The 1α,25-dihydroxy Vitamin D3 receptor preferentially recruits the coactivator SRC-1 during up-regulation of the osteocalcin gene (original) (raw)
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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.
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.
Biochemical Journal, 2002
1α,25-Dihydroxyvitamin D3-mediated transcriptional control of the bone-specific osteocalcin (OC ) gene requires the integration of regulatory signals at the vitamin D-responsive element (VDRE) and flanking tissue-specific sequences. The 1α,25-dihydroxyvitamin D3 receptor (VDR) is a member of the nuclear receptor superfamily and forms a heterodimeric complex with the receptor for 9-cis retinoic acid (RXR) that binds to the VDRE sequence. We have demonstrated previously that changes in chromatin structure at the VDRE region of the rat OC gene promoter accompany transcriptional enhancement in i o, suggesting a requirement for chromatin remodelling. Here we show that the VDRE in the distal region of the OC gene promoter is refractory to binding of the VDR-RXR complex when organized in a nucleosomal context. Addition of the ligand 1α,25-dihy-
Molecular and Cellular Biology, 2003
p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D 3 via the vitamin D 3 receptor complex. Therefore, we addressed p300 control of basal and vitamin D 3 -enhanced activity of the OC promoter. We find that transient overexpression of p300 results in a significant dose-dependent increase of both basal and vitamin D 3 -stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, we show that the endogenous Runx2/ Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. We also demonstrate by chromatin immunoprecipitation assays that p300, Runx2/Cbfa1, and 1␣,25-dihydroxyvitamin D 3 receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, as a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, we propose that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D 3 .
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,
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.
Proceedings of the National Academy of Sciences, 1993
In the osteocalcin (OC) gene promoter, both independent positive and negative regulatory elements, as well as others with contiguous [TATA/glucocorticoid-responsive elements (GRE)] or overlapping [TATA/GRE, vitamin D-responsive enhancer elements (VDRE)/AP-1, and OC box/AP-1] domains, are sites for modifications in protein-DNA interactions. In the present studies, we have examined nuclear protein extracts from fetal rat calvarial cells that undergo a developmental sequence of bone cell differentiation. Our results demonstrate modifications in protein-DNA interactions that relate to the developmental stages of the osteoblast and support developmental regulation of OC gene transcription. Basal expression of the OC gene is associated with sequence-specific protein-DNA interactions at the OC box, VDRE, and TATA/GRE box. Distinct differences are observed in proliferating osteoblasts, where the OC gene is not transcribed compared to postproliferative, differentiated osteoblasts that transc...
Proceedings of the National Academy of Sciences, 1994
The steroid hormone vitamin D is a principal mediator of skeletal homeostasis. 1,25-Dihydroxyvitamin D3 treatment of ROS 17/2.8 osteoblast-like cells results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. This transcriptional upregulation requires the positive cis-acting vitamin D responsive element (VDRE). We have used the ligation-mediated polymerase chain reaction to demonstrate that protein occupancy of the VDRE within the intact cell correlates with increased synthesis of osteocalcin transcripts. These protein-DNA contacts were not present in the absence of vitamin D or in osteosarcoma cells (ROS 24.1) lacking the vitamin D receptor. Our results establish in intact cells the requirement for both ligandand receptor-dependent occupancy of the VDRE for vitamin D responsive enhancement of osteocalcin gene transcription.