centre (Vancouver, Canada) where 100-300 bp fragments were isolated from 12% polyacrylamide gels for library construction and 36 bp single-end reads were generated by Illumina sequencing. Each sample was sequenced in two lanes and the data for the two lanes were pooled for final analysis.