A Downstream Intergenic Cluster of Regulatory Enhancers Contributes to the Induction of CYP24A1 Expression by 1α,25-Dihydroxyvitamin D3

Mark B Meyer,Paul D Goetsch,J Wesley Pike

doi:10.1074/jbc.m110.119958

Mark B Meyer, Paul D Goetsch + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.119958

Copy DOI

Abstract

CYP24A1 expression is up-regulated by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via a vitamin D receptor (VDR)/retinoid X receptor (RXR) heterodimer that binds to two vitamin D response elements (VDREs) located near the proximal promoter. Interestingly, although 1,25(OH)(2)D(3) induced VDR/RXR binding to the VDRE-containing proximal promoter, the VDR/RXR heterodimer also localized to a cluster of at least four potential enhancers located in intergenic regions 50-69 kb downstream of the human CYP24A1 gene and 35-45 kb downstream of the mouse Cyp24a1 gene as revealed by ChIP-chip and ChIP-seq analyses. To address whether this downstream region and potential VDREs located within mediated CYP24A1 induction, we constructed recombinant wild-type and mutant bacterial artificial chromosome clones that spanned mouse and human loci and contained luciferase reporters inserted into their 3'-untranslated regions. The activity of these clones in stably transfected cells revealed that both the proximal and the putative downstream elements contributed to CYP24A1 up-regulation by 1,25(OH)(2)D(3). Further analysis using transfected enhancer fragments led to the identification of contributing regulatory elements in several of these downstream regions. Additional studies of coregulator recruitment using ChIP-chip analysis revealed both similarities and differences between the region located proximal to and those located downstream of the promoter. Recruitment of these coregulators was likely responsible for the increase in RNA polymerase II and histone H4 acetylation, which was also observed in response to 1,25(OH)(2)D(3) at the enhancer sites across the locus. We conclude that a more complex mechanism is responsible for the striking CYP24A1 up-regulation induced by the vitamin D hormone in target cells.

Highlights

The maintenance of active levels of hormone is a dynamic process responsive to a multitude of systemic signals that monitor the mineral status of the organism. 1,25(OH)2D3 production in the kidney is regulated positively by many factors, including parathyroid hormone [3] and regulated negatively by others [4], including the phosphate hormone FGF23 [5, 6]
The cloning of the structural gene provided an entree into studies of structure/function, the cloning of the chromosomal gene enabled studies focused upon understanding the underlying mechanisms important to the regulation of CYP24A1 expression by 1,25(OH)2D3 and other modulators. 1,25(OH)2D3 is known in general terms to regulate gene transcription via the vitamin D receptor (VDR), a nuclear protein that forms a heterodimer with retinoid X receptor (RXR) at specific DNA sequences termed vitamin D response elements (VDREs) and participates directly in the recruitment of complex molecular machinery that is essential for changing the level of expression of target genes [13, 14]
It is well established that CYP24A1 is regulated in most species through an activation event that is mediated in trans by a VDR/ RXR heterodimer and its associated cofactors and facilitated in cis through the two VDREs located near the CYP24A1 proximal promoter

Summary

Introduction

The maintenance of active levels of hormone is a dynamic process responsive to a multitude of systemic signals that monitor the mineral status of the organism. 1,25(OH)2D3 production in the kidney is regulated positively by many factors, including parathyroid hormone [3] and regulated negatively by others [4], including the phosphate hormone FGF23 [5, 6]. Similar ChIP samples derived from either vehicle or 1,25(OH)2D3-treated LS180 cells were prepared and subjected to massively parallel DNA sequencing methods as described under “Experimental Procedures.” As is evident, mappable tags for the VDR were largely absent at the VDRE-containing CYP24A1 proximal promoter following vehicle treatment, these tags were strongly induced in this region in the presence of 1,25(OH)2D3.

Results

Conclusion