Synergistic up-regulation of muscle LIM protein expression in C2C12 and NIH3T3 cells by myogenin and MEF2C

Although the role of muscle LIM protein (MLP, also known as CRP3), a LIM-only protein of LIM domain-containing protein family, is well-characterized, the mechanism by which the MLP gene expresses remains unclear. Herein, we demonstrate that myogenin and myocyte enhancer factor 2C (MEF2C) cooperate in activating the MLP gene in myogenesis. RT-PCR, real-time PCR and Western blotting showed that overexpression of myogenin or myogenin plus MEF2C led to induction of the MLP gene in differentiating C2C12 and NIH3T3 fibroblasts. By contrary, knocking-down of myogenin by RNA interference (RNAi) suppressed MLP expression in differentiating C2C12. Deletion and reporter enzyme assay revealed that the promoter activity was determined largely by the region extending from −260 to −173, which containing three E-box (CANNTG motif) candidates. Site-directed mutagenesis demonstrated that the E-box at position −186 to −180 was crucial for activating the promoter by myogenin. Furthermore, MEF2C could enhance myogenin-mediated activation of the promoter. In addition, chromatin immunoprecipitation (ChIP) and re-ChIP showed that myogenin and MEF2C were associated with the activated MLP promoter. Together, these results suggest that myogenin and MEF2C cooperate in the MLP gene activation. The linking of the MLP gene activation with myogenin and MEF2C may facilitate myogenin-mediated differentiation of striated muscle.