ACTIVITY OF THE RAT OSTEOCALCIN BASAL PROMOTER IN OSTEOBLASTIC CELLS IS DEPENDENT UPON HOMEODOMAIN AND CP1 BINDING MOTIFS

A detailed analysis of the transcriptional machinery responsible for o steoblast-specific gene expression should provide tools useful for und erstanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATT GTGT) present in the region between -200 and -170 that binds a multisu bunit CP1/NFY/CBF-like CAAT factor complex contributes significantly t o high level basal activity and presumably functions as the CAAT box f or the rat OC promoter. We show that the region -121 to 32 is sufficie nt to confer osteoblastic cell type specificity in transient transfect ion assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-1 00 to -74) contains a CAAT motif, we could not detect CP1-like CAAT fa ctor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-en d of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognize s this motif appears to be present in osteoblastic ROS 17/2.8 cells, w hich produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-ri ch DNA cognates of the homeodomain-containing POU family of transcript ion factors. Taken together, these data suggest that a ubiquitous CP1- like CAAT factor and a cell type-restricted homeodomain containing (Ms x or POU family) transcription factor interact with the proximal rat O C promoter to direct appropriate basal OC transcription in osteoblasti c cells.