Yang Xinran, Mei Chugang, Raza Sayed Haidar Abbas, Ma Xinhao, Wang Jianfang, Du Jiawei, Zan Linsen
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China; National Beef Cattle Improvement Center, Northwest A&F University, Yangling 712100, Shaanxi, China.
Int J Biol Macromol. 2022 Dec 31;223(Pt A):916-930. doi: 10.1016/j.ijbiomac.2022.11.081. Epub 2022 Nov 12.
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.
DNA甲基化（5mC）和mRNA N - 甲基腺苷（mA）在基因转录调控中发挥着至关重要的作用。DNA甲基化已被充分证实参与骨骼肌发育。在多种生物学过程中，已发现DNA甲基化与mRNA mA修饰之间存在相互作用的调控机制。然而，mA对骨骼肌分化的影响及其潜在机制仍不清楚。在骨骼肌生成过程中，DNA甲基化与mRNA mA修饰之间是否存在相互作用也尚不清楚。在本研究中，我们使用mA - IP - qPCR、LC - MS/MS和斑点杂交分析确定，在牛骨骼肌成肌细胞分化过程中，DNA去甲基化酶基因TET1的mA水平升高，而mRNA表达降低。双荧光素酶报告基因分析和RIP实验表明，METTL3通过以mA - YTHDF2依赖的方式调节TET1 mRNA稳定性来抑制TET1表达。此外，TET1介导自身、MYOD1和MYOG的DNA去甲基化，从而刺激它们的表达以促进肌源性分化。TET1的异位表达挽救了METTL3敲低对肌管减少的影响。相反，TET1敲低损害了METTL3过表达促进的肌源性分化。此外，ChIP实验发现TET1可以结合并使METTL3 DNA去甲基化，从而增强METTL3表达。此外，TET1敲低降低了mA水平。ChIP分析还表明，TET1敲低有助于H3K4me3和H3K27me3与METTL3 DNA结合。我们的结果揭示了成肌细胞分化过程中TET1和METTL3之间的负反馈调节环，揭示了骨骼肌生成过程中DNA甲基化、RNA甲基化和组蛋白甲基化之间的相互作用。
