Identification of a homeodomain binding element in the bone sialoprotein gene promoter that is required for its osteoblast-selective expression

Bone sialoprotein is a 70-kDa extracellular matrix component that is intima tely associated with biomineralization, yet the cis-acting elements of the Bsp gene that restrict its expression to mineralizing cells remain uncharac terized. To identify such elements, we analyzed a 2472-base pair fragment o f the murine promoter that directs osteoblast-selective expression of a luc iferase reporter gene and found that the region between -338 and -178 relat ive to the transcriptional start is crucial for its osteoblast-selective ac tivity. We identified an element within this region that binds a protein co mplex in the nuclear extracts of osteoblastic cells and is required for its transcriptional activity. Introduction of a mutation that disrupts a homeo domain binding site within this sequence eliminates both its in vitro bindi ng and nearly all of the osteoblastic-selective activity of the 2472-base p air promoter. We further found that the Dlx5 homeoprotein, which is able to regulate the osteoblast-specific osteocalcin promoter, can bind this eleme nt and stimulate its enhancer activity when overexpressed in COS7 cells. Th ese data represent the first description of an osteoblast-specific element within the bone sialoprotein promoter and demonstrate its regulation by a m ember of a family of factors known to be involved in skeletogenesis.