Interactive regulation of DNA demethylase gene TET1 and mA methyltransferase gene METTL3 in myoblast differentiation.

DNA methylation (5mC) and mRNA N-methyladenosine (mA) play an essential role in gene transcriptional regulation. DNA methylation has been well established to be involved in skeletal muscle development. Interacting regulatory mechanisms between DNA methylation and mRNA mA modification have been identified in a variety of biological processes. However, the effect of mA on skeletal muscle differentiation and the underlying mechanisms are still unclear. It is also unknown whether there is an interaction between DNA methylation and mRNA mA modification in skeletal myogenesis. In the present study, we used mA-IP-qPCR, LC-MS/MS and dot blot assays to determine that the DNA demethylase gene, TET1, exhibited increased mA levels and decreased mRNA expression during bovine skeletal myoblast differentiation. Dual-luciferase reporter assays and RIP experiments demonstrated that METTL3 suppressed TET1 expression by regulating TET1 mRNA stability in a mA-YTHDF2-dependent manner. Furthermore, TET1 mediated DNA demethylation of itself, MYOD1 and MYOG, thereby stimulating their expression to promote myogenic differentiation. Ectopic expression of TET1 rescued the effect of METTL3 knockdown on reduced myotubes. In contrast, TET1 knockdown impaired the myogenic differentiation promoted by METTL3 overexpression. Moreover, ChIP experiments found that TET1 could bind and demethylate METTL3 DNA, which enhanced METTL3 expression. In addition, TET1 knockdown decreased mA levels. ChIP assays also showed that TET1 knockdown contributed to the binding of H3K4me3 and H3K27me3 to METTL3 DNA. Our results revealed a negative feedback regulatory loop between TET1 and METTL3 in myoblast differentiation, which unveiled the interplay among DNA methylation, RNA methylation and histone methylation in skeletal myogenesis.