Expression screening of factors binding to the osteocalcin bone-specific promoter element OC Box I: Isolation of a novel osteoblast differentiation-specific factor

Contributing to bone-specific expression of the osteocalcin gene is the pro moter element OC Box I (-99 to -76), which binds both Hox proteins and anot her nonhomeodomain factor (designated OCBP for osteocalcin-box binding prot ein) (Hoffmann et al. [1996] J Cell Biochem 61:310-324). OCBP correlates wi th increased promoter activity and may, therefore, be important to developm ent or maintenance of the osteoblast phenotype. To identify OCBP candidates , we used a multimerized OC Box I sequence to screen a gamma gt11 cDNA expr ession library, constructed from the rat osteosarcoma osteoblastic ROS 17/2 .8 cell line, for cDNA clones encoding factors that recognize this element. Mutant OC Box I sequences that do not bind OCBP and/or homeodomain protein s were used to counterscreen the cDNA isolates. Clones showing binding spec ificity were sequenced and further characterized for patterns of expression in different tissues and cell lines. Among the characterized nonhomeodomai n-related isolates, we identified a nucleolin, a clone encoding rCAP2 that is related to myogenic differentiation and more importantly, a cDNA clone c ontaining a previously uncharacterized gene that has been designated as a c ell differentiation-related factor (DRF). DRF mRNA is highly expressed in R OS 17/2.8 cells and in a developmentally regulated pattern during osteoblas t differentiation, being upregulated at the postproliferative maturation tr ansition and coinciding with the induction of osteocalcin gene expression. The 7.7-kb transcript encoded by clone 44 is abundantly expressed in osteob lasts, but the mRNA was not present at detectable levels in bone and soft t issues by Northern blot analysis. However, related expressed sequence tags were recently reported in cDNA libraries of rat lung and mouse sympathetic ganglion. The identification of DRF represents a novel osteoblast different iation-specific marker related to osteocalcin expression. The identificatio n of DRF may further facilitate definition of gene regulatory mechanisms me diating the final stages of bone cell differentiation (C) 2000 Wiley-Liss, Inc.