This project will continue to address the central hypotheses that the nuclear matrix contributes to transcriptional control of cell growth and bone-specific gene expression during progression of osteoblast differentiation, and that components of nuclear matrix are developmentally regulated in bone cells to support osteoblast maturation and bone formation. We postulate that the nuclear matrix supports osteoblast proliferation and differentiation by facilitating gene localization, as well as the concentration and localization of transactivation factors. These features of nuclear architecture, together with structural properties of the genome including chromatin structure and nucleosome organization, can influence transcriptional control of genes associated with cell growth and differentiation of osteoblasts. In this manner, the spatial organization of gene promoters within the osteoblast nucleus may support the integration of regulatory activities at multiple, independent cis-acting elements, thereby contributing to positive and negative control of transcription by a broad spectrum of physiological mediators that are developmentally and steroid hormone responsive. This renewal application will pursue the molecular mechanisms mediating transcription factor association with the nuclear matrix, which regulates osteoblast specific gene expression and skeletogenesis.
Our specific aims will experimentally address: (1) the precise molecular specificity of nuclear matrix targeting, by characterizing the critical amino-acids required for nuclear matrix association in AML-3 (NMP-2) and YYI (NMP-I), two transcription factors regulating bone-relating genes (Specific Aim 1); (ii) the physiological role of transcription factor association with the nuclear matrix in regulating osteoblast differentiation and bone formation in murine modes, by preparing genetic lesions in the NMTS of AML-3 (Specific Aim 2); (iii) the functional contribution of the nuclear matrix to physiological control of osteoblast-specific gene transcription (Specific Aim 3); (iv) the identity of the nuclear matrix acceptor protein fNMAP) of AML-3 using yeast two hybrid assays (Specific Aim 4); (v) the tissue-specificity, developmental plasticity and dynamics of intranuclear trafficking, as well as the spatial distribution of AML-3 and YYI transcription factors and their cognate nuclear matrix docking sites during bone cell phenotype development (Specific Aim 5) by high resolution in situ immunofluorescence microscopy analysis.
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