Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter
Heinrichs, Arianne A.J. ; Bortell, Rita ; Rahman, Shamim ; Stein, Janet L. ; Alnemri, Emad S. ; Litwack, Gerald ; Lian, Jane B. ; Stein, Gary S.
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UMass Chan Affiliations
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Keywords
Base Sequence
Binding Sites
Binding, Competitive
Calcitriol
Cloning, Molecular
DNA
DNA-Binding Proteins
Deoxyribonuclease I
Dexamethasone
Gene Expression Regulation, Neoplastic
Humans
Kinetics
Molecular Sequence Data
Nuclear Proteins
Osteocalcin
Osteosarcoma
*Promoter Regions, Genetic
Rats
Receptors, Glucocorticoid
Recombinant Proteins
Sulfuric Acid Esters
TATA Box
Transfection
Tumor Cells, Cultured
Cell Biology
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Embargo Expiration Date
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Abstract
The biosynthesis of osteocalcin (OC), a bone-specific, noncollagenous protein, is stringently regulated during differentiation of the osteoblast phenotype. Glucocorticoids, and also 1,25(OH)2D3, mediate the developmental regulation of OC gene transcription. In this study, we established that the -1097 to +23 promoter (pOCZCat) of the rat OC gene confers glucocorticoid responsiveness to both basal and vitamin D-induced OC expression. The presence of multiple glucocorticoid receptor (GR) binding sites in the proximal rat OC gene promoter was determined by the combined use of DNase I footprinting, dimethyl sulfate fingerprinting, and gel mobility shift analysis with glucocorticoid receptor protein. One glucocorticoid receptor binding element (GRE) resides immediately downstream of the TATA box (-16 to -1). In vivo activity was established by cotransfection of ROS 17/2.8 osteosarcoma cells with an OC-CAT construct in the presence of cloned GRE sequences (wild type or mutant) as competitors. A putative second, less protected GR binding site is located further upstream in the OC gene basal promoter within the region overlapping the TATA box. This is in direct contrast to the organization of GREs in the human OC proximal promoter wherein GR binding at the upstream GRE overlapping the TATA is stronger than at the downstream GRE. In addition, we detected sequence-specific binding of GR protein to another basal promoter element, the OC box (-99 to -76), which contains a central CCAAT motif. The presence of multiple GR binding sites in the rat OC gene proximal promoter indicates that regulation of basal and vitamin D-enhanced transcription by glucocorticoids may involve the integrated activities of multiple, independent GREs.
Source
Biochemistry. 1993 Oct 26;32(42):11436-44.