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

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.