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 promoter element OC Box I (-99 to -76), which binds both Hox proteins and another nonhomeodomain factor (designated OCBP for osteocalcin-box binding protein) (Hoffmann et al. [1996] J Cell Biochem 61:310-324). OCBP correlates with increased promoter activity and may, therefore, be important to development or maintenance of the osteoblast phenotype. To identify OCBP candidates, we used a multimerized OC Box I sequence to screen a γgt11 cDNA expression 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 proteins were used to counterscreen the cDNA isolates. Clones showing binding specificity were sequenced and further characterized for patterns of expression in different tissues and cell lines. Among the characterized nonhomeodomain-related isolates, we identified a nucleolin, a clone encoding rCAP2 that is related to myogenic differentiation and more importantly, a cDNA clone containing a previously uncharacterized gene that has been designated as a cell differentiation-related factor (DRF). DRF mRNA is highly expressed in ROS 17/2.8 cells and in a developmentally regulated pattern during osteoblast differentiation, being upregulated at the postproliferative maturation transition and coinciding with the induction of osteocalcin gene expression. The 7.7-kb transcript encoded by clone 44 is abundantly expressed in osteoblasts, but the mRNA was not present at detectable levels in bone and soft tissues 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 differentiation-specific marker related to osteocalcin expression. The identification of DRF may further facilitate definition of gene regulatory mechanisms mediating the final stages of bone cell differentiation. (C) 2000 Wiley-Liss, Inc.