Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation

Abstract

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (Dnmt3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain. Here we show, with the use of crystallography, that the carboxy-terminal domain of human Dnmt3L interacts with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and Dnmt3L showed further dimerization through Dnmt3a-Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a-Dnmt3L interface or the Dnmt3a-Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA-Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.

Figure 1. Structure of the Dnmt3a–Dnmt3L (3a–3L) C-terminal domain complex

a, Side view of the tetramer Dnmt3L (blue)–Dnmt3a (green) –Dnmt3a (green)–Dnmt3L (blue), with the bound AdoHcy shown as a stick model. The inset shows the two pairs of phenylalanine residues (F728 and F768 of Dnmt3a, and F261 and F301 of Dnmt3L) in the centre of the Dnmt3a–Dnmt3L interface. b, Top view showing the Dnmt3a–Dnmt3a interface with two pairs of salt bridges formed between R881 and D872 (enlarged). c, Activities of Dnmt3a2 and its point mutant F728A in the presence and absence of Dnmt3L. Error bars represent s.d. calculated from two independent experiments. d, A network of polar interactions between two Dnmt3a molecules (coloured blue and purple) involves R881–D872, D872–H869, H869–E851 and E851–R881.

Figure 2. Model of Dnmt3a–Dnmt3L tetramer with DNA

a, Surface representation of the Dnmt3a–Dnmt3L tetramer, with two short DNA molecules adopted by superimposition of the HhaI–DNA complex structure onto individual Dnmt3a-C. b, Two views of the Dnmt3a–Dnmt3L tetramer with one contiguous curved DNA molecule, approximately 25 base pairs in length, covering two active sites. c, Cooperative formation of large protein–DNA complexes (red arrow) after incubation of a 146-base-pair DNA with increasing concentrations of Dnmt3a-C or Dnmt3a-C–Dnmt3L-C complex. The lower band (black arrow) corresponds to one Dnmt3L-C bound to the DNA as a result of the presence of some free Dnmt3L-C in the mixture (Supplementary Fig. 5a).

Figure 3. Periodicity of Dnmt3a-C activity on long DNA substrates

a, Two DNA fragments (from λ phage (top; 520 base pairs, 40 CpGs) and the CpG island upstream of the human SUHW1 gene (bottom; 420 base pairs, 56 CpGs)) were used for methylation. A total of 119 clones (64 from λ phage, 55 from the human CpG island) were sequenced to determine their methylation pattern. b, Autocorrelation of CpG methylation. By averaging two substrates, the P values for the correlation factors observed for distances of eight to ten base pairs (arrow) are in the range (3–7) × 10⁻⁴. This periodicity is detectable for correlation of methylation of individual CpG sites as well as for correlation of the absence of methylation at two sites (Supplementary Fig. 6).

Figure 4. Periodicity of CpG sites in maternally imprinted DMRs

a, Frequencies of CpG pairs at a given distance with respect to all pairs, for 11 maternally imprinted DMRs. The slope of the graph in the inset is 9.5. b, The autocorrelation of peaks for 11 maternally imprinted DMRs. One sequence (Impact) was excluded from the plot because it contains repeats (see d), with each CpG site separated from the second subsequent one by ten base pairs; thus, it follows the same pattern seen by the other 11 DMRs, bringing the total number to 12 DMRs. c, Autocorrelation of peaks for ten CpG islands randomly selected from human chromosome 21. d, A region of DMR sequences of the U2af1-rs1 and Impact genes.