Groisman R, Masutani H, Leibovitch M P, Robin P, Soudant I, Trouche D, Harel-Bellan A
Laboratoire de Biologie des Tumeurs Humaines, CNRS URA 1156, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France.
J Biol Chem. 1996 Mar 1;271(9):5258-64. doi: 10.1074/jbc.271.9.5258.
Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. 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 transcription 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 physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.
肌肉特异性启动子在成肌细胞活跃增殖期间受到抑制，此时会被开启，而至少一些与增殖相关的启动子，如在细胞分裂期间活跃的c-fos启动子，则会被关闭。MyoD和肌细胞生成素是来自碱性螺旋-环-螺旋（bHLH）家族的转录因子，它们参与这两个过程，上调肌肉基因并下调c-fos。另一方面，血清反应因子（SRF）参与肌肉特异性基因如c-fos的激活，以及对有丝分裂原作出反应的一部分基因的上调。在终末分化时，这些不同转录因子的活性可能通过形成不同的蛋白质-蛋白质复合物而受到调节。在此，我们研究了一种假说，即SRF和/或MyoD及肌细胞生成素的功能可能通过这些转录因子之间的物理结合而受到调节。我们发现，正在分化的成肌细胞中的肌细胞生成素能特异性结合SRF。使用谷胱甘肽S-转移酶下拉试验的体外分析表明，SRF-肌细胞生成素相互作用仅发生在肌细胞生成素-E12异二聚体之间，而不发生在分离的肌细胞生成素之间。使用酵母中的三杂交方法也能在体内证明肌细胞生成素、E12和SRF之间存在物理相互作用。对这些蛋白质的各种突变体进行的谷胱甘肽S-转移酶下拉分析表明，肌细胞生成素和E12的bHLH结构域对于观察到这种相互作用是必要且充分的。MyoD也能与SRF特异性结合。相反，Id是肌源性bHLH蛋白的天然抑制剂，在任何测试情况下都不与SRF结合。这些数据表明，一方面SRF，另一方面肌源性bHLH，可以通过形成异三聚体复合物来相互调节彼此的活性。
