The vitamin D system is unique in that distinct calcium homeostatic functions and cell growth regulatory activities are mediated through a single ligand, calcitriol, acting through a specific receptor exhibiting ubiquitous tissue expression, the vitamin D receptor (VDR). The VDR is a member of a superfamily of nuclear steroid hormone receptors which regulate gene transcription by interacting with response elements in gene promoters. Structure-function analysis of the VDR protein has defined distinct domains involved in DNA binding, ligand binding, receptor dimerisation and gene transactivation, including a C-terminal activation function domain (AF-2) that is important for cofactor interaction. A model for regulation of gene transcription by the VDR is evolving and proposes VDR interaction with various components of the basal transcriptional machinery, including newly defined coactivators and corepressors, which may act to regulate gene transcription by altering histone acetylation and chromatin structure. This review describes the vitamin D endocrine system and the role of the VDR in regulating this system, including the molecular basis for the diverse actions of synthetic calcitriol analogues in the treatment of autoimmune disease and cancer.

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Hardeman, E. C. (1998) Mol. Cell. Biol. 18, 6641-6652) was identified in a yeast one-hybrid screen as a protein that binds within an upstream enhancer-containing region of the skeletal muscle-specific gene, TNNI1 (human troponin I slow; hTnI slow ). It has been proposed that hMus-TRD1␣1 may play an important role in fiber-specific muscle gene expression by virtue of its ability to bind to an Inr-like element (nucleotides ؊977 to ؊960) within the hTnI slow upstream enhancer-containing region that is necessary for slow fiber-specific expression. In this study we demonstrate that both MEF2C, a known regulator of slow fiber-specific genes, and hMusTRD1␣1 regulate hTnI slow through the Inr-like element. Co-transfection assays in C2C12 cells and Cos-7 cells demonstrate that hMusTRD1␣1 represses hTnI slow transcription and prevents MEF2C-mediated activation of hTnI slow transcription. Gel shift analysis shows that hMusTRD1␣1 can abrogate MEF2C binding to its cognate site in the hTnI slow enhancer. Glutathione S-transferase pull-down assays demonstrate that hMusTRD1␣1 can interact with both MEF2C and the nuclear receptor co-repressor. The data support the role of hMusTRD1␣1 as a repressor of slow fiber-specific transcription through mechanisms involving direct interactions with MEF2C and the nuclear receptor co-repressor.

The vitamin D receptor (VDR) is a ligand-dependent transcription factor that heterodimerizes with retinoid X receptor (RXR) and interacts with the basal transcription machinery and transcriptional cofactors to regulate target gene activity. The p160 coactivator GRIP1 and the distinct coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically enhance ligand-dependent VDR transcriptional activity. Both coregulators bind directly to and form a ternary complex with VDR, with GRIP1 contacting the activation function-2 (AF-2) domain and SKIP/NCoA-62 interacting through an AF-2 independent interface. It was previously reported that SKIP/NCoA-62 interaction with VDR was independent of the heterodimerization interface (specifically, helices H10/H11). In contrast, the present study defines specific residues within a conserved and surface-exposed region of VDR helix H10 that are required for interaction with SKIP/NCoA-62 and for full ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR helix H10 mutants at reduced levels compared with wild type in the absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/ NCoA-62 interacts with VDR at a highly conserved region not previously associated with coregulator binding to regulate transactivation by a molecular mechanism distinct from that of p160 coactivators. The vitamin D receptor (VDR)1 is a ligand-dependent transcription factor important in the regulation of calcium homeostasis, development, cell growth, and differentiation. VDR belongs to the nuclear receptor superfamily, members of which share a common modular structure including a highly conserved DNA binding domain (DBD) and a conserved ligand binding domain (LBD). The LBD has a predominantly ␣-helical structure with an activation function-2 (AF-2) domain in the COOH-terminal helix (H12). The LBD is the main site of VDR interaction with its heterodimer partner retinoid X receptor (RXR) and basal transcription factors, and the AF-2 domain in combination with the hydrophobic cleft forms an interaction surface for transcriptional corepressors and coactivators (1, 2). In the absence of its ligand 1,25-dihydroxyvitamin D 3

Human MusTRD1alpha1 was isolated as a result of its ability to bind a critical element within the Troponin I slow upstream enhancer (TnIslow USE) and was predicted to be a regulator of slow fiber-specific genes. To test this hypothesis in vivo, we generated transgenic mice expressing hMusTRD1alpha1 in skeletal muscle. Adult transgenic mice show a complete loss of slow fibers and a concomitant replacement by fast IIA fibers, resulting in postural muscle weakness. However, developmental analysis demonstrates that transgene expression has no impact on embryonic patterning of slow fibers but causes a gradual postnatal slow to fast fiber conversion. This conversion was underpinned by a demonstrable repression of many slow fiber-specific genes, whereas fast fiber-specific gene expression was either unchanged or enhanced. These data are consistent with our initial predictions for hMusTRD1alpha1 and suggest that slow fiber genes contain a specific common regulatory element that can be targeted by MusTRD proteins.

Synthetic ligands for the vitamin D receptor (VDR) are potential therapeutic agents for metabolic, neoplastic, and autoimmune disorders. Some of these ligands have similar or more potent antiproliferative, yet reduced hypercalcemic actions, than calcitriol. However, the mechanisms for these differential actions have not been clearly defined. We hypothesized that these gene-and tissue-specific effects may relate to ligand-directed selective recruitment of transcriptional coactivators. To identify key elements in ligand structure that facilitate VDR-coactivator interactions, the current studies assessed the ability of the VDR to recruit the coactivators GRIP1

A human MusTRD (muscle TFII-I repeat domain (RD)-containing protein) isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow [O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol. Cell. Biol. 18, 6641-6652]. MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs [Roy (2001) Gene 274, 1-13]. The human gene encoding MusTRD, GTF2IRD1 ( WBSCR11 / GTF3 ), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams-Beuren Syndrome [Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al. (1999) Genomics 57, 279-284; Franke, Peoples and Francke (1999) Cytogenet. Cell. Genet. 86, 296-304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures [Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles. These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription-PCR on RNA from various adult and embryonic muscles. The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3' region of the primary transcript of Gtf2ird1, the homologous mouse gene. The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms. Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters. This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.

The vitamin D(3) receptor (VDR) is a ubiquitously expressed nuclear hormone receptor, and its ligand, calcitriol, has diverse biological effects. The extent to which transcriptional coactivators are involved in modulating tissue-specific functions of the VDR is unclear. Hence, the current studies investigated the role of p160 coactivators in regulating VDR function and interaction with RXR. Two p160 coactivators, glucocorticoid receptor-interacting protein-1 (GRIP1) and receptor-associated coactivator-3 (RAC3), which are expressed in an inverse fashion in cell lines representative of calcitriol target tissues, interacted directly with the VDR, both in vitro and in yeast cells, but only in the presence of calcitriol. Deletional analyses of VDR indicated that GRIP1 and RAC3 required an intact VDR activation function (AF-2) domain for efficient interaction as well as additional but distinct regions of the VDR. Coexpression experiments in yeast cells indicated that both GRIP1 and RAC3 coassemble with the VDR to form an active transcriptional complex. They also form ternary complexes with VDR homodimers and VDR:RXRalpha heterodimers. In mammalian cells, GRIP1 augmented VDR activation of the osteocalcin promoter, whereas RAC3 enhanced VDR activation indirectly through RXR. These data suggest different coactivators regulate VDR function via distinct mechanisms and support the hypothesis that the VDR recruits different coactivators depending on specific gene and cellular contexts.