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...

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.

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting.