The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations

Abstract

One of the most fundamental questions in the control of gene expression in mammals is how the patterns of epigenetic modifications of DNA are generated, recognized, and erased. This includes covalent cytosine methylation of DNA and its associated oxidation states. An array of AdoMet-dependent methyltransferases, Fe(II)- and α-ketoglutarate-dependent dioxygenases, base excision glycosylases, and sequence-specific transcription factors is responsible for changing, maintaining, and interpreting the modification status of specific regions of chromatin. This review focuses on recent developments in characterizing the functional and structural links between the modification status of two DNA bases 5-methylcytosine and thymine (5-methyluracil).

Keywords: DNA 5mC oxidation; DNA demethylation; DNA enzyme; DNA methylation; DNA methyltransferase; DNA-binding protein; DNA-protein interaction; Tet dioxygenases; base excision DNA glycosylases; modification-dependent and sequence-specific transcription factors; transcription factor.

FIGURE 1.

General methylation and demethylation in histone lysines and nucleic acids. a, examples of post-synthetic methylation of both components of a nucleosome: DNA and histones. Major histone lysine methylation occurs at five residues on H3 (green) and one on H4 (blue), and primary enzymes responsible for demethylation are shown. DNA modifications occur at CpG and non-CpG (i.e. CpA) dinucleotides. b, overview of protein lysine methylation by AdoMet-dependent methyltransferases (top) and demethylation reactions catalyzed by LSD1/2 and Jumonji dioxygenases (bottom). The hydroxymethyl intermediate (N-CH₂OH) decomposes to release formaldehyde and the (one methyl group reduced) demethylated lysine. c, demethylation of N³-methylcytosine (N3mC) by AlkB dioxygenase (involved in the direct reversal of alkylation damage) results in the production of formaldehyde and unmodified cytosine. d, model of the mRNA N⁶-adenine methylation by methyltransferase-like METTL3/14 heterodimer, generating N6mA, and demethylation reaction by ALKBH5 and fat mass and obesity-associated protein (FTO) (two of the nine human homologs of AlkB), resulting in a release of formaldehyde and unmodified adenine. e, Tet dioxygenases convert 5mC to 5hmC, 5fC, and 5caC in three consecutive Fe(II)- and α-ketoglutarate-dependent oxidation reactions without release of formaldehyde. f, Tet dioxygenases convert thymine (5mU) to 5hmU, and potentially to 5fU and 5caU, without release of formaldehyde. g, Dnmt3A and Dnmt3B can methylate the cytosine in the context of the CpA/TpG dinucleotide. Tet dioxygenases can oxidize both 5mC and T (5mU). The diagram shows the potential fate of a single CpA/TpG site that is fully hydroxymethylated during DNA replication. After strand synthesis, the hemi-hydroxymethylated (5hmC/T) site could be modified by Tet enzymes to become fully hydroxymethylated (top). On the other hand, the hemi-hydroxymethylated (C/5hmU) site would require two reactions, methylation by Dnmt3 and oxidation by Tet, to become fully hydroxymethylated (bottom).

FIGURE 2.

Structures of the Tet enzymes. a, schematic representation of human Tet1 (hTet1) C-terminal catalytic domain and NgTet1. b, structure of NgTet1-DNA complex (Protein Data Bank (PDB) 4LT5). The NgTet1 protein folds in a three-layered jelly-roll structure. c, sequence alignment of human TET1, TET2, and TET3 (NP_085128.2, NP_001120680.1, and NP_001274420.1), mouse Tet1, Tet2, and Tet3 (NP_081660.1, NP_001035490.2, and NP_898961.2), honey bee (A. mellifera) AmTet (GB52555 in BeeBase OSGv3.2), and NgTet1 (XP_002667965.1). d, structure of human TET2-DNA complex (PDB 4NM6). The secondary structure elements are labeled according to NgTet1 structure (panel b). Note the large insertion in human TET2 between strands 8 and 9 (magenta), which is indicated by a magenta arrow in panel c.

FIGURE 3.

A methyl-Arg-G triad forms during recognition of methyl-CpG and TpG dinucleotides in double-stranded DNA. a, MeCP2 forms two 5mC-Arg-G triads to bind the palindromic fully methylated CpG duplex symmetrically (PDB 3C2I). b, Kaiso recognizes either a specific unmethylated DNA element containing a TpG dinucleotide (top) or a methylated CpG dinucleotide (bottom) (PDB 4F6M and 4F6N). In both cases, a methyl-Arg-G triad is involved. c, Zfp57 uses a pair of methyl-Arg-G triads to recognize the TpG dinucleotide on the top strand and a methyl-CpG on the bottom strand (PDB 4GZN). A third methyl-Arg-G triad recognizes the TpG on the bottom strand (not shown). d, example of transcription factor recognition sequences containing a CpA/TpG site (taken from Ref. 93). e–i, examples of protein domains with specificity for unmodified cytosine (e), 5mC = M (f), 5hmC = H (g), 5fC = F (h), or 5caC (i). The Tet3 CXXC domain binds to an unmodified cytosine in any sequence context (94), which is distinct from the CXXC domains of MLL (95), CFP1 (96), and Dnmt1 (97), which are restricted to unmodified CpG sites. CHTOP (chromatin target of PRMT1) binds to 5hmC (98). The modification of the CG dinucleotide (underlined) of TGACGCAA to 5mC, 5fC, or 5caC enhances DNA binding by the basic leucine zipper protein C/EBPβ (CCAAT-enhancer-binding protein β), whereas modification to 5hmC inhibits binding of C/EBPβ (99). The carboxylation of cytosine (5caC) in a CpG dinucleotide adjacent to the consensus recognition sequence (underlined) of the basic-helix-loop-helix transcription factor proteins Tcf3/Ascl1 (CGCANNTG) enhanced binding of the heterodimer by ∼10-fold (64).

FIGURE 4.

DNA glycosylases involved in removing modified bases from native Watson-Crick base pairs. ROS1 excises 5mC or 5hmC from its native base pairing with guanine; TDG excises 5fC or 5caC from base pairing with guanine; and SMUG1 excises 5hmU or 5fU from its native base pairing with an adenine. The base excision repair pathway removes the resulting abasic site and restores the unmodified C or T status.