title: G/C element contributes to the cell line-specific expression of the proximal osteocalcin promoter.
abstract: Sequential activation of cell type-specific genes occurs during osteoblast development. The promoter of one such gene, osteocalcin, has been widely studied, but the DNA sequences that govern osteoblast-specific expression have not been defined. The proximal osteocalcin promoter linked to pTKCAT directs strong promoter activity in osteoblast-like ROS17/2.8 cells and comparatively weak promoter activity in nonosteoblastic NIH3T3 cells. To identify sequences important in conferring cell-specific expression of the osteocalcin gene, a deletion series of the human proximal promoter was constructed and the activities assessed in ROS17/2.8 and NIH3T3 cells. These studies identified a 30 bp sequence within the proximal promoter (osteocalcin repressor element-1 [ORE-1]) which is responsible for repressing the transcriptional activity in NIH3T3 cells. In electrophoretic mobility shift assays from both NIH3T3 and ROS17/2.8 cells, a protein complex bound to the ORE-1 that was related to a complex which binds the G/C-rich repressor element in the collagen type I (alpha 1) promoter. In addition, there was a second complex from NIH3T3 cells but not ROS17/2.8 cells that bound the ORE-1 fragment. The presence of this additional factor in NIH3T3 cells parallels the observation that constructs carrying the ORE-1 sequence have repressed promoter activity relative to the analogous constructs lacking the ORE-1 when transfected into NIH3T3 and suggests that the NIH3T3-specific factor is a repressor. These data indicate that the G/C element in the ORE-1 contributes to the repression of osteocalcin gene transcription in a nonosteoblast cell line. The high homology between the ORE-1 sequence and a related sequence in the collagen type I (alpha 2) proximal promoter suggests that homologous regions in other osteoblast-expressed genes may function similarly.
authors.affiliation: Bone and Mineral Research Division, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Goldberg
authors.forename: E
authors.initials: E
authors.lastname: Gardiner
authors.forename: N A
authors.initials: NA
authors.lastname: Morrison
authors.forename: J A
authors.initials: JA
authors.lastname: Eisman
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D003596:Cytosine; D015870:Gene Expression; D006147:Guanine; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010006:Osteoblasts / Q000378:metabolism; D015675:Osteocalcin / Q000096:biosynthesis* / Q000235:genetics*; D012516:Osteosarcoma; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013937:Thymidine Kinase / Q000096:biosynthesis
chemical_list: C000716127:COL1A1 protein, human; D000090602:Collagen Type I, alpha 1 Chain; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015675:Osteocalcin; D006147:Guanine; D003596:Cytosine; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase