The Journal of Steroid Biochemistry and Molecular Biology
ReviewFundamentals of vitamin D hormone-regulated gene expression
Introduction
The vitamin D hormone 1,25(OH)2D3 exerts its diverse biological effects in target tissues by regulating gene expression [1]. Research over the past several decades focused upon several specific genes has shown that this activity is mediated the vitamin D receptor (VDR) which, following activation by 1,25(OH)2D3, binds as a retinoid X receptor (RXR) heterodimer directly to specific nucleotide sequences (vitamin D response elements or VDREs) located in regulatory regions of DNA [2], [3], [4], [5], [6]. This binding triggers the recruitment of diverse sets of enzymatic coregulatory complexes that remodel chromatin, facilitate the epigenetic modification of histones and influence the local concentration of RNA polymerase II (RNA pol II) at target gene promoters [7]. While this mechanism represents a general characterization drawn from the existing collection of gene targets, numerous gene-specific variations on this theme exist, perhaps the most important being the means through which the vitamin D hormone suppresses gene expression. Here, few clear examples exist and much less information is currently available. In the case of the cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) and parathyroid hormone (PTH) genes, their downregulation appears to involve the ability of the VDR to interact directly with and to nullify the activity of a prebound transcription factor that is essential for the expression of these genes [8], [9], [10]. Other negative regulatory mechanisms are likely to exist, however, as VDREs that permit negative regulation have been suggested and other modes of suppression that directly influence DNA structure are also possible [11]. However, the role of the ligand in VDR activation under many of these circumstances remains ill-defined. Finally, the DNA-independent interaction of the VDR with numerous transcription factors that are the regulatory end points of various signaling pathways defines additional sets of general mechanisms for both positive and negative signal integration through which the VDR modulates gene expression (reviewed in [12]). As is clear from this brief summary, while the central role of the VDR is quite clear, the underlying actions of the vitamin D hormone to modulate gene expression are indeed complex and much remains to be learned.
Delineation of the mechanism of action of 1,25(OH)2D3 defined over the past several decades has relied upon a cohort of standard molecular biological and biochemical methodologies in vitro [2]. These include mapping the activities of wildtype and mutant gene promoter/reporter plasmids in response to 1,25(OH)2D3 following transient transfection into cultured cells, determining the influence of various transcription factors including the VDR on reporter plasmids following co-transfection of plasmids overexpressing these factors, and examining the ability of these factors to interact with each other as well as with DNA sequences using biochemical interaction assays. While these and other assays have provided considerable insight into how genes are regulated, the methods display considerable limitations. First, the cloning and analysis of segments of genetic material is highly biased toward short regions of DNA (1–3 kb) located near gene promoters despite considerable genetic and clinical evidence that regulatory regions can occur distal to these transcriptional start sites. Second, segments of cloned DNA are restricted in size, rarely contain the entire span of DNA that constitutes an entire boundary-limited gene locus, and, following transient transfection, are unlikely to be properly chromatinized or to contain the appropriate epigenetic marks that characterize regulatory and other key landmark features of endogenous gene loci. Biochemical interaction assays are also flawed for a multitude of reasons, the least of which is the consequence of gene overexpression and the flagrant use of exceptionally high concentrations of reactant proteins. Many of these analytical difficulties have been overcome recently through the development of chromatin immunoprecipitation (ChIP) assays which now permit the detection and localization of transcription factors and both epigenetic DNA and histone modifications at specific sites on genomes without significant cellular modification both in vitro and in vivo [13], [14]. Coupled to tiled microarrays (ChIP-chip analysis) [15], [16] and now almost exclusively to the use of Next Generation Sequencing (NGS) methods (ChIP-seq analysis) [17], [18], chromatin immunoprecipitation is capable of providing detailed, unbiased transcription factor as well as epigenetic data on a genome-wide scale. The use of these techniques permits overarching assessment of the consequences of vitamin D hormone action at target cell genomes at the level of VDR DNA binding (termed the VDR cistrome), requirements for RXR co-localization at VDR binding sites, differences that emerge as a result of activation vs. suppression, and identification of numerous additional features of vitamin D action not previously approachable [19], [20]. Perhaps as important, these mechanistic data sets can be linked directly through DNA microarray or RNA sequencing methods [21] to parallel genome-wide measurements of the transcriptome under basal and induced conditions such as those which occur during treatment with 1,25(OH)2D3. They also permit a reassessment of the tenets of 1,25(OH)2D3 action that have emerged through single gene studies conducted over the past several decades. This review, generally of our own research, provides a summary of the results of such genome-wide studies which highlight features of vitamin D hormone action that are consistent with earlier tenets and features that are novel.
Section snippets
Chromatin immunoprecipitation
Chromatin immunoprecipitation was conducted as previously described [22], [23].
Tiled microarray (ChIP-chip) analysis
ChIP-chip analysis was conducted as described [24], [25].
DNA sequencing and ChIP-seq analysis
ChIP-seq analysis and NGS was carried out as documented in Meyer et al. [23].
Bioinformatic processing and analyses
Bioinformatic analyses were carried out as previously described [23].
The VDR and RXR cistromes
ChIP-chip and ChIP-seq analyses have been conducted in target cell lines to determine the number of DNA binding sites occupied by the VDR (the cistrome) in the absence and presence of 1,25(OH)2D3 (Table 1) [22], [23], [26], [27], [28], [29], [30]. In several of these studies, co-occupancy at these sites by several of the isoforms of RXR has also been determined [22], [23]. These analyses have revealed that despite the potential for 105–106 binding sites on either the mouse or human genomes
Conclusions
Early studies of a few selected target genes provided significant insight into how 1,25(OH)2D3 functions to regulate gene expression. The advent of ChIP, linked ultimately to unbiased methodologies that include the use of tiled microarrays and deep sequencing analysis, has revolutionized our approach to the study of gene regulation and provided new insight into how 1,25(OH)2D3 as well as other systemic and local factors operate to control the expression of genes. These unbiased methods have
Conflict of interest
The authors state they have no conflict of interest.
Acknowledgements
We thank members of the Pike Lab for their contributions to this review. This work was supported in part by research grants DK-72281, DK-73995, and DK-74993 from NIDDK and AR-45173 from NIAMS to JWP.
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