Impact of chromatin organization on the regulation of vitamin D receptor target genes expression in human prostate cells

Vitamin D Receptor (VDR) belongs to the superfamily of nuclear receptors that are in total 48 ligand-activated transcription factors that bind to the DNA and are involved in the regulation of gene expression. The active form of vitamin D3, hormone 1α,25-dihydroxyvitaminD3 (1α,25(OH)2D3), is a natural ligand for VDR. The generally described physiological functions of 1α,25(OH)2D3-activated VDR target genes are stimulation of metabolism as well as differentiation and inhibition of inflammation and cellular proliferation. In this study we focus primarily on the target genes of VDR that could potentially have a positive role in cancer prevention. Microarrays were performed of human non-malignant prostate RWPE1 cells after 4 h and 24 h treatment with 100 nM 1α,25(OH)2D3 and among several hundred responsive genes, we identified multiple members of the kallikrein (KLK) gene family as putative primary VDR targets. The KLKs are serine proteases that have been shown to be deregulated in various cancers. The 15 genes in the KLK family cluster together on human chromosome 19 span roughly 270 kB. KLK6 was the most responsive gene (16-fold induction after 4h treatment), followed by its neighboring KLK5, KLK7, KLK8 and KLK9. This effect of 1α,25(OH)2D3 was confirmed by real-time quantitative PCR and loss-of-function experiments. Interestingly, the VDR-mediated induction of the KLK genes was less pronounced in the cancer than in non-malignant prostate cells. In addition, we analyzed the genomic sequence of the KLK cluster in silico and identified a number of putative VDR binding sites (VDREs) as well as putative insulator CTCF binding sites. Chromatin regions containing putative binding sites were analyzed by chromatin immunoprecipitation assays to assess their functionality in RWPE1 cells. Six VDREs were associated with VDR whereas validation of detected CTCF binding sites showed increased occupancy of CTCF upon 1α,25(OH)2D3 stimulation. These results allow describing the changes in chromatin architecture on the KLK locus after VDR activation. Taken together, our study shows that 1α,25(OH)2D3-activated VDR has an impact on the regulation of the whole KLK gene cluster in prostate cells.