Maintenance DNA Methyltransferase Activity in the Presence of Oxidized Forms of 5-Methylcytosine: Structural Basis for Ten Eleven Translocation-Mediated DNA Demethylation

Abstract

A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein-DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.

Changes in DNA methylation and imprinting disorders in E9.5 mouse fetuses and placentas derived from vitrified eight‐cell embryos

“…In fetuses, DNMT1 and DNMT3b showed a significant increase in the VET group compared with the IVC group (Figure a). DNA methylation marks are introduced by de novo methyltransferases DNMT3a/b and are subsequently preserved by the maintenance of DNMT1 (Seiler et al, ). Thus, these results could partly explain the increased global methylation level observed in fetuses of the VET group.…”

“…Thus, these results could partly explain the increased global methylation level observed in fetuses of the VET group. The TET family of genes is thought to play a role in active genomic demethylation and the resetting of the epigenome (Inoue & Zhang, ; Seiler et al, ; Skiles et al, ). According to our results, TET2 and TET3 were increased in fetuses of the IVC group compared with two other groups, which revealed the reason for the decreased global methylation level of the IVC group.…”

Active DNA demethylation—The epigenetic gatekeeper of development, immunity, and cancer

“…20 One potential problem with this model is that DNMT1 activity is affected by the oxidation state of the hemi-methylated strand-its activity is only about a third on the Cs opposite to 5hmCs compared to those Cs opposite to 5mC. 21…”

Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N‐Demethylation

“…This pathway involves TET‐mediated oxidation of 5mC to generate 5‐hydroxymethylcytosine (5hmC), the predominant product, along with 5‐formylcytosine (5fC) and 5‐carboxycytosine (5caC) . Each of these oxidized 5mC bases (ox‐mCs) can promote the passive, replication‐dependent loss of 5mC by antagonizing the maintenance DNA methyltransferase DNMT1 . 5fC and 5caC can also be excised as part of a base excision repair pathway to complete an active, but indirect, demethylation process …”

“…[4][5][6] Each of these oxidized 5mC bases (ox-mCs) can promote the passive, replication-dependent loss of 5mC by antagonizing the maintenance DNA methyltransferase DNMT1. [7] 5fC and 5caC can also be excised as part of a base excision repair pathway to complete an active, but indirect, demethylation process. [5,8] This indirect demethylation of 5mC by TET enzymes stands as a point of contrast to their closely related family members in the larger Fe II /a-ketoglutarate(aKG)-dependent dioxygenase family.…”