In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells (original) (raw)
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Journal of Cellular Biochemistry, 1994
Vitamin D responsive transcription of the bone-specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post-proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D-dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA-directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half-elements, whereas the V2 complex appears to recognize the proximal half-element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype. r 1994 ~i~e y-~i s s , Inc.
Proceedings of the National Academy of Sciences, 1992
The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nudeotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (a) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE,
The Journal of Steroid Biochemistry and Molecular Biology, 2010
The architectural organization of the genome and regulatory proteins within the nucleus supports gene expression in a physiologically regulated manner. In osteoblastic cells ligand activation induces a nuclear punctate distribution of the 1␣,25-dihydroxy vitamin D3 (1␣,25(OH) 2 D 3 ) receptor (VDR) and promotes its interaction with transcriptional coactivators such as SRC-1, NCoA-62/Skip, and DRIP205. Here, we discuss evidence demonstrating that in osteoblastic cells VDR binds to the nuclear matrix fraction in a 1␣,25(OH) 2 D 3 -dependent manner. This interaction occurs rapidly after exposure to 1␣,25(OH) 2 D 3 and does not require a functional VDR DNA binding domain. The nuclear matrix-bound VDR molecules colocalize with the also nuclear matrix-associated coactivator DRIP205. We propose a model where the rapid association of VDR with the nuclear matrix fraction represents an event that follows 1␣,25(OH) 2 D 3dependent nuclear localization of VDR, but that precedes 1␣,25(OH) 2 D 3 -dependent transcriptional upregulation at target genes.
Journal of Cellular Physiology, 2008
Binding of 1a,25-dihydroxy vitamin D 3 to the C-terminal ligand-binding domain (LBD) of its receptor (VDR) induces a conformational change that enables interaction of VDR with transcriptional coactivators such as members of the p160/SRC family or the DRIP (vitamin D receptor-interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. The p160/SRC members contain intrinsic histone acetyl transferase (HAT) activities that remodel chromatin at promoter regulatory regions, and the DRIP/Mediator complex may establish a molecular bridge between the VDR complex and the basal transcription machinery. Here, we have analyzed the rate of recruitment of these coactivators to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1a,25-dihydroxy vitamin D 3 . We report that in intact osteoblastic cells VDR, in association with SRC-1, rapidly binds to the OC promoter in response to the ligand. The recruitment of SRC-1 correlates with maximal transcriptional enhancement of the OC gene at 4 h and with increased histone acetylation at the OC promoter. In contrast to other 1a,25-dihydroxy vitamin D 3 -enhanced genes, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter only after several hours of incubation with 1a,25-dihydroxy vitamin D 3 , concomitant with the release of SRC-1. Together, our results support a model where VDR preferentially recruits SRC-1 to enhance bone-specific OC gene transcription.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1995
The effects of two vitamin D analogs, 1,25-dihydroxyvitamin D-2 and 24-epi-l,25-dihydroxyvitamin D-2, were examined on osteocalcin gene expression in the rat osteosarcoma cell line ROS 17/2.8. Our results indicate that these analogs are more transcriptionally active than 1,25-dihydroxyvitamin D-3, particularly the 24-epimer. Assessment of reporter gene chloramphenicol acetyltransferase (CAT) activity, using the vitamin D responsive element (VDRE) derived from the human osteocalcin gene promoter, revealed that both analogs stimulated CAT activity 5-to 10-fold. 1,25-Dihydroxyvitamin D-2 was slightly more active than 1,25-dihydroxyvitamin D-3, while the 24-epimer was twice as effective. 1,25-Dihydroxyvitamin D-3 also stimulated osteocalcin mRNA accumulation by 2-fold over vehicle-treated cells, 1,25-dihydroxyvitamin D-2 by 2.5-fold, and 24-epi-l,25-dihydroxyvitamin D-2 by 4-fold. Electrophoretic mobility shift assays using the osteocalcin vitamin D responsive element revealed no increase in DNA binding with either analog when compared to 1,25-(OH)2D 3. Examination of CAT activity using the rat 24-hydroxylase VDRE indicated no significant difference in transcription with these compounds, suggesting that the vitamin D-2 analogs preferentially activate osteocalcin gene expression.
Endocrinology, 1999
Responsiveness of genes to steroid hormones is a complex process involving synergistic and/or antagonistic interactions between specific receptors and other nonreceptor transcription factors. Thus, DNA recognition elements for steroid hormone receptors are often located among binding sites for other transacting factors. The hormonal form of vitamin D, 1,25-dihydroxyvitamin D 3 , stimulates transcription of the tissue-specific osteocalcin (OC) gene in osteoblastic cells. The rat OC vitamin D response element contains an internal acitvating protein-1 (AP-1) site. Here, we report for the first time that this AP-1 site is critical for the transcriptional enhancement of rat osteocalcin gene expression mediated by vitamin D. Precise mutations were introduced either in the steroid half-elements or in the internal AP-1 sequences. One mutation within the internal AP-1 site retained vitamin D receptor/retinoid X receptor binding equivalent to that of the wild-type sequence, but resulted in complete loss of vitamin D inducibility of the OC promoter. These results suggest a functional interaction between the hormone receptor and nuclear oncoproteins at the rat OC vitamin D response element. This cooperation of activities may have important consequences in physiological regulation of osteocalcin transcription during osteoblast differentiation and bone tissue development in vivo. (Endocrinology 140: 63-70, 1999)
Steroids, 2001
The vitamin D response element in the bone tissue-specific osteocalcin gene has served as a prototype for understanding molecular mechanisms regulating physiologic responsiveness of vitamin D-dependent genes in bone cells. We briefly review factors which contribute to vitamin D transcriptional control. The organization of the vitamin D response element (VDRE), the multiple activities of the vitamin D receptor transactivation complex, and the necessity for protein-protein interactions between the VDR-RXR heterodimer activation complex and DNA binding proteins at other regulatory elements, including AP-1 sites and TATA boxes, provide for precise regulation of gene activity in concert with basal levels of transcription. We present evidence for molecular mechanisms regulating vitamin D-dependent mediated transcription of the osteocalcin gene that involve chromatin structure of the gene and nuclear architecture. Modifications in nucleosomal organization, DNase I hypersensitivity and localization of vitamin D receptor interacting proteins in subnuclear domains are regulatory components of vitamin D-dependent gene transcription. A model is proposed to account for the inability of vitamin D induction of the osteocalcin gene in the absence of ongoing basal transcription by competition of the YY1 nuclear matrix-associated transcription factor for TFIIB-VDR interactions. Activation of the VDR-RXR complex at the OC VDRE occurs through modifications in chromatin mediated in part by interaction of OC gene regulatory sequences with the nuclear matrix-associated Cbfa1 (Runx2) transcription factor which is required for osteogenesis.
Molecular Endocrinology, 1997
The sequences in the rat osteocalcin gene that lie 3 to the vitamin D response element (VDRE) have been shown to augment transcriptional activation by 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ]. These DNA sequences, however, are unable to bind the VDR or mediate 1,25-(OH) 2 D 3 responsiveness independently of the VDRE. To further characterize this region, the functional properties of a series of mutant oligonucleotides were examined in transiently transfected ROS 17/2.8 cells. When these mutant oligonucleotides were expressed upstream of the heterologous herpes simplex virus thymidine kinase promoter, the bases between ؊420 and ؊414 of the rat osteocalcin gene were identified as critical for maximal transactivation by 1,25-(OH) 2 D 3. Furthermore, mutation of these sequences in the context of the native osteocalcin promoter and enhancer totally abolished the ability of the VDRE to mediate 1,25-(OH) 2 D 3 responsiveness. These bases, which are essential for the 1,25-(OH) 2 D 3 responsiveness of the rat osteocalcin gene, are also present in a similar position, relative to the VDRE, in the human osteocalcin gene. To explore whether these sequences could enhance transactivation by other inducible transcription factors, they were examined for their ability to synergize with the chick vitellogenin estrogen response element and the rat somatostatin cAMP response element. When placed upstream to the herpes simplex virus thymidine kinase promoter and transfected into ROS 17/2.8 cells, these sequences were able to enhance transcriptional responsiveness to 17-estradiol and forskolin, respectively, demonstrating that they also contribute to transactivation by other inducible transcription factors. (Molecular Endocrinology 11: 210-217, 1997)
Endocrinology, 1999
Responsiveness of genes to steroid hormones is a complex process involving synergistic and/or antagonistic interactions between specific receptors and other nonreceptor transcription factors. Thus, DNA recognition elements for steroid hormone receptors are often located among binding sites for other transacting factors. The hormonal form of vitamin D, 1,25-dihydroxyvitamin D 3 , stimulates transcription of the tissue-specific osteocalcin (OC) gene in osteoblastic cells. The rat OC vitamin D response element contains an internal acitvating protein-1 (AP-1) site. Here, we report for the first time that this AP-1 site is critical for the transcriptional enhancement of rat osteocalcin gene expression mediated by vitamin D. Precise mutations were introduced either in the steroid half-elements or in the internal AP-1 sequences. One mutation within the internal AP-1 site retained vitamin D receptor/retinoid X receptor binding equivalent to that of the wild-type sequence, but resulted in complete loss of vitamin D inducibility of the OC promoter. These results suggest a functional interaction between the hormone receptor and nuclear oncoproteins at the rat OC vitamin D response element. This cooperation of activities may have important consequences in physiological regulation of osteocalcin transcription during osteoblast differentiation and bone tissue development in vivo. (Endocrinology 140: 63-70, 1999)
Journal of Cellular Physiology, 2010
1α,25-dihydroxy vitamin D 3 (Vitamin D 3 ) has an important role during osteoblast differentiation as it directly modulates the expression of key bone-related genes. Vitamin D 3 binds to the Vitamin D 3 receptor (VDR), a member of the superfamily of nuclear receptors, which in turn interacts with transcriptional activators to target this regulatory complex to specific sequence elements within gene promoters. Increasing evidence demonstrates that the architectural organization of the genome and regulatory proteins within the eukaryotic nucleus support gene expression in a physiological manner. Previous reports indicated that the VDR exhibits a punctate nuclear distribution that is significantly enhanced in cells grown in the presence of Vitamin D 3 . Here, we demonstrate that in osteoblastic cells, the VDR binds to the nuclear matrix in a vitamin D 3dependent manner. This interaction of VDR with the nuclear matrix occurs rapidly after vitamin D 3 addition and does not require a functional VDR DNA binding domain. Importantly, nuclear matrix-bound VDR colocalizes with its transcriptional coactivator DRIP205/TRAP220/MED1 which is also matrix-bound. Together these results indicate that after ligand stimulation the VDR rapidly enters the nucleus and associates with the nuclear matrix preceding vitamin D 3transcriptional upregulation.