title: DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45.
abstract: Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.
authors.affiliation: Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Kunal
authors.initials: K
authors.lastname: Rai
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Huggins
authors.forename: Smitha R
authors.initials: SR
authors.lastname: James
authors.forename: Adam R
authors.initials: AR
authors.lastname: Karpf
authors.forename: David A
authors.initials: DA
authors.lastname: Jones
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Cairns
mesh_terms: D000818:Animals; D002460:Cell Line; D003564:Cytidine Deaminase / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D045647:DNA Glycosylases / Q000378:metabolism*; D019175:DNA Methylation; D004625:Embryo, Nonmammalian / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000378:metabolism*; D015854:Up-Regulation; D015027:Zebrafish / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism*; D000097642:GADD45 Proteins
chemical_list: D003564:Cytidine Deaminase; D004248:DNA (Cytosine-5-)-Methyltransferases; D045647:DNA Glycosylases; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D044502:Thymine DNA Glycosylase; D029961:Zebrafish Proteins; D000097642:GADD45 Proteins; C000600492:MBD4 protein, zebrafish