Structural Basis of DNMT1 and DNMT3A-Mediated DNA Methylation

Abstract

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. The establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases (DNMTs): DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-domain proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1 and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

Keywords: DNA methyltransferase; DNMT1; DNMT3A; allosteric regulation; autoinhibition; de novo DNA methylation; maintenance DNA methylation.

Figure 1

Domain architectures of human DNA methyltransferases: DNMT1, DNMT3A and DNMT3B, and regulator DNMT3L, with individual domains marked by residue numbers.

Figure 2

Structure of mDNMT1-DNA productive complex. (A) Structural overview of mDNMT1 (amino acids 731–1602) covalently bound to hemimethylated DNA (Protein Data Base (PDB) 4DA4). The zinc ions are shown in purple spheres. 5fC and another flipped-out cytosine from the template strand are colored in purple and blue, respectively. (B) The DNA cavity vacated by the base flipping is filled with mDNMT1 residues M1235 and K1537. (C) The flipped-out 5fC is surrounded by active site residues through covalent linkage or hydrogen bonding interactions. (D) Residues from the target recognition domain (TRD) loop II form a hydrophobic groove harboring the methyl group from 5mC. (E) CpG-specific interactions by the TRD loop I and the catalytic loop. 5mC: 5-methylcytosine; 5fC: 5-fluorocytosine.

Figure 3

Structural analysis of the CXXC domain-mediated DNMT1 autoinhibition. (A) Structural overview of mDNMT1 (amino acids 650–1602) bound to a 19-mer DNA duplex containing unmethylated CpG sites (PDB 3PT6). (B) Surface views of the CXXC domain and the autoinhibitory linker in the complex of mDNMT1 with unmethylated CpG DNA. (C) Base-specific interactions between the CXXC domain and the CpG site. The hydrogen bonding interactions are depicted as dashed lines. (D) The MTase-DNA interactions in the autoinhibitory complex. (E) Structural overlay between the active (light blue) (PDB 4DA4) and autoinhibitory (pink) (PDB 3PT6) complexes of mDNMT1, with the catalytic loops highlighted in the expanded view.

Figure 4

Structural analysis of the replication foci-targeting sequence (RFTS) domain-mediated DNMT1 autoinhibition. (A) Structural overview of hDNMT1 (amino acids 351–1602) (PDB 4WXX). (B) The intramolecular interactions between the RFTS (green) and MTase (aquamarine) domains. The hydrogen bonding interactions are depicted as dashed lines. The water molecules are shown as purple spheres. (C) Structural overlap between the CXXC (PDB 3PT6) and RFTS (PDB 4WXX) mediated autoinhibitory complexes, with the autoinhibitory linkers colored in blue and light magenta, respectively. The repositioning of the CXXC domain is indicated by a red arrow. (D) The interaction of the autoinhibitory linker (magenta) with both the RFTS (green) and MTase domains (aquamarine).

Figure 5

A model for the allosteric regulation of DNMT1-mediated maintenance DNA methylation. Hemimethylated DNA and histone H3K9me3 serve as epigenetic signals to promote UHRF1-mediated ubiquitination of histone H3, which in turn shifts the conformation of DNMT1 from the autoinhibitory state into an active state for maintenance DNA methylation. UHRF1 P656, which occupies the H3K9me3-binding cage of the tandem Tudor domain (TTD) in the closed UHRF1 conformation, is indicated by the letter P. The active site of DNMT1 is marked by a filled red circle.

Figure 6

Structural analysis of the productive complex of the DNMT3A-DNMT3L tetramer with CpG DNA. (A) Structural overview of the DNMT3A-DNMT3L tetramer covalently bound to a 25-mer DNA duplex containing two CpG/ZpG sites (Z: Zebularine) (PDB 5YX2). The flipped-out zebularines are colored in purple. (B) Structural overlap between the DNA-bound and free DNMT3A-DNMT3L tetramer (PDB 2QRV). The TRD loops, which undergo disorder-to-order transition upon DNA binding, are colored in blue. (C) The DNA interactions involving the catalytic loop and other catalytic residues. (OG: Orphan guanine). The hydrogen bonding interactions are depicted as dashed lines. (D) Residues T834 and R836 from the TRD loop (blue) engage in base-specific recognition of the CpG site. The water molecules are shown as purple spheres.

Figure 7

Structural analysis of the Atrx-Dnmt3-Dnmt3l (ADD) domain-mediated DNMT3A autoinhibition. (A) Structural overview of the DNMT3A-DNMT3L tetramer, with the DNMT3A fragment comprised of both the ADD and MTase domains (PDB 4U7P). (B) Intramolecular interactions between the ADD loop (blue) and the MTase domain (aquamarine) of DNMT3A. The hydrogen bonding interactions are depicted as dashed lines. (C) The ADD-binding site of the DNMT3A MTase overlaps with its DNA binding site. (D) Structure of the DNMT3A-DNMT3L tetramer bound to the histone H3K4me0 peptide (PDB 4U7T), with the interaction between H3K4me0 and the ADD domain shown in an expanded view (PDB 3A1B).

Figure 8

Structures of DNMT3A/3B Pro-Trp-Trp-Pro (PWWP) domains. (A) Crystal structure of the DNMT3A PWWP domain (PDB 3LLR). (B) Crystal structure of DNMT3B PWWP bound to the histone H3K36me3 peptide (PDB 5CIU), with the side chains of H3K36me3 and the cage residues of the PWWP domain shown in stick representation. (C) Electrostatic surface view of the DNMT3B PWWP domain bound to the histone H3K36me3 peptide. The putative DNA binding surface is boxed with dotted lines.

Figure 9

Structural comparison of mDNMT1-DNA and DNMT3A-DNA interactions. (A) Recognition of hemimethylated CpG DNA by mDNMT1 (green) (PDB 4DA4). The hemimethylated CpG site, containing a 5-methyl group (green sphere), is colored in yellow or purple (5-fluorocytosine, 5fC′). The flipped-out cytosine on the template strand is colored in blue and the rest of the DNA is colored in wheat. (B) Detailed interactions between mDNMT1 and the hemimethylated CpG site. (C) Recognition of unmodified CpG DNA by DNMT3A (light blue). (D) Detailed interactions between DNMT3A and the unmodified CpG site. The hydrogen bonds are depicted as dashed lines.