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

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.

Figure 1

Overall structures of ADD_3A in the unliganded and H3-bound forms. (A) Ribbon representation of the ligand-free ADD_3A. 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 ADD_3A (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 ADD_3A is represented by a yellow dotted line. ADD_3A, ADD domain of DNMT3A; GATA1, GATA binding protein 1; PHD, plant homeodomain.

Figure 2

Calorimetric study of the binding of ADD_3A to H3_1–19 peptides containing non-methylated (A), dimethylated (B) or trimethylated (C) H3K4 analogues. ΔS, ΔH and K_D values are the means of two experiments that used different peptide and protein concentrations. H3_1–19, amino-terminal 19 amino acids of histone H3.

Figure 3

Recognition of non-methylated H3K4 by ADD_3A. Red dotted lines indicate hydrogen bonds (<3.3 Å) between H3 and ADD_3A residues. ADD_3A and H3 residues are represented as ball-and-stick models in green and yellow, respectively. (A) H3 peptide is shown with a F_o−F_c omit map contoured at 2.0 σ. (B) A close-up view of the recognition of the amino-terminus of the H3 tail by ADD_3A. (C) A close-up view of the Lys 4 binding pocket of ADD_3A. ADD_3A, ADD domain of DNMT3A.

Figure 4

Competitive binding of ADD_3A and CD_HP1α to the H3 tail. (A) GST pulldown assay analysed by SDS–PAGE and CBB staining. The GST–CD_HP1α bound to H3 K9me2 peptide in the absence of ADD_3A (lane 5) and was competed out by adding ADD_3A (lanes 6–9). (B) Reciprocal experiment of (A) using GST–ADD_3A, the H3 K9me3 peptide and CD_HP1α. (C) ¹H–¹⁵N HSQC spectra of the ¹⁵N-labelled CD_HP1α in the ligand-free (green) and H3 K9me3-bound (red) forms are superimposed (left panel). The CD_HP1α–H3 K9me3 complex was titrated with unlabelled ADD_3A (right panel). The cross-peak positions of ¹⁵N-labelled CD_HP1α 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 ADD_3A in complex with H3K9me2 (D) or H3K9me3 peptide (E) was titrated by unlabelled CD_HP1α. ADD, ATRX–DNMT3–DNMT3L; ADD_3A, ADD domain of DNMT3A; CBB, coomassie brilliant blue; CD_HP1α, chromodomain of HP1α; GST, glutathione-S-transferase; HP1α, heterochromatin protein 1α; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis.