Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.
Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.
Ducy
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Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Karsenty
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G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000502:physiology*; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015675:Osteocalcin