The objective of this research proposal is to begin to dissect the complex series of molecular events involved in osteoblast differentiation. The model system chosen to address these studies is the prechondroblastic MLB13 MYC clone 17 cell line which, when treated with bone morphogenic protein -2 (BMP-2), acquires markers of the osteoblastic phenotype, including expression of the endogenous osteocalcin gene. Transient expression of osteocalcin-CAT fusion genes will permit the identification of DNA sequences required for BMP-2 induction of this gene as the prechondroblasts acquire the osteoblastic phenotype. Characterization of the factor that interacts with these DNA sequences and studies of its regulation will help to unravel the series of molecular events that occur between BMP-2 treatment and the acquisition of the osteoblastic phenotype. The First Specific Aim involves identification of the DNA sequences required for induction of osteocalcin by BMP-2. Osteocalcin-CAT fusion genes with progressive deletions and mutations of the 5' regulatory region will be examined for their induction by BMP-2 in transient gene expression as says. The second Specific Aim involves using gel retardation assays to characterize the DNA-protein interactions of these sequences with nuclear extracts from cells which do and do not express the endogenous osteocalcin gene, including untreated and BMP-2 treated MLB13MYC clone 17 cells. Mutagenesis and methylation interference experiments will be undertaken to define the precise protein-DNA contact sites. Purification and/or expression cloning of the cognate protein will be undertaken if time permits.
