EXPRESSION AND ACTIVITY OF SERUM RESPONSE FACTOR IS REQUIRED FOR EXPRESSION OF THE MUSCLE-DETERMINING FACTOR MYOD IN BOTH DIVIDING AND DIFFERENTIATING MOUSE C2C12 MYOBLASTS

To understand the mechanism by which the serum response factor (SRF) i s involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the exp ression of the muscle-determining factor MyoD. Inhibition of SRF activ ity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the e ndogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF con taining the DNA-binding domain of SRF, which acts as a dominant negati ve mutant in vivo), or purified anti-SRF antibodies rapidly prevents t he expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in prol iferating myoblasts but also in myoblasts cultured under differentiati ng conditions. Additionally, by using a cellular system expressing a g lucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have show n that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofl uorescence and Northern blot analysis. Taken together these data demon strate that SRF expression and activity are required for the expressio n of the muscle-determining factor MyoD.