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...

Mutations in the ATRX protein are associated with the alpha-thalassemia and mental retardation X-linked syndrome (ATR-X). Almost half of the disease-causing mutations occur in its ATRX-Dnmt3-Dnmt3L (ADD) domain. By employing peptide arrays, chromatin pull-down and peptide binding assays, we show specific binding of the ADD domain to H3 histone tail peptides containing H3K9me3. Peptide binding was disrupted by the presence of the H3K4me3 and H3K4me2 modification marks indicating that the ATRX-ADD domain has a combined readout of these two important marks (absence of H3K4me2 and H3K4me3 and presence of H3K9me3). Disease-causing mutations reduced ATRX-ADD binding to H3 tail peptides. ATRX variants, which fail in the H3K9me3 interaction, show a loss of heterochromatic localization in cells, which indicates the chromatin targeting function of the ADD domain of ATRX. Disruption of H3K9me3 binding may be a general pathogenicity pathway of ATRX mutations in the ADD domain which may explain the clustering of disease mutations in this part of the ATRX protein.

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.