Multifunctional Enhancers Regulate Mouse and Human Vitamin D Receptor Gene Transcription

Abstract

AbstractThe vitamin D receptor (VDR) mediates the endocrine actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and autoregulates the expression of its own gene in target cells. In studies herein, we used chromatin immunoprecipitation-chip analyses to examine further the activities of 1,25(OH)2D3 and to assess the consequences of VDR/retinoid X receptor heterodimer binding at the VDR gene locus. We also explored mechanisms underlying the ability of retinoic acid, dexamethasone, and the protein kinase A activator forskolin to induce VDR up-regulation as well. We confirmed two previously identified intronic 1,25(OH)2D3-inducible enhancers and discovered two additional regions, one located 6 kb upstream of the VDR transcription start site. Although RNA polymerase II was present at the transcription start site in the absence of 1,25(OH)2D3, it was strikingly up-regulated at both this site and at individual enhancers in its presence. 1,25(OH)2D3 also increased basal levels of H4 acetylation at these enhancers as well. Surprisingly, many of these enhancers were targets for CCAAT enhancer-binding protein-β and runt-related transcription factor 2; a subset also bound cAMP response element binding protein, retinoic acid receptor, and glucocorticoid receptor. Unexpectedly, many of these factors were resident at the Vdr gene locus in the absence of inducer, suggesting that they might contribute to basal Vdr gene expression. Indeed, small interfering RNA down-regulation of CCAAT enhancer-binding protein-β suppressed basal VDR expression. These regulatory activities of 1,25(OH)2D3, forskolin, and dexamethasone were recapitulated in MC3T3-E1 cells stably transfected with a full-length VDR bacterial artificial chromosome (BAC) clone-luciferase reporter gene. Finally, 1,25(OH)2D3 also induced accumulation of VDR and up-regulated H4 acetylation at conserved regions in the human VDR gene. These data provide important new insights into VDR gene regulation in bone cells.