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

The myosin heavy chain (MHC) IIB gene is selectively expressedin skeletal muscles, imparting fast contractile kinetics. Why the MHCIIB gene product is expressed in muscles like the tibialis anterior(TA) and not expressed in muscles like the soleus is currently unclear.It is shown here that the mutation of an E-box within the MHC IIBpromoter decreased reporter gene activity in the fast-twitch TA muscle90-fold as compared with the wild-type promoter. Reporter geneexpression within the TA required this E-box for activation of aheterologous construct containing upstream regulatory regions of theMHC IIB promoter linked to the basal 70-kDa heat shock protein TATApromoter. Electrophoretic mobility shift assays demonstrated thatmutation of the E-box prevented the binding of both MyoD and myogeninto this element. In cotransfected C₂C₁₂myotubes and Hep G2 cells, MyoD preferentially activated the MHC IIBpromoter in an E-box-dependent manner, whereas myogenin activated theMHC IIB promoter to a lesser extent, and in an E-box-independent manner. A time course analysis of hindlimb suspension demonstrated thatthe unweighted soleus muscle activated expression of MyoD mRNA beforethe de novo expression of MHC IIB mRNA. These data suggest a possiblecausative role for MyoD in the observed upregulation of MHC IIB in theunweighted soleus muscle.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

MiR-499/PRDM16 axis modulates the adipogenic differentiation of mouse skeletal muscle satellite cells

Obesity is associated with increased risks of diverse diseases; brown adipose tissue (BAT) can increase energy expenditure and protect against obesity by increasing the decomposition of white adipose tissue (WAT) to enhance the non-coupled oxidative phosphorylation of fatty acid in adipocytes and contributes to weight loss. However, BAT is abundant in only small rodents and newborn humans, but not in adults. PRDM16 is a key factor that induces the differentiation of skeletal muscle precursors to brown adipocytes and simultaneously inhibits myogenic differentiation. In the present study, we set insulin-induced skeletal muscle satellite cells (SMSCs) adipogenic differentiation model, as confirmed by the contents of adipogenic markers PRDM16, UCP1 and PGC1α and myogenic markers MyoD1 and MyoG. We selected miR-499 as candidate miRNA, which might regulate PRDM16 to affect SMSCs adipogenic differentiation. Possibly through directly binding to PRDM16 3′-UTR, miR-499 negatively regulated PRDM16 expression and hindered SMSCs adipogenic differentiation by reducing adipogenic markers PRDM16, UCP1 and PGC1α and increasing myogenic markers MyoD1 and MyoG. PRDM16 overexpression could partially reverse the effect of miR-499 on the above markers and SMSCs adipogenic differentiation. Taken together, miR-499/PRDM16 axis can affect the balance between SMSC myogenic and adipogenic differentiation, targeting miR-499 to rescue PRDM16 expression, thus promoting SMSCs adipogenic differentiation may be a promising strategy for obesity treatment.