Mammalian DNA methyltransferases: new discoveries and open questions

Abstract

As part of the epigenetic network, DNA methylation is a major regulator of chromatin structure and function. In mammals, it mainly occurs at palindromic CpG sites, but asymmetric methylation at non-CpG sites is also observed. Three enzymes are involved in the generation and maintenance of DNA methylation patterns. DNMT1 has high preference for hemimethylated CpG sites, and DNMT3A and DNMT3B equally methylate unmethylated and hemimethylated DNA, and also introduce non-CpG methylation. Here, we review recent observations and novel insights into the structure and function of mammalian DNMTs (DNA methyltransferases), including new structures of DNMT1 and DNMT3A, data on their mechanism, regulation by post-translational modifications and on the function of DNMTs in cells. In addition, we present news findings regarding the allosteric regulation and targeting of DNMTs by chromatin modifications and chromatin proteins. In combination, the recent publications summarized here impressively illustrate the intensity of ongoing research in this field. They provide a deeper understanding of key mechanistic properties of DNMTs, but they also document still unsolved issues, which need to be addressed in future research.

Keywords: DNA methylation; DNA methyltransferases; enzymology; molecular epigenetics.

Figure 1. Cycle of DNA methylation in human cells and during development (adapted from [11]).

DNA methylation patterns are generated during development and germ cell differentiation by de novo methyltransferases and kept through DNA replication by maintenance methylation. DNA methylation can be lost through passive or active demethylation. Typically, Dnmt1 is considered to be a maintenance enzyme, whereas Dnmt3a and Dnmt3b are regarded as de novo methyltransferases (TET, ten eleven translocation enzyme; TDG, thymine–DNA glycosylase).

Figure 2. General features of DNMTs.

(A) Domain structure of the mammalian DNMT enzymes. The human DNMT1, DNMT3A, DNMT3B and DNMT3L proteins consist of 1616, 912, 853 and 387 amino acid residues, respectively. DMAPD, DNA methyltransferase-associated protein 1-interacting domain; PBD, PCNA-binding domain; NLS, nuclear localization signal; RFTD, replication foci targeting sequence (RFTS) domain; CXXC, CXXC domain; BAH1 and BAH2, bromo-adjacent homology domains 1 and 2; GK_n, glycine lysine repeats; PWWP, PWWP domain; ADD, ATRX-DNMT3-DNMT3L domain (reprinted with permission from [6]). (B) Catalytic mechanism of cytosine C5 DNA methyltransferases (adapted from [12]). The catalytic glutamate residue from ENV motif (motif IV) is colored brown, the catalytic cysteine residue from PCQ motif (motif IV) is colored green, AdoMet is blue with the methyl group shown in red. The base for final proton abstraction (shown in orange) is not identified; it may be a water molecule.

Figure 3. Structures of DNMT1.

(A) Structure of the DNMT1 apoenzyme [15] ( pdb 3AV4). The RFTS domain is shown in green, CXXC in red, BAH domains in orange and purple and the catalytic domain in blue. (B) Structure of a truncated C-terminal domain of DNMT in complex with hemimethylated DNA [21] ( pdb 4DA4). SAH is shown in yellow and DNA in light green. (C) Interaction of the flipped target base (Cyt) with the catalytic residues in the active site pocket of DNMT1 (C1339, E1369 and R1315).

Figure 4. Interaction of DNMTs with chromatin modifications.

(A) Structure of the DNMT1 RFTS domain (green) in complex with the H3 peptide (shown in yellow) ubiquitinated at K18 and K23 [39] ( pdb 5WVO). The ubiquitin moieties are colored in red and light red. For illustration, the RFTS domain is overlaid with the structure of the DNMT1 apoenzyme colored and oriented as in Figure 3A. (B) Binding of the DNMT3A ADD domain to the catalytic DNMT3A domain (blue) in the DNMT3A/3L heterotetramer [55] ( pdb 4U7P and 4U7T). The ADD domain is shown in the autoinhibitory conformation (orange) and with bound peptide (yellow) in the catalytically active allosteric conformation (red).

Figure 5. Structure of DNMT3A/3L in complex with DNA [58] ( pdb 6BRR).

(A) Overview of the structure. DNMT3A subunits are shown in blue and cyan, DNMT3L subunits in red and orange. SAH is shown in yellow, DNA in green. (B) Interaction of the flipped target base (Cyt) with the catalytic residues in the active site pocket of DNMT3A.