Abstract
In this report, we explore the interaction of the vitamin D receptor (VDR) at regulatory sites within both the Cyp24a1 and the Trpv6 genes using chromatin immunoprecipitation techniques in a mouse model in vivo. We show that exogenous 1,25(OH)(2)D(3) induces rapid VDR and RXR (retinoid X receptor) binding to the Cyp24a1 gene in both the kidney and the intestine and to the Trpv6 gene in the intestine. Separate studies of Trpv6 in vitro suggest that VDR binding occurs directly to VDR response elements located -2 and -4 kb upstream of the TSS. VDR binding is dose-dependent, demonstrating EC(50) values that are comparable with those for the induction of both Cyp24a1 and Trpv6 mRNA. Importantly, interaction of the VDR with these targets results in rapid changes in histone 4 acetylation as well as the recruitment of RNA polymerase II. The presence of both VDR and RNA polymerase II at these sites declines between 3-6 h, whereas the changes observed in acetylation decrease more slowly. Finally, we show that whereas mediator protein 1 is recruited to the Cyp24a1 promoter in the intestine, this coactivator is apparently not required for Trpv6 activation. These studies provide the first evidence for 1,25(OH)(2)D(3)-induced VDR interaction at key target genes in vivo, revealing the consequences of that interaction on the Cyp24a1 and Trpv6 genes.
Highlights
When activated by 1,25(OH)2D3 binds DNA, recruits coregulators, and modulates transcriptional output
Using chromatin immunoprecipitation (ChIP), we show that 1,25(OH)2D3 rapidly induces localization of both vitamin D receptor (VDR) and retinoid X receptor (RXR) at these gene targets in a time- and dose-dependent manner
We utilized ChIP to explore the nature of the early events that occur at the Cyp24a1 and the Trpv[6] genes following treatment of mice with a single dose of 1,25(OH)2D3 in vivo
Summary
Reagents—1,25(OH)2D3 was obtained from Solvay (da Weesp, The Netherlands). Minimum Eagle’s medium was purchased from Cellgro (Herndon, VA). Total RNA was prepared from isolated kidneys and subjected to quantitative reverse transcriptase PCR using primers to Cyp24a1. Groups of mice (3/treatment) were injected with either vehicle or a single dose of 1,25(OH)2D3 (10 ng/g bw), and the kidneys were harvested 1 h later and subjected to ChIP analysis. Precipitated DNA was subjected to qPCR analysis, quantitated using an external standard curve, and corrected for input variations. Each point represents the average quantity/input for the three samples Ϯ S.E. with an unpaired t test analysis compared with vehicle (*, p Ͻ 0.05). These results are representative of at least six similar studies
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