Mammalian DNA methyltransferases: a structural perspective

Abstract

The methylation of mammalian DNA, primarily at CpG dinucleotides, has long been recognized to play a major role in controlling gene expression, among other functions. Given their importance, it is surprising how many basic questions remain to be answered about the proteins responsible for this methylation and for coordination with the parallel chromatin-marking system that operates at the level of histone modification. This article reviews recent studies on, and discusses the resulting biochemical and structural insights into, the DNA nucleotide methyltransferase (Dnmt) proteins 1, 3a, 3a2, 3b, and 3L.

Figure 1. DNA Methylation

(A) DNA cytosine methylation at ring carbon C5. See the text for a summary of the mechanism. The question mark indicates possible activity of DNA demethylases (Kress et al., 2006; Vairapandi, 2004). (B) Members of the DNMT family. Schematic representation of Dnmt1 and Dnmt3. Dnmt2 is a tRNA^Asp MTase (Goll et al., 2006) (insert). Roman numerals refer to conserved motifs of DNA MTases (Kumar et al., 1994); motif IV includes the Cys nucleophile that forms a transient covalent bond to C6 of the target cytosine. Other details are explained in the text or in work by Goll and Bestor (2005). (C) Maintenance versus de novo methylation. As described in the text, the roles of the Dnmts are not completely distinct in this respect. The pale-blue segments are substrate sequences (usually CpG), and the turquoise shapes represent methyl groups on the cytosines. After replication or repair, the duplex is methylated on one strand only.

Figure 2. Domain Structures of the Dnmt3 Family

(A) The PWWP domain structure of Dnmt3b, shown as a GRASP output (Nicholls et al., 1991), is rich in basic residues (Qiu et al., 2002). Selected charged, surface-exposed residues are indicated. (B) Structure of Dnmt3L with a bound histone H3 N-terminal tail (orange) (Ooi et al., 2007). (C) A surface representation of the Dnmt3a-C/3L-C tetramer, with two short DNA molecules adopted by superimposition of the HhaI-DNA complex structure (Klimasauskas et al., 1994) onto individual Dnmt3a-C molecules. The figure was adapted from work by Jia et al. (2007). (D) The Dnmt3a-C/3L-C tetramer with one contiguous curved DNA molecule covering two active sites. The figure was adapted from work by Jia et al. (2007). (E) The Dnmt3a dimer could, in theory, methylate two CpGs separated by one helical turn in one binding event.

Figure 3. A Model for Interaction between a Dnmt3a-3L Tetramer and a Nucleosome

A nucleosome is shown, docked to a Dnmt3L-3a-3a-3L tetramer (3a-C in green; 3L full length in gray). The position of a peptide derived from the sequence of the histone H3 amino terminus (purple) is shown and is taken from a cocrystal structure with this peptide bound to Dnmt3L (Ooi et al., 2007). When the tetramer is wrapped around the nucleosome, the two Dnmt3L molecules could bind both histone tails from one nucleosome. The amino-proximal portion of Dnmt3a is shown in cartoon form, with domains labeled as N (for the variable region at the amino terminus), PWWP (which may be involved in nonspecific DNA binding), and CXXC (a Cys-rich 3-Zn-binding domain). By analogy to Dnmt3L, the CXXC domain of Dnmt3a might interact with histone tails from neighboring nucleosomes.

Figure 4. The 3a-3L Interface May Contribute to the Stability of Both AdoMet Binding and the Active-Site Loop Conformation

(A) Effects on the active-site loop. Left panel: superimposition of Dnmt3a and the protein component of the M.HhaI-DNA complex (PDB code: 1mht; gray). This superimposition has a root-mean-square deviation of 2.3 Å (comparing 220 pairs of Cα atoms by DALI) or 0.8 Å (manually aligning 53 pairs of Cα atoms forming the central 7 β strands). Right panel: superimposition of Dnmt3a and M.HhaI in the absence of DNA (PDB code: 1hmy). The M.HhaI active-site loop (gray) is shown from both structures: the closed, catalytically competent form adopted in the presence of DNA, and the open form adopted in its absence. (B) Effects on AdoMet binding. Dnmt3a-C is shown in green, Dnmt3L-C is shown in cyan, and the active-site loop of Dnmt3a is shown in magenta. AdoHcy (yellow) and residues involved in interactions are shown as stick models, and the corresponding ICF mutations in human Dnmt3b are shown in red (see Figure 6C). The panel was adopted from Supplemental Data in Jia et al. (2007).

Figure 5. Structures of the 3a-3L and 3a-3a Interfaces

(A) Structure of the Dnmt3a-3L interface (PDB code: 2QRV). (B) Structure of the Dnmt3a-3a interface (PDB code: 2QRV). (C) Structure of the Dnmt3L-3L interface (PDB code: 2PV0). (D) Size-exclusion chromatography of wild-type Dnmt3a2 (black trace) or of an F728A mutant (red trace) (adopted from Supplemental Data in Jia et al. [2007]). The cartoons indicate the interpretation of oligomerization; the small yellow ovals indicate the Dnmt3a DNA-binding region (though it is absent altogether in Dnmt3L), and the other oval indicates Phe (dark green, WT) or Ala (red, mutant).

Figure 6. Sequences of the 3a-3L and 3a-3a Interfaces

(A) The Dnmt3a-3L interface is shown; secondary structural elements are indicated in light blue. Numbering above the sequences corresponds to the mouse ortholog. White-on-black residues are conserved in Dnmt3a, Dnmt3b, and Dnmt3L, whereas gray-highlighted positions are conserved (with ≤ 1 substitution) between 3a and 3b. The red highlighted C is the cysteine nucleophile of the MTase active site, whereas human Dnmt3b amino acids shown in red are associated with the disease ICF when altered in specific ways (see text). Positions highlighted in yellow are responsible for interactions between Dnmt3a and Dnmt3L (indicated by lines). The range of vertebrates with obvious Dnmt3L orthologs is much more restricted than that for Dnmt3a or Dnmt3b. (B) The Dnmt3a-3a interface is shown; the sequence is highlighted as in (A). The block arrows underneath indicate reciprocal salt bridges between the conserved Arg and Asp, as shown at Figure 5B. (C) Distribution of human ICF mutations on Dnmt3b (in parentheses). The panel was adopted from the Supplemental Data in Jia et al. (2007).