The Mammalian de Novo DNA Methyltransferases DNMT3A and DNMT3B Are Also DNA 5-Hydroxymethylcytosine Dehydroxymethylases

Che-Kun James Shen,Chun-Chang Chen,Keh-Yang Wang

doi:10.1074/jbc.c112.406975

Abstract

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.

Highlights

The pathways of DNA dehydroxymethylation and demethylation are yet to be better defined
We found that the mammalian DNA methyltransferases (DNMTs), in particular DNMT3A and DNMT3B, but not DNMT1, could effectively convert 5-hydroxymethyl C (5-hmC) to C
In vitro oxidation assays were carried out using 5-hmC- or 5-methyl C (5-mC)-containing double-stranded DNA oligonucleotides incubated with nuclear extracts prepared from 293T cells transfected with plasmids expressing the mouse TET1 or DNMT3A, respectively

Summary

Background

The pathways of DNA dehydroxymethylation and demethylation are yet to be better defined. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are redox-dependent DNA dehydroxymethylases. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation. In contrast to our understanding of DNA methylation by the enzymatic activities of the vertebrate DNA methyltransferases (DNMTs), such as the de novo enzymes DNMT3A/DNMT3B. 5-mC residues on the paternal chromosomes have been found to be converted to 5-hmC ahead of DNA replication These 5-hmC residues might contribute to DNA demethylation by interfering with the substrate recognition of DNMT1 [18, 19] or by serving as intermediates for active demethylation process(es) in the paternal pronucleus [20, 21]. We present in vitro evidence supporting that DNMT3A and DNMT3B could function as active DNA 5-hmC dehydroxymethylases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION