title: Identification of multiple glucocorticoid receptor binding sites in the rat osteocalcin gene promoter.
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.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
authors.forename: A A
authors.initials: AA
authors.lastname: Heinrichs
authors.forename: R
authors.initials: R
authors.lastname: Bortell
authors.forename: S
authors.initials: S
authors.lastname: Rahman
authors.forename: J L
authors.initials: JL
authors.lastname: Stein
authors.forename: E S
authors.initials: ES
authors.lastname: Alnemri
authors.forename: G
authors.initials: G
authors.lastname: Litwack
authors.forename: J B
authors.initials: JB
authors.lastname: Lian
authors.forename: G S
authors.initials: GS
authors.lastname: Stein
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002117:Calcitriol / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D003907:Dexamethasone / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics*; D012516:Osteosarcoma; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D016385:TATA Box; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D013463:Sulfuric Acid Esters; D015675:Osteocalcin; D003907:Dexamethasone; D004247:DNA; D003850:Deoxyribonuclease I; D002117:Calcitriol; C007482:dimethyl sulfate