PHYSICAL INTERACTION BETWEEN THE MITOGEN-RESPONSIVE SERUM RESPONSE FACTOR AND MYOGENIC BASIC-HELIX-LOOP-HELIX PROTEINS

Terminal differentiation of muscle cells results in opposite effects o n gene promoters: muscle-specific promoters, which are repressed durin g active proliferation of myoblasts, are turned on, whereas at least s ome proliferation-associated promoters, such as c-fos, which are activ e during cell division, are turned off. MyoD and myogenin, transcripti on factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-f os. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens . Upon terminal differentiation, the activity of these various transcr iption factors could be modulated by the formation of distinct protein -protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a phys ical association between these transcription factors. We show that myo genin from differentiating myoblasts specifically binds to SRF. In, vi tro analysis, using the glutathione S-transferase pull-down assay, ind icates that SRF-myogenin interactions occur only with myogenin-E12 het erodimers and not with isolated myogenin. A physical interaction betwe en myogenin, E12, and SRF could also be demonstrated in vivo using a t riple-hybrid approach in yeast. Glutathione S-transferase pull-down an alysis of vari ous mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for t he interaction to be observed. Specific binding to SRF was also seen w ith MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH protei ns, did not bind SRF in any of the situations tested. These data sugge st that SRF, on one hand, and myogenic bHLH, on the other, could modul ate each other's activity through the formation of a heterotrimeric co mplex.