Multiple enhancer regions located at significant distances upstream of the transcriptional start site mediate RANKL gene expression in response to 1,25-dihydroxyvitamin D3

One of the primary regulators of receptor activator of NF-κB ligand (RANKL) is 1,25-dihydoxyvitamin D3 (1,25(OH)2D3). To elucidate the mechanism whereby 1,25(OH)2D3 activates RANKL expression we screened some 300 kb 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 70 kb 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)2D3. The region −76 kb upstream from the TSS, termed D5, was capable of mediating VDR-dependent transcriptional output in response to 1,25(OH)2D3 in luciferase assays. The identified VDRE in this region was able to confer dramatic 1,25(OH)2D3 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.