Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
J Biol Chem. 2011 Jan 28;286(4):2517-25. doi: 10.1074/jbc.M110.163709. Epub 2010 Nov 15.
Gain- and loss-of-function experiments have illustrated that the family of myogenic regulatory factors is necessary and sufficient for the formation of skeletal muscle. Furthermore, MyoD required cellular aggregation to induce myogenesis in P19 embryonal carcinoma stem cells. To determine the mechanism by which stem cells can be directed into skeletal muscle, a time course of P19 cell differentiation was examined in the presence and absence of exogenous MyoD. By quantitative PCR, the first MyoD up-regulated transcripts were the premyogenic mesoderm factors Meox1, Pax7, Six1, and Eya2 on day 4 of differentiation. Subsequently, the myoblast markers myogenin, MEF2C, and Myf5 were up-regulated, leading to skeletal myogenesis. These results were corroborated by Western blot analysis, showing up-regulation of Pax3, Six1, and MEF2C proteins, prior to myogenin protein expression. To determine at what stage a dominant-negative MyoD/EnR mutant could inhibit myogenesis, stable cell lines were created and examined. Interestingly, the premyogenic mesoderm factors, Meox1, Pax3/7, Six1, Eya2, and Foxc1, were down-regulated, and as expected, skeletal myogenesis was abolished. Finally, to identify direct targets of MyoD in this system, chromatin immunoprecipitation experiments were performed. MyoD was observed associated with regulatory regions of Meox1, Pax3/7, Six1, Eya2, and myogenin genes. Taken together, MyoD directs stem cells into the skeletal muscle lineage by binding and activating the expression of premyogenic mesoderm genes, prior to activating myoblast genes.
获得和丧失功能的实验表明，肌调节因子家族对于骨骼肌的形成是必要和充分的。此外，MyoD 要求细胞聚集以诱导 P19 胚胎癌细胞干细胞的肌生成。为了确定可以将干细胞定向为骨骼肌的机制，在存在和不存在外源性 MyoD 的情况下检查了 P19 细胞分化的时间过程。通过定量 PCR，在分化的第 4 天，首先上调的 MyoD 转录物是原始中胚层因子 Meox1、Pax7、Six1 和 Eya2。随后，上调了成肌细胞标记物 myogenin、MEF2C 和 Myf5，导致骨骼肌生成。Western blot 分析证实了这些结果，显示出 Pax3、Six1 和 MEF2C 蛋白在 myogenin 蛋白表达之前上调。为了确定显性负性 MyoD/EnR 突变体可以在哪个阶段抑制肌生成，创建了稳定的细胞系并进行了检查。有趣的是，原始中胚层因子 Meox1、Pax3/7、Six1、Eya2 和 Foxc1 下调，并且如预期的那样，骨骼肌生成被废除。最后，为了鉴定该系统中 MyoD 的直接靶标，进行了染色质免疫沉淀实验。观察到 MyoD 与 Meox1、Pax3/7、Six1、Eya2 和 myogenin 基因的调节区域结合并激活其表达。总之，MyoD 通过与原始中胚层基因的调节区域结合并激活其表达，将干细胞定向为骨骼肌谱系，然后激活成肌细胞基因。
