title: Multiple enhancer regions located at significant distances upstream of the transcriptional start site mediate RANKL gene expression in response to 1,25-dihydroxyvitamin D3.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA. skim@biochem.wisc.edu
authors.forename: Sungtae
authors.initials: S
authors.lastname: Kim
authors.forename: Miwa
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Lee A
authors.initials: LA
authors.lastname: Zella
authors.forename: Mark B
authors.initials: MB
authors.lastname: Meyer
authors.forename: Jackie A
authors.initials: JA
authors.lastname: Fretz
authors.forename: Nirupama K
authors.initials: NK
authors.lastname: Shevde
authors.forename: J Wesley
authors.initials: JW
authors.lastname: Pike
mesh_terms: D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D051379:Mice; D011485:Protein Binding; D053245:RANK Ligand / Q000235:genetics*; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics; D047488:Retinoid X Receptors / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D053245:RANK Ligand; D018167:Receptors, Calcitriol; D047488:Retinoid X Receptors; C118756:dihydroxy-vitamin D3; D014807:Vitamin D