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Study on the transcriptional regulatory mechanism of the MyoD1 gene in Guanling bovine

机译：关岭牛MyoD1基因转录调控机制的研究

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The MyoD1 gene plays a key role in regulating the myoblast differentiation process in the early stage of skeletal muscle development. To understand the functional elements of the promoter region and transcriptional regulation of the bovine MyoD1 gene, we cloned eight fragments from the sequence region of the MyoD1 gene promoter and inserted them into eukaryotic expression vectors for cotransfection with the mouse myoblast cell line C2C12 and Madin-Darby bovine kidney (MDBK) line. A variety of transcription factor binding sites in the longest 5′-flanking fragment from Guanling cattle MyoD1-P1 were predicted by using the online software TFSEARCH and ALGGEN PROMO as well as validated by the promoter-binding TF profiling assay II and yeast one-hybrid (Y1H) assay, including MyoD, VDR, MEF1, MEF2, SF1, and Myf6. Myf6 strongly activated the MyoD1 promoter, while MyoD1 was also capable of efficiently activating the expression of its own promoter. The transcription factors MEF2A, SF1, and VDR were further confirmed to be capable of binding to MyoD1 by Y1H system experiments. The effects of the Guanling cattle MyoD1 gene on the mRNA expression of the MEF2A, SF1, and VDR genes were determined by using a lentivirus-mediated overexpression technique, confirming that overexpression of the MyoD1 gene upregulated the mRNA expression of MEF2A as well as downregulated the expression of SF1 and VDR in the process of muscle myogenesis. Our study revealed the effects of transcription factors including MEF2A, SF1 and VDR on regulatory aspects of MyoD1, providing abundant information for transcriptional regulation of MyoD1 in muscle differentiation.
机译：MyoD1基因在骨骼肌发育的早期阶段，在调节成肌细胞分化过程中起关键作用。为了了解牛MyoD1基因启动子区域的功能元件和转录调控，我们从MyoD1基因启动子的序列区域中克隆了8个片段，并将它们插入到真核表达载体中，以便与小鼠成肌细胞C2C12和Madin-达比牛肾（MDBK）品系。使用在线软件TFSEARCH和ALGGEN PROMO预测了关岭牛MyoD1-P1最长5'侧翼片段中的多种转录因子结合位点，并通过启动子结合TF谱分析II和酵母一杂交进行了验证（Y1H）分析，包括MyoD，VDR，MEF1，MEF2，SF1和Myf6。 Myf6强烈激活了MyoD1启动子，而MyoD1也能够有效激活其自身启动子的表达。 Y1H系统实验进一步证实了转录因子MEF2A，SF1和VDR能够与MyoD1结合。利用慢病毒介导的过表达技术确定了关岭牛MyoD1基因对MEF2A，SF1和VDR基因的mRNA表达的影响，证实MyoD1基因的过表达上调了MEF2A的mRNA表达，并下调了MEF2A的mRNA表达。肌新生过程中SF1和VDR的表达我们的研究揭示了包括MEF2A，SF1和VDR在内的转录因子对MyoD1调控的作用，为MyoD1在肌肉分化中的转录调控提供了丰富的信息。

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