Cooperative Interactions between RUNX2 and Homeodomain Protein-binding Sites Are Critical for the Osteoblast-specific Expression of the Bone Sialoprotein Gene

摘要: The bone sialoprotein (Bsp) gene provides an excellent model for studying mechanisms controlling osteoblast-specific expression. Although the RUNX2 transcription factor directly regulates many osteoblast-related genes, its function in Bsp expression remains uncertain. By using chromatin immunoprecipitation (ChIP) analysis MC3T3-E1 (clone MC-4) preosteoblast cells, was shown to bind a fragment containing proximal promoter. Two putative RUNX2-binding sites (R1 and R2) were identified within this region of mouse, rat, human genes vitro vivo (by ChIP assay). Site-specific mutagenesis established that both act as transcriptional enhancers together account nearly two-thirds total promoter activity. In addition, functional cooperativity observed between R2 site adjacent homeodomain protein-binding previously characterized by laboratory (the C site). All three (R1, R2, C) are necessary maximal activity osteoblasts. DLX5 MC-4 cell nuclear extracts binds vitro. Furthermore, assays revealed is selectively associated with vicinity only when transcriptionally active. Finally, co-immunoprecipitation detected physical complex RUNX2. Taken together, our data show direct regulator osteoblasts it functions cooperation or related activate