Runx2 is an essential transcription factor for osteoblast development and bone formation. It also contributes to T cell lymphomogenesis and breast cancer-associated osteolysis and skeletal disease. Runx2 is both an activator and represser of gene expression. Its activity is regulated by interactions with other proteins, including co-activators and co-repressors. Histone deacetylases (Hdacs) are among the co-repressors that associate with Runx2. We have identified four Hdacs (Hdac3, Hdac4, Hdac6, Hdac7) and the Hdac- associated co-repressor, mSinSA, as Runx2 interacting proteins. Our recent studies indicate that these interactions may be important for bone formation because Hdac inhibitors and Hdac3 shRNAs accelerate osteoblast maturation in vitro. Thus, we know the identity of several enzymes that negatively regulate Runx2 transcriptional activity, but we do not know what dictates their interactions with Runx2, whether they cooperate to modify Runx2 activity during bone formation, or if these co-repressors are important regulators of bone formation in vivo. Our next steps in pursuit of our long-term goal of understanding the molecular mechanisms whereby Runx2 regulates gene expression are to determine if Hdac3 delays osteoblast maturation in vivo as it does in vitro, to continue defining the multi-protein Runx2-repression complexes in vitro, and to begin identifying the molecular """"""""switches"""""""" that convert Runx2 from a represser to an activator by preventing its interactions with co-repressors. The objectives of this application are to determine how Hdac7 modifies Runx2 activity on its own, in cooperation with Hdac3 and in response to an osteogenic factor, and to determine if Hdac3 depletion accelerates osteoblast maturation and bone formation in vivo as it does in vitro. The central hypothesis of this study is that Hdac3 and Hdac7 are components of a multi-protein complex that regulates Runx2 activity and bone formation and is responsive to extracellular signals. The three specific aims we will pursue to test our hypothesis and accomplish the objectives of this application are to: 1) Define the role of Hdac3 in osteoblast maturation and bone formation in vivo, 2) Determine how Hdac7 regulates and interacts with Runx2, and 3) Identify stimuli and mechanisms that modify interactions between Runx2 and Hdac7 complexes. The proposed work is innovative because it will explore how multi-component represser complexes regulate Runx2 activity and determine how the co-repressors Hdac3 and Hdac7 contribute to bone formation in vivo and in vitro. These results will have collective impact because they are expected to define how Hdacs regulate Runx2 activity and osteoblast maturation. Our previous work indicates that Hdac inhibitors are potential anabolic agents. Thus, it is our expectation that this work will provide new perspectives and knowledge into treatment of bone formation disorders. ? ? ?
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