The transcription factor hierarchy controlling osteoblast gene expression during development is emerging. Runx2 (Cbfal/Osf2) confers osteogenic potential. Rb, Smads, and the osteoblast homeoproteins (Msx2, Msx1) function as Runx2 coregulators of progenitor proliferation, lineage allocation, and the temporospatial kinetics of matrix mineralization. Msx2 controls cell cycle and mesenchymal cell lineage as well as the matrix gene expression associated with terminal differentiation. Msx2 suppresses osteocalcin (OC) gene expression via (a) a Ku antigen complex that enhances Runx2-dependent OC transcription; and (b) MINT (Msx2 interacting nuclear target), an RRM domain nuclear matrix protein that we discovered by interaction cloning. A fundamental understanding of the mechanisms whereby Msx2, Runx2, and coregulators control calvarial osteoblast gene expression will provide insights into (a) the pathobiology of craniofacial dysmorphias; and (b) osteoprogenitor growth, lineage allocation, & differentiation. Our limited understanding of Msx2 function in osteoblast molecular biology represents a major deficiency. We hypothesize that Msx2 selectively regulates chromatin composition of specific Runx2-activated promoters during osteoblast differentiation (OC, MMP13 vs. osteopontin).
Specific aims are:
Aim1 : To identify the molecular interactions between Msx2, Runx2, and Ku70 that control calvarial osteoblast gene expression, using the OC promoter as a model for study. Runx2 and Msx2 physically interact, as do Runx2 and Ku70.
This aim will identify structural features of Runx2 required for inhibition by Msx2 and augmentation by Ku70. Interactions that inhibit (Msx2-Runx2) or promote (Runx2- Ku) OC & MMP 13 transcription will be characterized in vitro and in native chromatin context. Msx2-regulated nuclear localization of Runx2 will be detailed by chromatin immunoprecipitation assay & confocal microscopy. Ku70 structure-function relationships for Runx2-dependent OC transcription will be studied in CV 1 cells & Ku70 -/- calvarial osteoblasts (profound OC expression deficit).
Aim2 : To detail the role and regulation of MINT in calvarial osteoblast gene expression, using the OC gene as a model for study, The goal is to characterize MINT's role in osteoblast transcription & mRNA metabolism. MINT structure-functions studies will detail the regulation of OC gene expression in concert with Msx2.
With Aim1, Aim2 will test if Msx2 inhibits OC expression in part via targeting of OC chromatin to the nucleosomal remodeling & deacetylase (NuRD) complex. MINT structure-function studies will be extended to future studies of craniofacial development, gene expression, and RNA metabolism in MINT -/- mice & cultured cells.
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