Abstract

The vitamin D receptor (VDR), the nuclear receptor for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), controls gene expression by binding discrete DNA sequences in promoter regions of target genes, referred to as 1alpha,25(OH)2D3 response elements (VDREs). Although these elements are well characterized in vitro, the function of VDREs in living cells in the context of chromatin is still largely unknown. To resolve this issue, 7 to 8 kB of the promoter regions of the primary 1alpha,25(OH)2D3 target genes CYP24, cyclin C and p21(Waf1/Cip1) were studied by chromatin immunoprecipitation (ChIP) assays using antibodies against acetylated histone H4 (to assess the global chromatin status) and various other components of VDR-dependent gene activation, such as VDR, retinoid X receptor (RXR), coactivator (CoA) and corepressor proteins. This approach identified three to four functional VDREs per gene promoter. In parallel, the extended analysis of the gene areas, of all six members of the insulin-like growth factor binding protein (IGFBP) family (i.e., 10 kB of promoter, introns, exons and 10 kB of the downstream region) were screened in silico for putative VDR-binding sites. Gel shift, reporter gene and ChIP assays identified, in total, ten functional VDREs in the genes IGFBP1, 3 and 5 and real-time PCR confirmed that these genes are primary VDR targets. Taken together, these results suggest that a reasonable proportion of all VDR target genes, if not all, are under the control of multiple VDREs. These results will have an impact on the development of therapeutic regimes for diseases, such as cancer, that use 1alpha,25(OH)2D3 and its analogs.