Controlling the chromatin organization of vitamin D target genes by multiple vitamin D receptor binding sites

An essential prerequisite for the direct modulation of transcription by 1alpha,25-dihydroxy vitamin D(3) (1alpha,25(OH)(2)D(3)) is the location of at least one activated vitamin D receptor (VDR) protein close to the transcription start site of the respective primary 1alpha,25(OH)(2)D(3) target gene. This is achieved through the specific binding of VDR to a 1alpha,25(OH)(2)D(3) response element (VDRE). 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, approximately 8kB of the promoter regions of the primary 1alpha,25(OH)(2)D(3) target genes CYP24, cyclin C and p21((Waf1/Cip1)) were screened by chromatin immunoprecipitation (ChIP) assays for VDR binding sites using antibodies against VDR and its partner proteins. This approach identified three to four functional VDREs per gene promoter. In parallel, in silico screening of the extended gene areas (i.e. 10kB of promoter, introns, exons and 10kB of the downstream region) of all six members of the insulin-like growth factor binding protein (IGFBP) gene family was performed. Gel shift, reporter gene and ChIP assays identified in total 10 functional VDREs in the genes IGFBP1, IGFBP3 and IGFBP5. Taken together, both screening approaches suggest that a reasonable proportion of all VDR target genes, if not all, are under the control of multiple VDREs.