title: Virion protein 16 induces demethylation of DNA integrated within chromatin in a novel mammalian cell model.
abstract: DNA methylation and demethylation play important roles in mediating epigenetic regulation. So far, the mechanism of DNA demethylation remains elusive and controversial. Here, we constructed a plasmid, named with pCBS-luc, that contained an artificial CpG island, eight Gal4 DNA-binding domain binding site, an SV40 promoter, and a firefly luciferase reporter gene. The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells. The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained. The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification. The methylation status could be maintained even after 15 passages. The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs. Using our me-pCBS-luc model, we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells. Altogether, we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid, and in this model we found that VP16 could lead to DNA demethylation. We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification.
authors: Key Laboratory of Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, China.
authors: Lu
authors: L
authors: Yang
authors: Huijun
authors: H
authors: Wang
authors: Xin
authors: X
authors: Luo
authors: Pengliang
authors: P
authors: Mao
authors: Weidong
authors: W
authors: Tian
authors: Yujiang
authors: Y
authors: Shi
authors: Guoying
authors: G
authors: Huang
authors: Jin
authors: J
authors: Zhang
authors: Duan
authors: D
authors: Ma
mesh_terms: D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D044127:Epigenesis, Genetic; D005260:Female; D017930:Genes, Reporter; D057809:HEK293 Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D014981:Xenopus
chemical_list: D002843:Chromatin; D017931:DNA Primers; D017405:Herpes Simplex Virus Protein Vmw65; D008156:Luciferases