DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells

Biswanath Chatterjee, Miao Hsia Lin, Chun Chang Chen, Kai Lin Peng, Mu Sheng Wu, Mei Chun Tseng, Yu Ju Chen, Che Kun James Shen

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca2+ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca2+ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca2+-dependent physiological processes.

Original languageEnglish
Pages (from-to)1048-1061
Number of pages14
JournalBiochimica et Biophysica Acta - Gene Regulatory Mechanisms
Issue number11
Publication statusPublished - Nov 2018
Externally publishedYes


  • Cell culture
  • DNA demethylation
  • DNA methyltransferases
  • DNA transfection
  • LC-MS/MS
  • M.SssI
  • Na-bisulfite sequencing
  • pEGFP-N2
  • S-adenosyl-L-methionine

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics


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