title: The mouse RANKL gene locus is defined by a broad pattern of histone H4 acetylation and regulated through distinct distal enhancers.
abstract: RANKL is a stromal cell-derived tumor necrosis factor (TNF)-like factor that plays a primary role in osteoclast formation and function. Recent studies suggest that 1,25(OH)(2) D(3) induces Rankl expression via vitamin D receptor (VDR) interaction at several enhancers located up to 76  kb upstream of the gene's transcriptional start site (TSS). In the current studies, we explored these interactions further using ChIP-chip and RNA analysis. We confirm VDR and RXR binding to the five enhancers described previously and identify two additional sites, one located within the Rankl coding region. We also show that RNA polymerase II is recruited to these enhancers, most likely through transcription factors TBP, TFIIB, and TAF(II) 250. Interestingly, the recruitment of these factors leads to the production of RNA transcripts, although their role at present is unknown. We also discovered that histone H4 acetylation (H4ac) marks many upstream Rankl enhancers under basal conditions and that H4ac is increased upon 1,25(OH)(2) D(3) treatment. Surprisingly, the hormone also induces C/EBPβ binding across the Rankl locus. C/EBPβ binding correlates directly with increased H4ac activity following 1,25(OH)(2) D(3) treatment. Finally, elevated H4ac is restricted to an extended region located between two potential insulator sites occupied by CTCF and Rad21. These data suggest a mechanism whereby 1,25(OH)(2) D(3) functions via the VDR and C/EBPβ to upregulate Rankl expression.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Martowicz
authors.forename: Mark B
authors.initials: MB
authors.lastname: Meyer
authors.forename: J Wesley
authors.initials: JW
authors.lastname: Pike
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000818:Animals; D050071:Bone Density Conservation Agents / Q000494:pharmacology; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism; D000076246:CCCTC-Binding Factor; D002117:Calcitriol / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins; D056426:Genetic Loci / Q000502:physiology*; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D038101:Insulator Elements / Q000502:physiology; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000096:biosynthesis* / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000502:physiology*; D035581:Transcription Factor TFIIB / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*
chemical_list: D050071:Bone Density Conservation Agents; D022782:CCAAT-Enhancer-Binding Protein-beta; D000076246:CCCTC-Binding Factor; D018797:Cell Cycle Proteins; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D010750:Phosphoproteins; D053245:RANK Ligand; C465862:Rad21 protein, mouse; D018167:Receptors, Calcitriol; D012097:Repressor Proteins; C505689:Tnfsf11 protein, mouse; D035581:Transcription Factor TFIIB; D012319:RNA Polymerase II; D002117:Calcitriol