MyoD Directly Up-regulates Premyogenic Mesoderm Factors during Induction of Skeletal Myogenesis in Stem Cells
MyoD Directly Up-regulates Premyogenic Mesoderm Factors during Induction of Skeletal Myogenesis in Stem Cells
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DOI：
10.1074/jbc.m110.163709
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发表时间：
2011-01-28
影响因子：
4.8
通讯作者：
Skerjanc, Ilona S.
中科院分区：
文献类型：
--
作者：
Gianakopoulos, Peter J.;Mehta, Virja;Skerjanc, Ilona S.
功能获得和功能丧失实验表明，成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需要细胞聚集来诱导P19胚胎癌干细胞中的肌生成。为了确定干细胞可以被引导到骨骼肌中的机制，在存在和不存在外源性MyoD的情况下检查P19细胞分化的时程。通过定量PCR，第一个MyoD上调的转录物是分化第4天的前肌源性中胚层因子Meox 1、Pax 7、Six 1和Eya 2。随后，成肌细胞标记物肌细胞生成素、MEF 2C和Myf 5上调，导致骨骼肌发生。Western印迹分析证实了这些结果，表明在肌细胞生成素蛋白表达之前，Pax 3、Six 1和MEF 2C蛋白上调。为了确定显性阴性MyoD/EnR突变体在什么阶段可以抑制肌发生，建立并检查了稳定的细胞系。有趣的是，前肌源性中胚层因子Meox 1、Pax 3/7、Six 1、Eya 2和Foxc 1下调，并且正如预期的那样，骨骼肌发生被取消。最后，为了在该系统中鉴定MyoD的直接靶标，进行染色质免疫沉淀实验。观察到MyoD与Meox 1、Pax 3/7、Six 1、Eya 2和肌生成素基因的调节区相关。总之，MyoD通过在激活成肌细胞基因之前结合并激活前肌源性中胚层基因的表达，将干细胞引导到骨骼肌谱系中。
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.