The Experts below are selected from a list of 732 Experts worldwide ranked by ideXlab platform
Roberto Paredes - One of the best experts on this subject based on the ideXlab platform.
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bone specific transcription factor runx2 interacts with the 1α 25 dihydroxyvitamin d3 receptor to up regulate rat osteocalcin gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Alejandro Villagra, Kaleem Zaidi, Andre J Van Wijnen, Gary S Stein, Jane B Lian, Juan Olate, Janet L SteinAbstract:Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1α,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1α,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1α,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1α,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a “master” transcriptional regulator of osteoblast differentiation, and 1α,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
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the runx2 transcription factor plays a key role in the 1α 25 dihydroxy vitamin d3 dependent upregulation of the rat osteocalcin oc gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Andre J Van Wijnen, Janet L Stein, Gary S Stein, Jane B Lian, Juan Olate, Martin MontecinoAbstract:Abstract Bone-specific transcription of the osteocalcin (OC) gene is principally regulated by the Runx2 transcription factor and further stimulated in response to 1α,25-dihydroxy Vitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A–C). Mutation of sites A and B, which flank the 1α,25-dihydroxy Vitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxy Vitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between VDR and Runx2 factors. Additionally, the transcriptional co-activator p300 is recruited to the OC promoter by Runx2 where it up-regulates both basal and 1α,25-dihydroxy Vitamin D3-enhanced OC expression. Here, we present an overview of how in osteoblastic cells expressing OC, Runx2 modulates the 1α,25-dihydroxy Vitamin D3-dependent stimulation of the OC promoter by first recruiting transcriptional co-activators and then by further stabilizing the interaction of the VDR with the VDRE.
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Interaction of the 1alpha,25-dihydroxyvitamin D3 receptor at the distal promoter region of the bone-specific osteocalcin gene requires nucleosomal remodelling.
2002Co-Authors: Roberto Paredes, José Gutiérrez, Soraya Gutierrez, Lizabeth Allison, Marcia Puchi, Maria Imschenetzky, Andre Van Wijnen, Jane Lian, Gary Stein, Janet SteinAbstract:1alpha,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 1alpha,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 vivo, 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 1alpha,25-dihydroxyvitamin D3 or the presence of other transcription factors, such as YY1 and Runx/Cbfa (core-binding factor alpha), which also bind to sequences partially overlapping or near the VDRE, is not sufficient to render the VDRE accessible. Thus the VDR-RXR, unlike other steroid receptors, such as glucocorticoid receptor, progesterone receptor and thyroid receptor, is unable to bind its target sequence within a nucleosomal context. Taken together these results demonstrate that nucleosomal remodelling is required for in vivo occupancy of binding sites in the distal region of the OC gene promoter by the regulatory factors responsible for 1alpha,25-dihydroxyvitamin D3-dependent enhancement of transcription.
Janet L Stein - One of the best experts on this subject based on the ideXlab platform.
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bone specific transcription factor runx2 interacts with the 1α 25 dihydroxyvitamin d3 receptor to up regulate rat osteocalcin gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Alejandro Villagra, Kaleem Zaidi, Andre J Van Wijnen, Gary S Stein, Jane B Lian, Juan Olate, Janet L SteinAbstract:Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1α,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1α,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1α,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1α,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a “master” transcriptional regulator of osteoblast differentiation, and 1α,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
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The Vitamin D Response Element in the Distal Osteocalcin Promoter Contributes to Chromatin Organization of the Proximal Regulatory Domain
2004Co-Authors: Soraya E. Gutierrez, Gary S Stein, Jane B Lian, Jilin Liu, Amjad Javed, Martin Montecino, Janet L SteinAbstract:Abstract Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR·retinoid X receptor directly impacts both the distal and proximal regulatory domains.
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the runx2 transcription factor plays a key role in the 1α 25 dihydroxy vitamin d3 dependent upregulation of the rat osteocalcin oc gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Andre J Van Wijnen, Janet L Stein, Gary S Stein, Jane B Lian, Juan Olate, Martin MontecinoAbstract:Abstract Bone-specific transcription of the osteocalcin (OC) gene is principally regulated by the Runx2 transcription factor and further stimulated in response to 1α,25-dihydroxy Vitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A–C). Mutation of sites A and B, which flank the 1α,25-dihydroxy Vitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxy Vitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between VDR and Runx2 factors. Additionally, the transcriptional co-activator p300 is recruited to the OC promoter by Runx2 where it up-regulates both basal and 1α,25-dihydroxy Vitamin D3-enhanced OC expression. Here, we present an overview of how in osteoblastic cells expressing OC, Runx2 modulates the 1α,25-dihydroxy Vitamin D3-dependent stimulation of the OC promoter by first recruiting transcriptional co-activators and then by further stabilizing the interaction of the VDR with the VDRE.
Carsten Carlberg - One of the best experts on this subject based on the ideXlab platform.
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Recruitment of VDR–RXR heterodimers and VDIR to the VDREs found in the gene promoter
2011Co-Authors: Mikko M. Turunen, Thomas W. Dunlop, Carsten Carlberg, Sami VäisänenAbstract:Copyright information:Taken from "Selective use of multiple vitamin D response elements underlies the 1 α,25-dihydroxyvitamin D-mediated negative regulation of the human gene"Nucleic Acids Research 2007;35(8):2734-2747.Published online 10 Apr 2007PMCID:PMC1885674.© 2007 The Author(s) Sequence and location of VDREs within the human promoter (). The hexameric core motifs within the DR3-type VDREs and the E-boxes within the nVDRE are shown in bold. Efficiency of translation of VDR, RXR and VDIR was examined by using S-labeled proteins that were analyzed on a 12% denaturing polyacrylamide gel (). Gel shift experiments were performed with -translated VDR, RXR and VDIR proteins in the presence or absence of 1 µM 1α,25(OH)D on the established DR4-type VDRE of the rat gene (core sequence 5′-gaAGTTCAtgagAGTTCA-3′, ), the nVDRE () and the putative VDREs 1, 2 and 3 (). Anti-VDR antibody was used to challenge the specificity of the protein–DNA complexes. The latter were resolved from free probe through 8% non-denaturing polyacrylamide gels. Representative gels are shown
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Regulation of the human cyclin C gene via multiple vitamin D 3-responsive regions in its promoter
2005Co-Authors: Lasse Sinkkonen, Marjo Malinen, Katri Saavalainen, Carsten CarlbergAbstract:The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3], but binding sites for the 1a,25(OH)2D3 receptor (VDR), so-called 1a,25(OH)2D3 response elements (VDREs), have not yet been identified in the promoter of this gene. We screened various cancer cell lines by quantitative PCR and found that the 1a,25(OH)2D3 inducibility of cyclin C mRNA expression, in relationship with the 24-hydroxylase (CYP24) gene, was best in MCF-7 humanbreastcancercells.Tocharacterize the molecu-lar mechanisms, we analyzed 8.4 kb of the cyclin C promoter by using chromatin immunoprecipitation assays (ChIP) with antibodies against acetylated his-tone 4, VDR and itspartner receptor, retinoid X receptor (RXR). The histone 4 acetylation status of all 23 invest-igated regions of the cyclin C promoter did not change significantly in response to 1a,25(OH)2D3, but four independent promoter regions showed a consistent, 1a,25(OH)2D3-dependent association with VDR and RXR over a time period of 240 min. Combined in silico/in vitro screening identified in each of these promoter regions a VDRE and reporter gene assays confirmedtheirfunctionality.Moreover,re-ChIPassays monitored simultaneous association of VDR with RXR, coactivator, mediator and RNA polymerase II proteins on these regions. Since cyclin C protein is associated with those mediator complexes that display transcrip-tional repressive properties, this study contributes to the understanding of the downregulation of a number of secondary 1a,25(OH)2D3-responding genes
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Regulation of the human cyclin C gene via multiple vitamin D3 - responsive regions in its promoter
2005Co-Authors: Lasse Sinkkonen, Sami Väisänen, Marjo Malinen, Katri Saavalainen, Carsten CarlbergAbstract:The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1a,25-dihydroxyvitamin D3 [1a,25(OH) 2D3], but binding sites for the 1a,25(OH)2D3 receptor (VDR), so-called 1a,25(OH)2D3 response elements (VDREs), have not yet been identified in the promoter of this gene. We screened various cancer cell lines by quantitative PCR and found that the 1a,25(OH)2D3 inducibility of cyclin C mRNA expression, in relationship with the 24-hydroxylase (CYP24) gene, was best in MCF-7 human breast cancer cells. To characterize the molecular mechanisms, we analyzed 8.4 kb of the cyclin C promoter by using chromatin immunoprecipitation assays (ChIP) with antibodies against acetylated histone 4, VDR and its partner receptor, retinoid X receptor (RXR). The histone 4 acetylation status of all 23 investigated regions of the cyclin C promoter did not change significantly in response to 1a,25(OH) 2D3, but four independent promoter regions showed a consistent, 1a,25(OH)2D3-dependent association with VDR and RXR over a time period of 240 min. Combined in silico/in vitro screening identified in each of these promoter regions a VDRE and reporter gene assays confirmedtheirfunctionality.Moreover,re-ChIPassays monitored simultaneous association of VDR with RXR, coactivator, mediator and RNA polymerase II proteins on these regions. Since cyclin C protein is associated with those mediator complexes that display transcriptional repressive properties, this study contributes to the understanding of the downregulation of a number of secondary 1a,25(OH)2D3-responding genes
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All natural DR3-type vitamin D response elements show a similar functionality in vitro.
2000Co-Authors: Andrea Toell, Patsie Polly, Carsten CarlbergAbstract:The vitamin D(3) receptor (VDR), which is the nuclear receptor for 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], acts primarily as a heterodimer with the retinoid X receptor (RXR) and binds preferentially to directly repeated arrangements of two hexameric binding sites with three spacing nucleotides [DR3-type vitamin D response elements (VDREs)]. In this study, all presently known natural DR3-type VDREs have been compared and classified on the basis of their complex-formation with VDR-RXR heterodimers and their ability to stabilize VDR-RXR heterodimer conformations. Based on the affinity of each VDRE for VDR-RXR heterodimers, the DR3-type VDREs were divided into three classes. The ligand sensitivity of this complex-formation and conformational stabilization was determined to be in the range of 0.1 nM. No significant differences in the 1alpha,25(OH)(2)D(3)-modulated interactions of the DR3-type VDRE-complexed VDR-RXR heterodimer with the co-activator SRC-1 (steroid receptor co-activator-1) or the co-repressor NCoR (nuclear receptor co-repressor) were found. Taken together, the affinity for VDR-RXR heterodimers appears to be the major discriminating parameter between natural DR3-type VDREs. This will not only facilitate further investigation of the principles of DR3-type-VDRE-mediated gene regulation, but also strongly suggests that DR3-type VDREs alone cannot explain the pleiotropic genomic action of 1alpha,25(OH)(2)D(3).
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The role of the T-box for the function of the vitamin D receptor on different types of response elements
1998Co-Authors: Marcus Quack, Karol Szafranski, Juha Rouvinen, Carsten CarlbergAbstract:The nuclear hormone 1α,25-dihydroxyvitamin D3 (VD) mainly functions through a heterodimer formed between the VD receptor (VDR) and the retinoid X receptor (RXR). This transcription factor complex specifically recognizes DNA sequences, referred to as VD response elements (VDREs), that are formed by two hexameric core binding motifs arranged either as direct repeats spaced by 3 nt (DR3) or inverted palindromes with nine intervening nucleotides (IP9). Gel shift clipping assays provided the first evidence that VDR‐RXR heterodimers form different conformations on these two types of VDREs. Since the T-box within the C-terminal extension of the receptor DNA binding domain (DBD) was previously shown to form a dimerization interface with the partner receptor DBD when bound to DR-type response elements, all six amino acid residues of the VDR T-box were investigated for their role in VDR‐RXR heterodimer complex formation on DR3- and IP9-type VDREs. Interestingly, the residue Phe93 (F93) was found to be critical on both types of VDREs, whereas the role of the residue Ile94 (I94) was found to depend on ionic strength of the binding reaction and the nature of the VDRE. However, under physiological conditions I94 was also shown to be critical on both VDRE types. The monitored differences between the two VDR-containing protein‐DNA complexes helps in an understanding of the differential action of the nuclear hormone VD and its therapeutically important analogues.
Jane B Lian - One of the best experts on this subject based on the ideXlab platform.
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bone specific transcription factor runx2 interacts with the 1α 25 dihydroxyvitamin d3 receptor to up regulate rat osteocalcin gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Alejandro Villagra, Kaleem Zaidi, Andre J Van Wijnen, Gary S Stein, Jane B Lian, Juan Olate, Janet L SteinAbstract:Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1α,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1α,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1α,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1α,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a “master” transcriptional regulator of osteoblast differentiation, and 1α,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
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The Vitamin D Response Element in the Distal Osteocalcin Promoter Contributes to Chromatin Organization of the Proximal Regulatory Domain
2004Co-Authors: Soraya E. Gutierrez, Gary S Stein, Jane B Lian, Jilin Liu, Amjad Javed, Martin Montecino, Janet L SteinAbstract:Abstract Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR·retinoid X receptor directly impacts both the distal and proximal regulatory domains.
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the runx2 transcription factor plays a key role in the 1α 25 dihydroxy vitamin d3 dependent upregulation of the rat osteocalcin oc gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Andre J Van Wijnen, Janet L Stein, Gary S Stein, Jane B Lian, Juan Olate, Martin MontecinoAbstract:Abstract Bone-specific transcription of the osteocalcin (OC) gene is principally regulated by the Runx2 transcription factor and further stimulated in response to 1α,25-dihydroxy Vitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A–C). Mutation of sites A and B, which flank the 1α,25-dihydroxy Vitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxy Vitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between VDR and Runx2 factors. Additionally, the transcriptional co-activator p300 is recruited to the OC promoter by Runx2 where it up-regulates both basal and 1α,25-dihydroxy Vitamin D3-enhanced OC expression. Here, we present an overview of how in osteoblastic cells expressing OC, Runx2 modulates the 1α,25-dihydroxy Vitamin D3-dependent stimulation of the OC promoter by first recruiting transcriptional co-activators and then by further stabilizing the interaction of the VDR with the VDRE.
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A central dinucleotide within vitamin D response elements modulates DNA binding and transactivation by the vitamin D receptor in cellular response to natural and synthetic ligands.
2002Co-Authors: Gert Jan C.m. Van Den Bemd, Gary S Stein, Jane B Lian, Mila Jhamai, Ada Staal, Andre J. Van Wijnen, Huibert A.p. Pols, Johannes P. T. M. Van LeeuwenAbstract:Abstract There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin D3(1,25-(OH)2D3)-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)2D3. Subsequently, we have shown that vitamin D receptor/retinoid X receptor α (VDR/RXRα) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRα with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)2D3. Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)2D3 in stimulating VDRE binding ofin vitro synthesized VDR/RXRα. Thus, the extent of 1,25-(OH)2D3- and KH1060-dependent binding of VDR/RXRα is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.
Gary S Stein - One of the best experts on this subject based on the ideXlab platform.
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bone specific transcription factor runx2 interacts with the 1α 25 dihydroxyvitamin d3 receptor to up regulate rat osteocalcin gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Alejandro Villagra, Kaleem Zaidi, Andre J Van Wijnen, Gary S Stein, Jane B Lian, Juan Olate, Janet L SteinAbstract:Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1α,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1α,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1α,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1α,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a “master” transcriptional regulator of osteoblast differentiation, and 1α,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
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The Vitamin D Response Element in the Distal Osteocalcin Promoter Contributes to Chromatin Organization of the Proximal Regulatory Domain
2004Co-Authors: Soraya E. Gutierrez, Gary S Stein, Jane B Lian, Jilin Liu, Amjad Javed, Martin Montecino, Janet L SteinAbstract:Abstract Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR·retinoid X receptor directly impacts both the distal and proximal regulatory domains.
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the runx2 transcription factor plays a key role in the 1α 25 dihydroxy vitamin d3 dependent upregulation of the rat osteocalcin oc gene expression in osteoblastic cells
2004Co-Authors: Roberto Paredes, Gloria Arriagada, Fernando Cruzat, Andre J Van Wijnen, Janet L Stein, Gary S Stein, Jane B Lian, Juan Olate, Martin MontecinoAbstract:Abstract Bone-specific transcription of the osteocalcin (OC) gene is principally regulated by the Runx2 transcription factor and further stimulated in response to 1α,25-dihydroxy Vitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A–C). Mutation of sites A and B, which flank the 1α,25-dihydroxy Vitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxy Vitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between VDR and Runx2 factors. Additionally, the transcriptional co-activator p300 is recruited to the OC promoter by Runx2 where it up-regulates both basal and 1α,25-dihydroxy Vitamin D3-enhanced OC expression. Here, we present an overview of how in osteoblastic cells expressing OC, Runx2 modulates the 1α,25-dihydroxy Vitamin D3-dependent stimulation of the OC promoter by first recruiting transcriptional co-activators and then by further stabilizing the interaction of the VDR with the VDRE.
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A central dinucleotide within vitamin D response elements modulates DNA binding and transactivation by the vitamin D receptor in cellular response to natural and synthetic ligands.
2002Co-Authors: Gert Jan C.m. Van Den Bemd, Gary S Stein, Jane B Lian, Mila Jhamai, Ada Staal, Andre J. Van Wijnen, Huibert A.p. Pols, Johannes P. T. M. Van LeeuwenAbstract:Abstract There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin D3(1,25-(OH)2D3)-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)2D3. Subsequently, we have shown that vitamin D receptor/retinoid X receptor α (VDR/RXRα) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRα with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)2D3. Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)2D3 in stimulating VDRE binding ofin vitro synthesized VDR/RXRα. Thus, the extent of 1,25-(OH)2D3- and KH1060-dependent binding of VDR/RXRα is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.