TET enzymes, TDG and the dynamics of DNA demethylation

Abstract

DNA methylation has a profound impact on genome stability, transcription and development. Although enzymes that catalyse DNA methylation have been well characterized, those that are involved in methyl group removal have remained elusive, until recently. The transformative discovery that ten-eleven translocation (TET) family enzymes can oxidize 5-methylcytosine has greatly advanced our understanding of DNA demethylation. 5-Hydroxymethylcytosine is a key nexus in demethylation that can either be passively depleted through DNA replication or actively reverted to cytosine through iterative oxidation and thymine DNA glycosylase (TDG)-mediated base excision repair. Methylation, oxidation and repair now offer a model for a complete cycle of dynamic cytosine modification, with mounting evidence for its significance in the biological processes known to involve active demethylation.

Figure 1. TET and TDG function in oxidation and excision of modified C bases

a, Schematic of mouse Tet enzymes, showing the double-stranded β-helix (DSβH) fold core oxygenase domain, a preceding cysteine (Cys)-rich domain and a CXXC domain in Tet1 and Tet3. b, Catalytic pathway for generation of 5hmC by Tet enzymes. An active site Fe(II) (left) is bound by conserved His–His–Asp residues in Tet and coordinates water and α-ketoglutarate (α-KG). A two-electron oxidation of α-KG by molecular oxygen yields CO₂ and enzyme-bound succinate, and results in a high-valent Fe(IV)-oxo intermediate (right). The intermediate reacts with 5mC to yield 5hmC, with a net oxidative transfer of the single oxygen atom to the substrate, resulting in regeneration of the Fe(II) species. c, TDG specifically accommodates oxidized C bases. Shown is the active site of TDG, bound to DNA, containing a substrate analogue of 5caC (PDB 3UOB). Critical residues of the enzyme are labelled. The 5caC analogue is highlighted in yellow. Heteroatoms are shown with nitrogen (blue), oxygen (red) and phosphorus (orange). The distance of hydrogen bonds (dashed red lines) are measured in Å. In addition to several interactions with the Watson–Crick face of the base from Asn 191 and His 151, the carboxylate substituent in the 5-position is well-accommodated by the active site with a binding pocket defined by Ala 145, and hydrogen bonds from Asn 157 and the backbone amide of Tyr 152.

Figure 2. A complete pathway for dynamic modifications of C

a, A biochemically validated pathway for modification of C within DNA is shown. 5mC bases, introduced by DNA methyltransferase (DNMT) enzymes, can be oxidized iteratively to 5hmC, 5fC and 5caC. In the pathway of active modification (AM) followed by passive dilution (PD), 5hmC is diluted in a replication-dependent manner to regenerate unmodified C. For clarity, PD of highly oxidized 5fC and 5caC is not depicted. In the pathway of AM followed by active restoration (AR), 5fC or 5caC is excised by TDG generating an abasic site as part of the base excision repair (BER) process that regenerates unmodified C. b, The individual reactions in the pathway are shown with all reactants depicted. The BER pathway involves excision of the abasic site, replacement of the nucleotide using unmodified deoxycytidine triphosphate (dCTP) by a DNA polymerase (generating pyrophosphate, PPi) and ligation to repair the nick. α-KG, α-ketoglutarate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

Figure 3. DNA methylation dynamics in pre-implantation embryos and primordial germ cells

a, Dynamics of 5mC and its oxidation products in pre-implantation embryos. Although the maternal DNA goes through passive demethylation, the paternal genome is demethylated in two steps. Tet3 first oxidizes the 5mC in the paternal genome, and the oxidation products are then diluted through a replication-dependent process. For clarity, although the absolute levels of 5hmC, 5fC and 5caC differ, the bases are schematically shown together (dotted line) given that their increase and subsequent depletion follow similar patterns. DNA methylation patterns are re-established by de novo DNMTs at the blastocyst stage. b, Illustration of the 5mC and 5hmC dynamics in primordial germ cells (PGCs) during their reprogramming. DNA demethylation in PGCs goes through three stages: loss of bulk DNA methylation in a Tet-independent manner; oxidation of remaining 5mC to 5hmC by Tet1 and potentially Tet2 proteins; and loss of 5hmC through replication-dependent passive dilution. 5fC and 5caC are not shown in this panel because no dynamic change in their levels was observed by immunostaining^–. Figure scale is shown in embryonic days post-fertilization.