Both combinatorial K4me0-K36me3 marks on sister histone H3s of a nucleosome are required for Dnmt3a-Dnmt3L mediated de novo DNA methylation

A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively.Here,taking advantage of the bivalent histone H3 system in yeast,we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L.The results show that lack of both K4me0 and K36me3 on one sister H3 tail,or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC,revealing a synergy of two sister H3s in DNA methylation regulation.Accordingly,the Dnmt3a or Dnmt3L mutation that disrupts the interaction of Dnmt3aADD domain-H3K4me0,Dnmt3LADD domain-H3K4me0,orDnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation.These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s,and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

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.     

The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation

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.     

The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase

The DNA methyltransferases, Dnmts, are the enzymes responsible for methylating DNA in mammals, which leads to gene silencing. Repression by DNA methylation is mediated partly by recruitment of the methyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associate and facilitate histone methylation at Lys9 of H3, which is a key epigenetic modification involved in gene silencing. Here, we show that endogenous Dnmt3a associates primarily with histone H3-K9 methyltransferase activity as well as, to a lesser extent, with H3-K4 enzymatic activity. The association with enzymatic activity is mediated by the conserved PHD-like motif of Dnmt3a. The H3-K9 histone methyltransferase that binds Dnmt3a is likely the H3-K9 specific SUV39H1 enzyme since we find that it interacts both in vitro and in vivo with Dnmt3a, using its PHD-like motif. We find that SUV39H1 also binds to Dnmt1 and, consistent with these interactions, SUV39H1 can purify DNA methyltransferase activity from nuclear extracts. In addition, we show that HP1β, a SUV39H1-interacting partner, binds directly to Dnmt1 and Dnmt3a and that native HP1β associates with DNA methyltransferase activity. Our data show a direct connection between the enzymes responsible for DNA methylation and histone methylation. These results further substantiate the notion of a self-reinforcing repressive chromatin state through the interplay between these two global epigenetic modifications.     

Zebra fish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development

DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organ-specific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.     

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

Np95 interacts with de novo DNA methyltransferases,Dnmt3a and Dnmt3b,and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.     

Solution NMR Structure and Histone Binding of the PHD Domain of Human MLL5

Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide ‘reader’ domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.     

DNA hypermethylation in Drosophila melanogaster causes irregular chromosome condensation and dysregulation of epigenetic histone modifications

The level of genomic DNA methylation plays an important role in development and disease. In order to establish an experimental system for the functional analysis of genome-wide hypermethylation, we overexpressed the mouse de novo methyltransferase Dnmt3a in Drosophila melanogaster. These flies showed severe developmental defects that could be linked to reduced rates of cell cycle progression and irregular chromosome condensation. In addition, hypermethylated chromosomes revealed elevated rates of histone H3-K9 methylation and a more restricted pattern of H3-S10 phosphorylation. The developmental and chromosomal defects induced by DNA hypermethylation could be rescued by mutant alleles of the histone H3-K9 methyltransferase gene Su(var)3-9. This mutation also resulted in a significantly decreased level of genomic DNA methylation. Our results thus uncover the molecular consequences of genomic hypermethylation and demonstrate a mutual interaction between DNA methylation and histone methylation.     

Histone H3 Inhibits Ubiquitin-Ubiquitin Intermolecular Interactions to Enhance Binding to DNA Methyl Transferase 1

DNA methyltransferase 1 (Dnmt1) is crucial for cell maintenance and preferentially methylates hemimethylated DNA. Recently, a study revealed that Dnmt1 is timely and site-specifically activated by several types of two-mono-ubiquitinated histone H3 tails (H3Ts). However, the molecular mechanism of Dnmt1 activation has not yet been determined, in addition to the role of H3T. Based on experimental data, two-mono-ubiquitinated H3Ts activate Dnmt1 by binding, with different binding affinities. In contrast, ubiquitin molecules unlinked with H3T do not bind to Dnmt1. Despite the existence of experimental data, it is unclear why the binding affinities for Dnmt1 are different. To obtain new insights into the activation mechanism of Dnmt1, we performed all-atom molecular dynamics (MD) simulations on three systems: (1) K14/K18, (2) K14/K23 mono-ubiquitinated H3Ts, and (3) two ubiquitin molecules unlinked with H3T. As an analysis of our MD trajectories, these ubiquitylation patterns modulated ubiquitin-ubiquitin intermolecular interactions. More specifically, the intermolecular contacts between a pair of ubiquitin molecules linked with H3T became weak in the presence of H3T, indicating that H3T makes a cleft between them to inhibit their intermolecular interactions. For these three systems, the intermolecular interactions between the ubiquitin molecules calculated by our MD simulations are in good agreement with the binding affinities for Dnmt1 experimentally measured in a previous study. Therefore, we conclude that H3T acts as a spacer to inhibit ubiquitin-ubiquitin intermolecular interactions, enhancing binding to Dnmt1.     

PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression

Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHD(UHRF1)), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHD(UHRF1) bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHD(UHRF1) binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.     

The multi-domain protein Np95 connects DNA methylation and histone modification

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.