The actions of the vitamin D hormone 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) are mediated by the vitamin D receptor (VDR), a ligand-activated transcription factor that functions to control gene expression. After ligand activation, the VDR binds directly to specific sequences located near promoters and recruits a variety of coregulatory complexes that perform the additional functions required to modify transcriptional output. Recent advances in transcriptional regulation, which permit the unbiased identification of the regulatory regions of genes, are providing new insight into how genes are regulated. Surprisingly, gene regulation requires the orchestrated efforts of multiple modular enhancers often located many kilobases upstream, downstream, or within the transcription units themselves. These studies are transforming our understanding of how 1,25(OH) 2 D 3 regulates gene transcription.
Research during the past 2 decades has established that the diverse biologic actions of 1,25-dihydroyxyvitamin D 3 (1,25(OH) 2 D 3 ) are initiated through precise changes in gene expression that are mediated by an intracellular vitamin D receptor (VDR). Activation of the VDR through direct interaction with 1,25(OH) 2 D 3 prompts the receptor’s rapid binding to regulatory regions of target genes, where it acts to nucleate the formation of large protein complexes whose functional activities are essential for directed changes in transcription. In most target cells, these actions trigger the expression of networks of target genes whose functional activities combine to orchestrate specific biologic responses. These responses are tissue-specific and range from highly complex actions essential for homeostatic control of mineral metabolism to focal actions that control the growth, differentiation, and functional activity of numerous cell types including those of the immune system, skin, the pancreas and bone, as well as many other targets that are described in this issue devoted to vitamin D. In these tissues, gene targets are numerous. New studies combined with new techniques are now revealing a surprising increase in mechanistic complexity wherein multiple regulatory regions, frequently located many kilobases upstream, within, or downstream of a target gene’s transcription unit, seem to participate in transcriptional modulation.
VDR structure and function
The VDR is Structurally Organized to Mediate Changes in Transcription in Response to 1,25(OH) 2 D 3
Despite nearly 2 decades of extensive biochemical characterization of the VDR after its discovery in 1974, it was the cloning of this receptor’s gene and the subsequent analysis of recombinant protein that led to key insights into its structure and its function. As depicted in Fig. 1 A, the VDR protein is comprised of 3 distinct regions, an N-terminal dual zinc finger DNA-binding domain, a C-terminal ligand-binding activity domain, and an extensive and unstructured region that links the 2 functional domains of this protein together. The C-terminal region of the molecule, whose three-dimensional structure has been solved by X-ray crystallography, is the most complex and is comprised of 12 α-helices as illustrated in Fig. 1 B. Amino acid contacts within a subset of these α-helices form a dynamic ligand-binding pocket, as shown in Fig. 1 C. Selective occupancy by 1,25(OH) 2 D 3 leads to the formation of 2 independent protein interaction surfaces on the VDR protein: 1 that facilitates interaction with a heterodimer partner required for specific DNA binding and 1 that is essential for the recruitment of large coregulatory complexes required for gene modulation. Additional studies suggest that the VDR can also be posttranslationally modified through phosphorylation, an alteration in the protein that may be capable of modulating and fine-tuning its transcriptional activity. Collectively, these domains within the VDR create a macromolecule receptive to physiologically relevant levels of circulating 1,25(OH) 2 D 3 and capable of directing cellular regulatory machinery to specific subsets of genes whose protein products are key to 1,25(OH) 2 D 3 response.
The VDR Specifies Target Genes Through its DNA-binding Properties
The zinc finger containing the DNA-binding domain of the VDR is typical of that found in all members of the steroid receptor gene family including those for estrogens, androgens, and glucocorticoids, as well as for thyroid hormone, retinoid acid, and other lipophilic regulators. The VDR is now known to recognize a specific DNA sequence or vitamin D response element (VDRE) comprised of 2 hexameric nucleotide half-sites separated by 3 base pairs (bp). Other response element structures also occur, although these appear much less frequently. The 2 DNA half-sites accommodate the binding of a heterodimer comprised of a VDR molecule and a retinoid X receptor (RXR) molecule. The latter forms a heterodimer with other members of the steroid receptor family as well, including receptors for retinoic acid and thyroid hormone, thus linking the activities of several different endocrine systems. Recent studies, described later, suggest that RXR is independently bound to many sites on the genome in the absence of an activating ligand, thereby marking potential regulatory sites for subsequent activation by 1,25(OH) 2 D 3 . 1,25(OH) 2 D 3 via its receptor also suppresses the transcriptional expression of numerous genes. The requirements for direct VDR DNA binding and for heterodimer formation with RXR in the suppression of gene activity are currently unclear.
The VDR Regulates Transcription Through its Ability to Recruit Coregulatory Complexes
Selective VDR DNA binding in a cell serves to highlight that subset of genes within a genome whose transcriptional activities are targeted under a specific set of conditions for modification by 1,25(OH) 2 D 3 . Changes in gene expression are not mediated directly via the VDR, however, but rather indirectly through the protein’s ability to facilitate through its transactivation domain the recruitment of large and diverse coregulatory machines that directly mediate such changes. This recruitment is often gene specific, suggesting a role for additional and as yet unidentified components. Coregulatory complexes generally contain 1 VDR-interacting component as well as many additional subunits, several of which can contain inherent enzymatic activity. These complexes include machines with ATPase-containing nucleosomal remodeling ability, enzymes such as acetyl- and deacetyltransferases and methyl- and demethyltransferases containing selective chromatin histone modifying capabilities, and complexes that play a role in RNA polymerase II (RNA pol II) recruitment and initiation such as Mediator, as documented in Fig. 2 . Each of these groups of proteins identifies a key step in the process of transcription regulation and many more are likely to be identified in the future. The details of how these machines operate to enhance or suppress the expression of these gene targets are only now beginning to emerge.
Vitamin D target genes
1,25(OH) 2 D 3 Regulates Networks of Genes in a Tissue/Cell-specific Fashion
As described earlier, the role of ligand-activated VDR is to direct cellular transcription machinery to specific sites on the genome where these complexes can influence the production of RNA, which encodes proteins that are integral to specific biologic activities. It is in this manner that 1,25(OH) 2 D 3 plays a central role in regulating mineral metabolism via its actions in intestinal and kidney epithelial cells and in specific bone cells. Although many target genes that play important roles in calcium and phosphorus homeostatic have been identified, additional targets important to these processes continue to be discovered. These include the calcium and phosphate transporters and their associated basolaterally located, energy-driven ion pumps in the intestine and kidney, and the osteoblast-synthesized osteoclastogenic differentiation factor receptor activator of NF-κB ligand (RANKL), which stimulates the activity of existing bone-resorbing osteoclasts, prolongs their lifespan, and induces the formation of new replacements. Vitamin D also regulates gene networks involved in bile acid metabolism in the colon, the degradation of xenobiotic compounds in several tissues, the differentiation of keratinocytes in skin, the development and cycling of dermal hair follicles, and the functions of key cell types involved in innate and adaptive immunity. The genes and gene networks that have been identified as responsible for these biologic actions of 1,25(OH) 2 D 3 are extensive. Indeed, many have emerged as a consequence of contemporary genome-wide analyses that are almost routinely conducted by investigators currently, and which are capable of measuring the effects of the hormone on entire cellular or tissue transcriptomes. Many of these gene networks are regulated by the hormone in a tissue-specific fashion. Perhaps most interesting is the intricate regulatory controls exerted directly by 1,25(OH) 2 D 3 and its receptor at genes involved in the vitamin D ligand’s production and degradation, actions that contribute to the maintenance of biologically active levels of intracellular 1,25(OH) 2 D 3 . Thus, as outlined in Fig. 3 , 1,25(OH) 2 D 3 suppresses the renal expression of Cyp27b1 , whose protein product is responsible for its synthesis, and induces Cyp24a1 , whose product is responsible for its degradation to calcitroic acid. In addition to these activities, 1,25(OH) 2 D 3 also autoregulates the expression of its own receptor gene (see Fig. 3 ), thus modulating not only levels of the ligand but also of the VDR. Some of the mechanistic details of this regulation are discussed later. Thus, 1,25(OH) 2 D 3 also contributes directly to the maintenance of the key signaling components essential for generating and mediating hormonal response.
Traditional Studies were Initiated by Identifying Target Genes and Defining Regions that Mediate Regulation by the Vitamin D Hormone
Identifying the site(s) of action of 1,25(OH) 2 D 3 at a target gene locus represents the first step in defining the molecular processes that are essential for altering a gene’s transcriptional output. This step is also important because it often leads to the identification of a region that is likely to provide important regulatory control after activation through other signaling pathways as well. Early studies of the osteocalcin gene and its regulation by 1,25(OH) 2 D 3 in bone cells provide an excellent example of this principle. Based on the ability of 1,25(OH) 2 D 3 to induce osteocalcin in bone cells, our early molecular studies, using a traditional human osteocalcin promoter-reporter plasmid approach coupled to classic protein-DNA interaction analyses, revealed the first DNA-binding site for the VDR. This site was located approximately 485 bp upstream of the human gene’s transcriptional start site (TSS) and was comprised of 2 directly repeated 6-bp sequences separated by 3 bp. Follow-on studies confirmed the general location and highly conserved nature of this vitamin D responsive region in the rat and mouse genes. The latter was functionally suppressed by 1,25(OH) 2 D 3 as a result of a strategic change in the regulatory element’s base structure thereby highlighting an important species-specific difference in vitamin D response. An extensive series of studies conducted more than a decade after these initial discoveries firmly established that this general region was a direct target for many different transcription factors some of which were activated by either separate or overlapping signal transduction pathways. The ability of these proteins to influence response to 1,25(OH) 2 D 3 and for the vitamin D hormone and its receptor to influence their actions was characterized. Perhaps the most important transcription factor to be discovered at the osteocalcin promoter was RUNX2, a regulatory protein now known to be essential to the formation and bone-forming activity of osteoblasts. During the ensuing years, many genes have been explored for the location of regulatory sites that are capable of mediating 1,25(OH) 2 D 3 action, binding the VDR and its heterodimer partner, and recruiting coregulatory complexes necessary for changes in transcriptional output. These include the genes for osteocalcin, osteopontin, bone sialoprotein, TRPV6 , PTH , PTHrp , Cyp24a1 , and Cyp27b1 as well as many others. In the case of Cyp24a1 , 2 sites located within 300 bp of the TSS were identified as significant mediators of the actions of 1,25(OH) 2 D 3 .
Vitamin D target genes
1,25(OH) 2 D 3 Regulates Networks of Genes in a Tissue/Cell-specific Fashion
As described earlier, the role of ligand-activated VDR is to direct cellular transcription machinery to specific sites on the genome where these complexes can influence the production of RNA, which encodes proteins that are integral to specific biologic activities. It is in this manner that 1,25(OH) 2 D 3 plays a central role in regulating mineral metabolism via its actions in intestinal and kidney epithelial cells and in specific bone cells. Although many target genes that play important roles in calcium and phosphorus homeostatic have been identified, additional targets important to these processes continue to be discovered. These include the calcium and phosphate transporters and their associated basolaterally located, energy-driven ion pumps in the intestine and kidney, and the osteoblast-synthesized osteoclastogenic differentiation factor receptor activator of NF-κB ligand (RANKL), which stimulates the activity of existing bone-resorbing osteoclasts, prolongs their lifespan, and induces the formation of new replacements. Vitamin D also regulates gene networks involved in bile acid metabolism in the colon, the degradation of xenobiotic compounds in several tissues, the differentiation of keratinocytes in skin, the development and cycling of dermal hair follicles, and the functions of key cell types involved in innate and adaptive immunity. The genes and gene networks that have been identified as responsible for these biologic actions of 1,25(OH) 2 D 3 are extensive. Indeed, many have emerged as a consequence of contemporary genome-wide analyses that are almost routinely conducted by investigators currently, and which are capable of measuring the effects of the hormone on entire cellular or tissue transcriptomes. Many of these gene networks are regulated by the hormone in a tissue-specific fashion. Perhaps most interesting is the intricate regulatory controls exerted directly by 1,25(OH) 2 D 3 and its receptor at genes involved in the vitamin D ligand’s production and degradation, actions that contribute to the maintenance of biologically active levels of intracellular 1,25(OH) 2 D 3 . Thus, as outlined in Fig. 3 , 1,25(OH) 2 D 3 suppresses the renal expression of Cyp27b1 , whose protein product is responsible for its synthesis, and induces Cyp24a1 , whose product is responsible for its degradation to calcitroic acid. In addition to these activities, 1,25(OH) 2 D 3 also autoregulates the expression of its own receptor gene (see Fig. 3 ), thus modulating not only levels of the ligand but also of the VDR. Some of the mechanistic details of this regulation are discussed later. Thus, 1,25(OH) 2 D 3 also contributes directly to the maintenance of the key signaling components essential for generating and mediating hormonal response.
Traditional Studies were Initiated by Identifying Target Genes and Defining Regions that Mediate Regulation by the Vitamin D Hormone
Identifying the site(s) of action of 1,25(OH) 2 D 3 at a target gene locus represents the first step in defining the molecular processes that are essential for altering a gene’s transcriptional output. This step is also important because it often leads to the identification of a region that is likely to provide important regulatory control after activation through other signaling pathways as well. Early studies of the osteocalcin gene and its regulation by 1,25(OH) 2 D 3 in bone cells provide an excellent example of this principle. Based on the ability of 1,25(OH) 2 D 3 to induce osteocalcin in bone cells, our early molecular studies, using a traditional human osteocalcin promoter-reporter plasmid approach coupled to classic protein-DNA interaction analyses, revealed the first DNA-binding site for the VDR. This site was located approximately 485 bp upstream of the human gene’s transcriptional start site (TSS) and was comprised of 2 directly repeated 6-bp sequences separated by 3 bp. Follow-on studies confirmed the general location and highly conserved nature of this vitamin D responsive region in the rat and mouse genes. The latter was functionally suppressed by 1,25(OH) 2 D 3 as a result of a strategic change in the regulatory element’s base structure thereby highlighting an important species-specific difference in vitamin D response. An extensive series of studies conducted more than a decade after these initial discoveries firmly established that this general region was a direct target for many different transcription factors some of which were activated by either separate or overlapping signal transduction pathways. The ability of these proteins to influence response to 1,25(OH) 2 D 3 and for the vitamin D hormone and its receptor to influence their actions was characterized. Perhaps the most important transcription factor to be discovered at the osteocalcin promoter was RUNX2, a regulatory protein now known to be essential to the formation and bone-forming activity of osteoblasts. During the ensuing years, many genes have been explored for the location of regulatory sites that are capable of mediating 1,25(OH) 2 D 3 action, binding the VDR and its heterodimer partner, and recruiting coregulatory complexes necessary for changes in transcriptional output. These include the genes for osteocalcin, osteopontin, bone sialoprotein, TRPV6 , PTH , PTHrp , Cyp24a1 , and Cyp27b1 as well as many others. In the case of Cyp24a1 , 2 sites located within 300 bp of the TSS were identified as significant mediators of the actions of 1,25(OH) 2 D 3 .
Related posts:
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
