Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene

P Ducy, G Karsenty - Molecular and cellular biology, 1995 - Am Soc Microbiol
P Ducy, G Karsenty
Molecular and cellular biology, 1995Am Soc Microbiol
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 …
Abstract
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 2147 and 234 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 264 and 247, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp 2110 and 283, bound a ubiquitously expressed factor. The C element, located between bp 2146 and 2132, 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.
American Society for Microbiology