Characterization of Osf1, an osteoblast-specific transcription factor binding to a critical cis-acting element in the mouse Osteocalcin promoters

To elucidate the mechanisms of osteoblast-specific gene expression we are s tudying the regulation of osteocalcin, the most osteoblast-specific gene. P revious studies of OG2, one of the two mouse osteocalcin genes, identified two osteoblast-specific cia-acting elements, OSE1 and OSE2, the latter bein g the binding site of Cbfa1, the only osteoblast-specific transcription fac tor known to date. Here we show that OSE1. is a cis-acting element as impor tant as OSE2 for the osteoblast-specific expression of OG2 in cell culture and transgenic mice. We also show that OSE1 is present in the promoter of s everal osteoblast-specific genes including Cbfa1 itself. These biological f eatures demonstrate the importance of OSE1 and led us to further characteri ze this site and the factor binding to it, provisionally termed Osf1, We fi rst defined the core OSE1 sequence, 5'-TTACATCA-3', which is necessary and sufficient for Osf1 binding do DNA. This sequence has no strong homology to any known transcription factor-binding sites. As a first step in identifyi ng Osf1, we performed an analytical purification of this protein using nucl ear extracts from two different osteoblastic cell lines, We purified Osf1 t o homogeneity through a five-step procedure including a renaturation experi ment and found that its apparent molecular mass is 40 kDa, In conclusion, t his study indicates the existence of multiple osteoblast-specific cia-actin g elements of equal importance in controlling OG2 promoter activity and pro vides the first biochemical characterization of Osf1, a novel osteoblast-sp ecific transcription factor.