TET enzymes, DNA modification, and DNA demethylation. (A) TET-mediated 5mC oxidation. TET uses reduced iron (Fe²⁺), oxygen, and α-ketoglutarate (αKG) to oxidize 5mC, generating the products CO₂, succinate, and the oxidized methylcytosine (oxi-mC) bases 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxylcytosine (5caC). The enzymatic activity of TET is modulated by the levels of αKG and is subject to product inhibition by succinate. Vitamin C enhances the enzymatic activity of TET, most likely by maintaining Fe²⁺ in its reduced state. Hypoxia and both enantiomers of the oncometabolite 2-hydroxyglutarate (2HG) inhibit TET activity. (B) TET2-DNA interaction.¹¹²  The diagram shows a portion of the TET2 catalytic domain (gray; amino acids [aa]1129-1480, 1844-1936) interacting with 5mC (orange) on double-stranded DNA (turquoise). The 5mC base is flipped and inserted into the TET2 active site with the methyl group adjacent to Fe²⁺ (blue) and αKG (magenta). Zinc ions are shown in yellow. (C) TET-mediated DNA demethylation. Unmodified cytosines in DNA are methylated by DNMTs to yield 5mC. TET proteins successively oxidize 5mC to 5hmC, 5fC, and 5caC. 5hmC is the most abundant of the oxi-mCs (∼1% to 10% of 5mC in most somatic cell types and often higher in neurons); 5fC and 5caC are ∼10- to 100-fold and ∼100- to 1000-fold less abundant, respectively, compared with 5hmC. Oxi-mCs present on the unreplicated DNA strand in the CpG sequence context are not recognized by the DNMT1/UHRF1 complex, which normally recognizes hemimethylated CpGs; this prevents the restoration of symmetrical methylation on the newly replicated strand and facilitates passive (replication-dependent) DNA demethylation (top arrow). TET can also facilitate DNA demethylation independently of DNA replication (bottom arrow), because 5fC and 5caC can both be excised by thymine DNA glycosylase (TDG) and replaced with unmodified cytosine through base-excision repair (BER). (D) TET2 mutations in DLBCL. Diagrammatic representation of the domain structure of TET2 including the cysteine-rich domain (cys-rich; aa1129-1312) and catalytic domain. Dotted line (aa1481-1843) represents the region replaced with low-complexity linker in the structural study in panel B (top).¹¹²  Hatch marks showing the distribution of TET2 mutations observed in diffuse large B-cell lymphoma (DLBCL; data from cBioPortal^113,114 ). In a total of 1295 DLBCL cases, 72 (∼5.6%) were observed to bear TET2 mutations, of which 34 were nonsense mutations predicted to produce a truncated TET2 protein and 46 were missense mutations predicted to give rise to single amino acid substitutions (middle and bottom). Note that TET1 and TET3 mutations are rare (∼0.2%).