Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin 1 . 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 2 . Here we show, with the use of crystallography, that the carboxyterminal 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.In both flowering plants and placental mammals, DNA methylation has a central role in imprinting, but in neither case is it clear how imprinted genes are targeted for methylation. Imprinted genes in mammals are often associated with differentially methylated regions (DMRs) 3 , which show DNA methylation patterns that depend on the parent of origin. How the imprinting machinery recognizes DMRs is unknown. The Dnmt3 family includes three members: two de novo CpG methyltransferases, namely Dnmt3a and Dnmt3b (ref. 4), and anCorrespondence and requests for materials should be addressed to A.J. [E-mail: a.jeltsch@jacobs-university.de] or X.C. [E-mail: xcheng@emory.edu]. * These authors contributed equally to this work.Author Information The X-ray structure of Dnmt3a-Dnmt3L C-terminal tetramer complex is deposited in the Protein Data Bank under ID code 2QRV. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. We ...

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post-translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome-wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.

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...