An E-box within the MHC IIB gene is bound by MyoD and is required for geneexpression in fast muscle

The myosin heavy chain (MHC) IIB gene is selectively expressed in skeletal muscles, imparting fast contractile kinetics. Why the MHC IIB gene product is expressed in muscles like the tibialis anterior (TA) and not expressed i n muscles like the soleus is currently unclear. It is shown here that the m utation of an E-box within the MHC IIB promoter decreased reporter gene act ivity in the fast-twitch TA muscle 90-fold as compared with the wild-type p romoter. Reporter gene expression within the TA required this E-box for act ivation of a heterologous construct containing upstream regulatory regions of the MHC IIB promoter linked to the basal 70-kDa heat shock protein TATA promoter. Electrophoretic mobility shift assays demonstrated that mutation of the E-box prevented the binding of both MyoD and myogenin to this elemen t. In cotransfected C2C12 myotubes and Hep G2 cells, MyoD preferentially ac tivated the MHC IIB promoter in an E-box-dependent manner, whereas myogenin activated the MHC IIB promoter to a lesser extent, and in an E-box-indepen dent manner. A time course analysis of hindlimb suspension demonstrated tha t the unweighted soleus muscle activated expression of MyoD mRNA before the de novo expression of MHC IIB mRNA. These data suggest a possible causativ e role for MyoD in the observed upregulation of MHC IIB in the unweighted s oleus muscle.