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The regulatory actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on target genes are mediated by the vitamin D receptor (VDR). Interestingly, one of the genomic targets of 1,25(OH)(2)D(3) action is the VDR gene itself; however, the mechanism underlying this regulation is unknown. We investigated VDR autoregulation by screening the mouse VDR locus from 20kb upstream of the transcriptional start site (TSS) to 10kb downstream of the last exon using chromatin immunoprecipitation (ChIP)-DNA microarray analysis (ChIP/chip). Three potential VDR binding sites were located within introns lying downstream of the TSS and their activities confirmed through direct ChIP analysis. Further exploration revealed that one of these intronic regions was capable of conferring 1,25(OH)(2)D(3) response to both a downstream heterologous promoter and the minimal VDR promoter. Importantly, this regulatory region contained a classic vitamin D response element and was highly conserved within the human gene. We also demonstrated using ChIP analysis that the binding of VDR is associated with co-localization of RXR and the enhanced entry of RNA polymerase II. Thus, each of these sites appears likely to contribute to VDR autoregulation. Our studies using ChIP/chip analysis coupled to more traditional approaches define a direct mechanism whereby the VDR gene is upregulated by 1,25(OH)(2)D(3).

Canonical Wnt signaling is essential for bone formation. Activation involves binding of secreted members of the Wnt family of proteins with a membrane receptor Frizzled on osteoblasts, an interaction that is facilitated by LRP5/LRP6 co-receptors. LRP5 is known to play a particularly important role in bone formation such that the loss of this protein results in a reduction in osteoblast number, a delay in mineralization and a reduction in peak BMD. During the course of a VDR ChIP-chip analysis we found that 1,25(OH)(2)D(3) could induce binding of the VDR to sites within the Lrp5 gene locus. Importantly, this interaction between 1,25(OH)(2)D(3)-activated VDR and the Lrp5 gene led to both a modification in chromatin structure within the Lrp5 locus and the induction of LRP5 mRNA transcripts in vivo as well as in vitro. One site within Lrp5 was discovered to confer 1,25(OH)(2)D(3) response to a heterologous promoter in osteoblastic cells, permitting both the identification and characterization of the component VDRE. While the regulatory region in Lrp5 was highly conserved in the human genome, the VDRE was not. Our studies show that 1,25(OH)(2)D(3) can enhance the expression of a critical component of the Wnt signaling pathway which is known to impact osteogenesis.

One of the primary regulators of receptor activator of NF-kappaB ligand (RANKL) is 1,25-dihydoxyvitamin D(3) (1,25(OH)(2)D(3)). To elucidate the mechanism whereby 1,25(OH)(2)D(3) activates RANKL expression we screened some 300kb of the RANKL gene locus using a ChIP on chip analysis and identified five potential regulatory regions lying significant distances upstream of the transcription start site (TSS), the farthest over 70kb from the TSS. A direct ChIP analysis confirmed the presence of the VDR/RXR heterodimer at these sites. The binding of the VDR was associated with histone modification and enhanced entry of RNA polymerase II, indicating an important functional consequence to the localization of these transcription factors in response to 1,25(OH)(2)D(3). The region -76kb upstream from the TSS, termed D5, was capable of mediating VDR-dependent transcriptional output in response to 1,25(OH)(2)D(3) in luciferase assays. The identified VDRE in this region was able to confer dramatic 1,25(OH)(2)D(3) sensitivity to heterologous promoters. This region was highly evolutionarily conserved and functionally active in the human RANKL gene as well. We propose that the RANKL gene is regulated via multiple enhancers that while located at significant distances from the TSS, likely form a chromatin hub centered on the RankL promoter.