Lau Kei M, Gong Amy G W, Xu Miranda L, Lam Candy T W, Zhang Laura M L, Bi Cathy W C, Cui D, Cheng Anthony W M, Dong Tina T X, Tsim Karl W K, Lin Huangquan
Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
Brain Res. 2016 Jul 1;1642:114-123. doi: 10.1016/j.brainres.2016.03.013. Epub 2016 Mar 26.
The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation.
乙酰胆碱酯酶（AChE）是一种在脊椎动物神经肌肉接头处水解神经递质乙酰胆碱的酶，其表达在肌生成过程中受到调控，这表明肌肉内在因子在控制该酶表达方面具有重要意义。DNA甲基化对于肌生成过程中肌源性基因表达的时间控制至关重要；然而，其在AChE调控中的作用尚不清楚。脊椎动物ACHE基因的启动子带有高度保守的富含CG的区域，这意味着它有可能被甲基化以进行表观遗传调控。在整个肌管形成过程中，将一种DNA甲基转移酶抑制剂5-氮杂胞苷（5-Aza）应用于C2C12细胞。当DNA甲基化受到抑制时，分化第3天后AChE的启动子活性、转录表达和酶活性显著增加，这表明了DNA甲基化的假定作用。通过亚硫酸氢盐焦磷酸测序发现，在C2C12细胞分化过程中，总体甲基化率在第3天达到峰值；位于小鼠ACHE基因上游-1826bp处的一个SP1位点被发现高度甲基化。本文阐述了转录因子SP1在AChE表观遗传调控中的作用：（i）在5-Aza处理的C2C12培养物中，SP1驱动的转录活性增加；（ii）DNA甲基化完全阻断了SP1与ACHE基因SP1位点的结合；（iii）通过染色质免疫沉淀试验沉淀了ACHE基因SP1位点侧翼的序列。这些发现表明了DNA甲基化在AChE转录调控中的作用，并为阐明肌肉分化过程中DNA甲基化介导的AChE表达调控机制提供了见解。
