| dc.description.abstract | FoxO1 belongs to the forkhead family that bind to their target sites by their forkhead DNA-binding domain. FoxO1 has been shown to play important roles in the regulation of cell growth, proliferation, differentiation, and longevity. Its chimeric fusion with Pax3 or Pax7, generating either Pax7-or Pax3- FoxO1, has been demonstrated as strong inhibitors of muscle cell differentiation and apoptosis. To further elucidate the roles of FoxO1 in myogenesis and its possible implication in tumorigenesis, FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which led to a consistently inhibitory effect on myogenic differentiation. The expression of myogenic regulatory factors, including MyoD, Myogenin and Myf5, was significantly reduced in C2C12-FoxO1 cells. In this study, we observed constitutive nuclear accumulation of FoxO1 in differentiating myoblasts. However, simultaneous nuclear exclusion of FoxO1 and MHC expression was observed in mononucleated cells ready for fusion into multinucleated myotubes. FoxO1 remains cytoplasmic in multinucleated myotubes and GPF-FoxO1 introduced into myotubes also stays in the cytoplasm, implying that nuclear exclusion of FoxO1 is prerequisite for terminal differentiation. The nuclear exclusion process of FoxO1 can be mimicked by insulin treatment, but they return to the nucleus 8 hr after the treatment. To further elucidate the roles of FoxO1 in myogenesis and its possible implication in tumorigenesis, FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which strongly inhibited myogenic differentiation. Both insulin treatment and over-expression of MyoD partially rescued myogenic differentiation of C2C12-FoxO1 cells, implying that direct targeting of FoxO1 on myogenic genes, including MyoD, is required for its inhibitory effect. In silico analysis and transient transfection promoter assays have identified putative FoxO1-binding sites in the 24 kb upstream regulatory region of MyoD. These putative FoxO1-binding sites will be confirmed by EMSA and chromatin immunoprecipitation assay. Our results suggest that FoxO1 inhibits myogenesis by direct targeting the regulatory elements of myogenic genes.
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