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. 2009 Nov;10(11):1235-41.
doi: 10.1038/embor.2009.218. Epub 2009 Oct 16.

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain

Junji Otani  1 , Toshiyuki Nankumo, Kyohei Arita, Susumu Inamoto, Mariko Ariyoshi, Masahiro Shirakawa
Affiliations

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain

Junji Otani et al. EMBO Rep. 2009 Nov.

Abstract

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Overall structures of ADD3A in the unliganded and H3-bound forms. (A) Ribbon representation of the ligand-free ADD3A. Three zinc ions and cysteine residues forming CxxC motifs are shown as magenta spheres and multicoloured stick models, respectively. GATA1-like and PHD fingers are shown in orange and green, respectively. The green dotted line represents the disordered H3N loop. (B) Crystal structure of ADD3A (green) bound to the H3 tail (yellow). H3 peptide (residues 1–9) is shown as a ball-and-stick model. The disordered linker peptide that connects the carboxy-terminus of the peptide and the amino-terminus of ADD3A is represented by a yellow dotted line. ADD3A, ADD domain of DNMT3A; GATA1, GATA binding protein 1; PHD, plant homeodomain.
Figure 2
Figure 2
Calorimetric study of the binding of ADD3A to H31–19 peptides containing non-methylated (A), dimethylated (B) or trimethylated (C) H3K4 analogues. ΔS, ΔH and KD values are the means of two experiments that used different peptide and protein concentrations. H31–19, amino-terminal 19 amino acids of histone H3.
Figure 3
Figure 3
Recognition of non-methylated H3K4 by ADD3A. Red dotted lines indicate hydrogen bonds (<3.3 Å) between H3 and ADD3A residues. ADD3A and H3 residues are represented as ball-and-stick models in green and yellow, respectively. (A) H3 peptide is shown with a Fo−Fc omit map contoured at 2.0 σ. (B) A close-up view of the recognition of the amino-terminus of the H3 tail by ADD3A. (C) A close-up view of the Lys 4 binding pocket of ADD3A. ADD3A, ADD domain of DNMT3A.
Figure 4
Figure 4
Competitive binding of ADD3A and CDHP1α to the H3 tail. (A) GST pulldown assay analysed by SDS–PAGE and CBB staining. The GST–CDHP1α bound to H3 K9me2 peptide in the absence of ADD3A (lane 5) and was competed out by adding ADD3A (lanes 6–9). (B) Reciprocal experiment of (A) using GST–ADD3A, the H3 K9me3 peptide and CDHP1α. (C) 1H–15N HSQC spectra of the 15N-labelled CDHP1α in the ligand-free (green) and H3 K9me3-bound (red) forms are superimposed (left panel). The CDHP1α–H3 K9me3 complex was titrated with unlabelled ADD3A (right panel). The cross-peak positions of 15N-labelled CDHP1α shifted from those of the H3-bound form to those of the free form during the titration. Selected spectral regions (i)–(iv) are magnified. (D,E) Reciprocally, 15N-labelled ADD3A in complex with H3K9me2 (D) or H3K9me3 peptide (E) was titrated by unlabelled CDHP1α. ADD, ATRX–DNMT3–DNMT3L; ADD3A, ADD domain of DNMT3A; CBB, coomassie brilliant blue; CDHP1α, chromodomain of HP1α; GST, glutathione-S-transferase; HP1α, heterochromatin protein 1α; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis.
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