The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.In mammals, DNA methylation plays important roles in differentiation, gene regulation, genomic imprinting, X chromosome inactivation, and disease-related processes (1-3). DNA methylation patterns are set during embryogenesis by the Dnmt3a and Dnmt3b DNA methyltransferases and their regulatory factor Dnmt3L (Dnmt3-like) (4 -6). However, the mechanisms guiding these enzymes to their target regions are not well understood. Dnmt3a and 3b consist of a C-terminal catalytic domain and an N-terminal part containing a PWWP domain and an ADD 2 domain (1,7,8). Biochemical studies provide evidence for a direct interaction of Dnmt3a and 3b with native nucleosomes (9), which could be mediated by the ADD domain or the PWWP domain. The ADD domains of Dnmt3L and Dnmt3a have been shown to interact with the histone 3 tail unmethylated at Lys 4 (10 -12), which can explain the anticorrelation of DNA methylation and the activating H3K4me3 mark as observed in many genome-wide DNA methylation studies (13-16). However, the ADD domain is not directly involved in heterochromatic targeting of Dnmt3a (17,18).PWWP domains belong to the Royal domain superfamily, members of which were identified to interact with histone tails in various modification states (19). The PWWP domains of Dnmt3a and 3b are essential for heterochromatic targeting (17,18). An S333P missense mutation in the Dnmt3a PWWP domain (numbering refers to murine Dnmt3a) led to the loss of chromatin targeting of Dnmt3a (18). This mutation corresponds to the S282P mutation in the PWWP domain of human Dnmt3b, which has been identified in immunodeficiency, centromeric heterochromatin instability, and facial anomalies syndrome patients (20).Here, we explore the possibility of the interaction of the Dnmt3a PWWP domain with histone peptides using...

By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins.Epigenetic regulation of gene expression by covalent modification of histone proteins and methylation of DNA controls development and disease processes 1 . Post-translational modification of histone proteins includes acetylation, phosphorylation and methylation. Many of these modifications occur on the N-terminal tails of the histone proteins that protrude from the nucleosome. Methylation of lysine residues in histone tails has been identified in histone H3 lysine residues 4, 9, 27 and 36; in histone H4 lysine 20; and in histone H1b lysine 25. Each of these methylations has different biological functions 1 .The first histone lysine methyltransferase was identified in 2000 (ref.2), and today about 30 different enzymes are known in different species 1 . Most protein lysine methyltransferases (PKMTs) contain a SET domain, which harbors the active center of the enzymes 3 . Here, we investigate the substrate sequence specificity of the human G9a PKMT, which is important for the euchromatic histone H3K9 methylation that is essential for early embryogenesis 4 , the propagation of imprints 5 and control of DNA methylation 6 . Knockout of G9a results in a decrease of global H3K9me1 and H3K9me2 levels 7 . In vitro G9a generates mainly H3K9me1 and H3K9me2 (ref. 8), as well as H3K9me3 after long incubation 9 . In contrast to the Dim-5 and Suv39H1 H3K9 methyltransferases, G9a methylates not only H3K9 but also H3K27 (ref. 10), which implicates different specificities in peptide recognition. To analyze the substrate specificity of PKMTs, we prepared peptide arrays on functionalized cellulose membranes using the first 21 residues of histone H3 as template 11 (Supplementary Methods online). The membranes were incubated with G9a in the presence of radioactively labeled [methyl-3 H]-S-adenosyl-L-methionine ( 3 H-AdoMet, 1), and the transfer of methyl groups to the immobilized peptides was detected by autoradiography ( Supplementary Fig. 1 online). To quantify the contribution of each amino acid to the recognition of the substrate and display it graphically, the discrimination factor of G9a at each position was calculated (Fig. 1a). The results showed that G9a interacts with H3 residues 6-11, which agrees with a report describing a heptapeptide of the histone H3 tail (TARKSTG) as the minimal substrate methylated by G9a (ref. 12). In addition to Lys9 (the target of methylation), Arg8 is the most important specificity determinant for G9a. Any other amino acid substituted at that position completely abolished the activity...

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.