title: Transcriptional activity of acetylcholinesterase gene is regulated by DNA methylation during C2C12 myogenesis.
abstract: 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.
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Kei M
authors.initials: KM
authors.lastname: Lau
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Amy G W
authors.initials: AGW
authors.lastname: Gong
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Miranda L
authors.initials: ML
authors.lastname: Xu
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Candy T W
authors.initials: CTW
authors.lastname: Lam
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Laura M L
authors.initials: LML
authors.lastname: Zhang
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Cathy W C
authors.initials: CWC
authors.lastname: Bi
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: D
authors.initials: D
authors.lastname: Cui
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Anthony W M
authors.initials: AWM
authors.lastname: Cheng
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Tina T X
authors.initials: TTX
authors.lastname: Dong
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China. Electronic address: botsim@ust.hk.
authors.forename: Karl W K
authors.initials: KWK
authors.lastname: Tsim
authors.affiliation: Division of Life Science and Center of Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China. Electronic address: linhuangquan@ust.hk.
authors.forename: Huangquan
authors.initials: H
authors.lastname: Lin
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D051379:Mice; D024510:Muscle Development; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D016329:Sp1 Transcription Factor; D000110:Acetylcholinesterase
keywords: AChE; DNA methylation; Epigenetics; Myogenesis; Transcription regulation