In vertebrates, acetylcholinesterase (AChE) is well-known for its role in hydrolyzing neurotransmitter acetylcholine at the neuromuscular junction (nmj), which is critical for proper function of our motor system. Previous studies evidenced that the expression of AChE was developmentally regulated during myogenesis, indicating the significance of intrinsic factors in regulating gene expression. However, these factors involved in the AChE regulation remained unclear. During myogenesis, DNA methylation is essential for temporal control of myogenic gene expression. Vertebrate AChE gene carries highly conserved CG-rich regions in its promoters, implying its likeliness to be methylated. Here, we aimed to investigate the regulation of AChE during myogenesis by DNA methylation. Myogenic...[ Read more ]

In vertebrates, acetylcholinesterase (AChE) is well-known for its role in hydrolyzing neurotransmitter acetylcholine at the neuromuscular junction (nmj), which is critical for proper function of our motor system. Previous studies evidenced that the expression of AChE was developmentally regulated during myogenesis, indicating the significance of intrinsic factors in regulating gene expression. However, these factors involved in the AChE regulation remained unclear. During myogenesis, DNA methylation is essential for temporal control of myogenic gene expression. Vertebrate AChE gene carries highly conserved CG-rich regions in its promoters, implying its likeliness to be methylated. Here, we aimed to investigate the regulation of AChE during myogenesis by DNA methylation. Myogenic differentiation of mouse C2C12 myoblast cells was triggered by serum reduction; the mRNA level, promoter activity, enzymatic activity and protein expression of AChE were up-regulated along with the myotube formation. To investigate the role of DNA methylation in AChE regulation, a DNA methyltransferase inhibitor, 5-Azacytidine (5-Aza), was applied throughout myogenesis. 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. The effect of 5-Aza led to further investigation on the potential methylated sites on AChE promoter during myogenesis. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 differentiation; a SP1 site on AChE promoter was revealed to be heavily methylated. Additionally, the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture. By gel mobility shift assay, the DNA methylation on the SP1 site, derived from AChE promoter, totally blocked the binding of SP1. The findings suggest the role of DNA methylation on AChE transcriptional regulation and provide insight for elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during myogenic differentiation.