Regulation of the Bone-Specific Osteocalcin Gene by p300 Requires Runx2/Cbfa1 and the Vitamin D3 Receptor but Not p300 Intrinsic Histone Acetyltransferase Activity

ABSTRACT 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 D3 via the vitamin D3 receptor complex. Therefore, we addressed p300 control of basal and vitamin D3-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 D3-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 1α,25-dihydroxyvitamin D3 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 D3.

[1]  G. Stein,et al.  Histone Acetylation in Vivo at the Osteocalcin Locus Is Functionally Linked to Vitamin D-dependent, Bone Tissue-specific Transcription* , 2002, The Journal of Biological Chemistry.

[2]  G. Stein,et al.  CCAAT/Enhancer-binding Proteins (C/EBP) β and δ Activate Osteocalcin Gene Transcription and Synergize with Runx2 at the C/EBP Element to Regulate Bone-specific Expression* , 2002, The Journal of Biological Chemistry.

[3]  R. Goodman,et al.  CBP/p300 in cell growth, transformation, and development. , 2000, Genes & development.

[4]  M. Lazar,et al.  The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes , 2000, Molecular and Cellular Biology.

[5]  L. Freedman,et al.  Mechanisms of gene regulation by vitamin D(3) receptor: a network of coactivator interactions. , 2000, Gene.

[6]  J. Wong,et al.  p300 Requires Its Histone Acetyltransferase Activity and SRC-1 Interaction Domain To Facilitate Thyroid Hormone Receptor Activation in Chromatin , 2000, Molecular and Cellular Biology.

[7]  A. Wolffe,et al.  Nuclear receptors: coactivators, corepressors and chromatin remodeling in the control of transcription. , 1999, Journal of molecular endocrinology.

[8]  J. T. Kadonaga,et al.  Biochemical Analysis of Distinct Activation Functions in p300 That Enhance Transcription Initiation with Chromatin Templates , 1999, Molecular and Cellular Biology.

[9]  J. Davie,et al.  Role of covalent modifications of histones in regulating gene expression. , 1999, Gene.

[10]  G. Stein,et al.  Multiple Cbfa/AML Sites in the Rat Osteocalcin Promoter Are Required for Basal and Vitamin D-Responsive Transcription and Contribute to Chromatin Organization , 1999, Molecular and Cellular Biology.

[11]  L. Freedman,et al.  Nuclear receptor cofactors as chromatin remodelers. , 1999, Current opinion in genetics & development.

[12]  Paul Tempst,et al.  Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex , 1999, Nature.

[13]  B. Frenkel,et al.  Chromatin hyperacetylation abrogates vitamin D-mediated transcriptional upregulation of the tissue-specific osteocalcin gene in vivo. , 1999, Biochemistry.

[14]  G. Stein,et al.  Regulated expression of the bone-specific osteocalcin gene by vitamins and hormones. , 1999, Vitamins and hormones.

[15]  B. Frenkel,et al.  AP-1 and vitamin D receptor (VDR) signaling pathways converge at the rat osteocalcin VDR element: requirement for the internal activating protein-1 site for vitamin D-mediated trans-activation. , 1999, Endocrinology.

[16]  H. Erdjument-Bromage,et al.  A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system. , 1998, Genes & development.

[17]  M. Ohki,et al.  Interaction and functional cooperation of the leukemia‐associated factors AML1 and p300 in myeloid cell differentiation , 1998, The EMBO journal.

[18]  David Newsome,et al.  Gene Dosage–Dependent Embryonic Development and Proliferation Defects in Mice Lacking the Transcriptional Integrator p300 , 1998, Cell.

[19]  C. Glass,et al.  Differential use of CREB binding protein-coactivator complexes. , 1998, Science.

[20]  C. Glass,et al.  Transcription factor-specific requirements for coactivators and their acetyltransferase functions. , 1998, Science.

[21]  G. Stein,et al.  Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone‐specific complex , 1997, Journal of cellular biochemistry.

[22]  S. Mundlos,et al.  Cbfa1, a Candidate Gene for Cleidocranial Dysplasia Syndrome, Is Essential for Osteoblast Differentiation and Bone Development , 1997, Cell.

[23]  Makoto Sato,et al.  Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts , 1997, Cell.

[24]  G. Karsenty,et al.  Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation , 1997, Cell.

[25]  M. Montminy,et al.  Role of CBP/P300 in nuclear receptor signalling , 1996, Nature.

[26]  B. Howard,et al.  A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A , 1996, Nature.

[27]  G. Stein,et al.  An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Thorsten Heinzel,et al.  A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors , 1996, Cell.

[29]  G. Stein,et al.  Changes in chromatin structure support constitutive and developmentally regulated transcription of the bone-specific osteocalcin gene in osteoblastic cells. , 1996, Biochemistry.

[30]  G. Karsenty,et al.  Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene , 1995, Molecular and cellular biology.

[31]  G. Stein,et al.  In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[32]  G. Stein,et al.  DNase I hypersensitive sites in promoter elements associated with basal and vitamin D dependent transcription of the bone-specific osteocalcin gene. , 1994, Biochemistry.

[33]  G. Stein,et al.  Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix , 1990, Journal of cellular physiology.

[34]  S. Rodan,et al.  Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. , 1980, Endocrinology.

[35]  Vlad I. Morariu,et al.  Expression , 2015, Principles of Molecular Virology.