西亚试剂：DNA Demethylation in Zebrafish Involves the Coupling of a D

DNA Demethylation in Zebrafish Involves the Coupling of a Deaminase, a Glycosylase, and Gadd45

Kunal Rai1,3,4,Ian J. Huggins4,Smitha R. James5,Adam R. Karpf5,David A. Jones1,2,4,,andBradley R. Cairns1,3,4,,

1 Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA
2 Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA
3 Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA
4 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
5 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

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 invivo 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 thedeaminase/glycosylase pair AID/Mbd4 invivo 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.

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