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  • Review Article
  • Published:

TET-mediated active DNA demethylation: mechanism, function and beyond

Key Points

  • Active DNA demethylation in mammals is achieved through TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution of oxidized 5mC or thymine DNA glycosylase (TDG)-mediated excision of 5fC and 5caC coupled with base excision repair.

  • Active DNA demethylation is regulated at various levels, including substrate and cofactor availability, post-transcriptional and post-translational regulation of TET and TDG, and genomic localization of the demethylation machinery.

  • Studies of tissue distribution, genomic distribution and the dynamics of oxidized 5mC provide insights into the mechanism and function of active DNA demethylation as well as the potential roles of oxidized 5mC.

  • Active DNA demethylation and oxidized 5mC are involved in pre-implantation embryo development, primordial germ cell development, pluripotency and differentiation, as well as neuronal functions. In certain biological contexts, such as in pre-implantation embryos, the biological meaning of TET-mediated oxidation is not fully understood. In some other biological contexts, such as neurons, the extent of active DNA demethylation and its function require further study.

  • TET may function in a catalytic-activity-independent manner. Further analysis is needed to distinguish the functions of the TET proteins themselves from the function of active DNA demethylation.

  • Emerging evidence suggests an interplay between TET, active DNA demethylation and genomic instability and the DNA damage response.

Abstract

In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodified cytosine (C) through TET dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair. In the past few years, biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA demethylation. Additionally, many regulatory mechanisms of this process have been identified. Technological advances in mapping and tracing the oxidized forms of 5mC allow further dissection of their functions. Furthermore, the biological functions of active DNA demethylation in various biological contexts have also been revealed. In this Review, we summarize the recent advances and highlight key unanswered questions.

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Figure 1: TET-mediated active DNA demethylation.
Figure 2: Processivity of TET at three different levels.
Figure 3: Mechanism of DNA demethylation in different biological contexts.

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Acknowledgements

The authors thank members of the Zhang laboratory and also H. Wu for their comments on the manuscript. X.W. was supported by a fellowship from the China Scholarship Council. Y.Z. is an investigator of the Howard Hughes Medical Institute. The authors apologize to colleagues whose work cannot be cited owing to space limitations.

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Glossary

TET

A family of methylcytosine dioxygenase enzymes that are involved in several steps of the oxidative demethylation of 5-methylcytosine. They are named after the cancer-associated 'ten–eleven translocation', which creates a fusion between mixed-lineage leukaemia (MLL; also known as KMT2A) on chromosome 11 and TET1 on chromosome 10.

Base excision repair

(BER). The repair of a damaged base through the following steps: excising the base to create an abasic site, generating a single-strand break (SSB) and repairing the SSB through short-patch or long-patch repair.

Genetic processivity

The genetic outcome of TET-mediated oxidation in cells. Genomic regions or CpG sites modified by 5-hydroxymethylcytosine (5hmC) but not 5-formylcytosine (5fc) or 5-carboxylcytosine (5caC) are regarded as having low genetic processivity, whereas genomic regions or CpG sites modified by 5fC or 5caC are regarded as having higher genetic processivity. Unlike physical and chemical processivity, which describe the biochemical behaviour of the enzyme, genetic processivity describes the outcome of TET-mediated oxidation in vivo and is determined by various factors, including enzymatic activity and local chromatin environment.

AP lyase

An enzyme that is capable of cleaving the 3′ side of an abasic (apurinic or apyrimidinic (AP)) site to create a 3′-terminal unsaturated sugar and 5′-deoxyribosephosphate. This activity generates a single-strand break to initiate the downstream steps of base excision repair.

CpG islands

Genomic regions with a high density of CpG dinucleotides.

Bivalent promoters

Promoters that are enriched for both the active mark histone H3 lysine 4 trimethylation (H3K4me3) and the repressive mark H3K27me3.

γH2AX

Phosphorylated histone H2AX, a marker of a DNA strand break. At the genomic site of a DNA double-strand break (DSB), histone variant H2AX becomes phosphorylated (at S139 for human H2AX). Although γH2AX is most commonly used as a marker for DSBs, single-strand breaks (SSBs) can also induce γH2AX.

Penetrance

The proportion of individuals or animals (with a particular genotype) manifesting the phenotype of interest.

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Wu, X., Zhang, Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet 18, 517–534 (2017). https://doi.org/10.1038/nrg.2017.33

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