The DNMT3A ADD domain is required for efficient de novo DNA methylation and maternal imprinting in mouse oocytes.

Establishment of a proper DNA methylation landscape in mammalian oocytes is important for maternal imprinting and embryonic development. De novo DNA methylation in oocytes is mediated by the DNA methyltransferase DNMT3A, which has an ATRX-DNMT3-DNMT3L (ADD) domain that interacts with histone H3 tail unmethylated at lysine-4 (H3K4me0). The domain normally blocks the methyltransferase domain via intramolecular interaction and binding to histone H3K4me0 releases the autoinhibition. However, H3K4me0 is widespread in chromatin and the role of the ADD-histone interaction has not been studied in vivo. We herein show that amino-acid substitutions in the ADD domain of mouse DNMT3A cause dwarfism. Oocytes derived from homozygous females show mosaic loss of CG methylation and almost complete loss of non-CG methylation. Embryos derived from such oocytes die in mid-to-late gestation, with stochastic and often all-or-none-type CG-methylation loss at imprinting control regions and misexpression of the linked genes. The stochastic loss is a two-step process, with loss occurring in cleavage-stage embryos and regaining occurring after implantation. These results highlight an important role for the ADD domain in efficient, and likely processive, de novo CG methylation and pose a model for stochastic inheritance of epigenetic perturbations in germ cells to the next generation.

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases.

In mammals, three major DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains. For instance, Dnmt3a and Dnmt3b each contain a Pro-Trp-Trp-Pro (PWWP) domain that recognizes the histone H3K36me2/3 mark, an Atrx-Dnmt3-Dnmt3l (ADD) domain that recognizes unmodified histone H3 tail, and a catalytic domain that methylates CpG sites. Dnmt1 contains an N-terminal independently folded domain (NTD) that interacts with a variety of regulatory factors, a replication foci-targeting sequence (RFTS) domain that recognizes the histone H3K9me3 mark and H3 ubiquitylation, a CXXC domain that recognizes unmodified CpG DNA, two tandem Bromo-Adjacent-homology (BAH1 and BAH2) domains that read the H4K20me3 mark with BAH1, and a catalytic domain that preferentially methylates hemimethylated CpG sites. In this chapter, the structures and functions of these domains are described.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Delayed male germ cell sex-specification permits transition into embryonal carcinoma cells with features of primed pluripotency.

Testicular teratomas result from anomalies in embryonic germ cell development. In 129 inbred mice, teratoma initiation coincides with germ cell sex-specific differentiation and the mitotic-meiotic switch: XX and XY germ cells repress pluripotency, XX germ cells initiate meiosis, and XY germ cells activate male-specific differentiation and mitotic arrest. Here, we report that expression of Nanos2, a gene that is crucial to male sex specification, is delayed in teratoma-susceptible germ cells. Decreased expression of Nanos2 was found to be due, in part, to the Nanos2 allele present in 129 mice. In teratoma-susceptible germ cells, diminished expression of genes downstream of Nanos2 disrupted processes that were crucial to male germ cell differentiation. Deficiency for Nanos2 increased teratoma incidence in 129 mice and induced developmental abnormalities associated with tumor initiation in teratoma-resistant germ cells. Finally, in the absence of commitment to the male germ cell fate, we discovered that a subpopulation of teratoma-susceptible germ cells transition into embryonal carcinoma (EC) cells with primed pluripotent features. We conclude that delayed male germ cell sex-specification facilitates the transformation of germ cells with naïve pluripotent features into primed pluripotent EC cells.

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

Total Synthesis and Structural Characterization of Caveolin-1.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis.

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity.

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7-null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7-deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity.

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1.

Posttranslational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitination is one such modification. We previously reported on the synthesis of ubiquitinated histone H3 with an isopeptide mimetic structure. In this report, we describe the preparation of ubiquitinated histone H3 peptides with a native isopeptide structure, which showed a slightly weaker effect on the enzymatic activity of DNA methyltransferase 1 than the previous ubiquitinated H3 peptide analogs. These findings show that a native structure is important for determining the mechanism of the function, although ubiquitinated H3 peptide analogs can mimic the role of the original ubiquitinated H3. We also report on the successful preparation of the ubiquitinated full length histone H3.

A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution.

Since the discovery of oxidative demethylation of methylcytosine (mC) by Tet enzymes, an analytical method has been urgently needed that would enable the identification of mC and hydroxymethylcytosine (hmC) at the single base resolution level, because their roles in gene regulation are quite different from each other. However, the bisulfite sequencing method, the gold standard for DNA methylation analysis at present, does not distinguish them. Recently reported alternative methods, such as oxBS-seq and TAB-seq, are not even capable of determining mC and hmC simultaneously. Here, we report a novel method for the direct identification of mC, hmC and unmodified cytosine (C) at a single base resolution. We named this method the Enzyme-assisted Identification of Genome Modification Assay (EnIGMA), and it was demonstrated to indeed have a highly efficient and reliable analytic capacity for distinguishing them. We also successfully applied this novel method to the analysis of the maintenance of the DNA methylation status of imprinted H19-DMR. Importantly, hydroxymethylation plays an ambivalent role in the maintenance of the genome imprinting memory in parental genomes essential for normal development, shedding new light on the epigenetic regulation in ES cells.

Interactions of HP1 Bound to H3K9me3 Dinucleosome by Molecular Simulations and Biochemical Assays.

Heterochromatin protein 1 (HP1), associated with heterochromatin formation, recognizes an epigenetically repressive marker, trimethylated lysine 9 in histone H3 (H3K9me3), and generally contributes to long-term silencing. How HP1 induces heterochromatin is not fully understood. Recent experiments suggested that not one, but two nucleosomes provide a platform for this recognition. Integrating previous and new biochemical assays with computational modeling, we provide near-atomic structural models for HP1 binding to the dinucleosomes. We found that the dimeric HP1α tends to bind two H3K9me3s that are in adjacent nucleosomes, thus bridging two nucleosomes. We identified, to our knowledge, a novel DNA binding motif in the hinge region that is specific to HP1α and is essential for recognizing the H3K9me3 sites of two nucleosomes. An HP1 isoform, HP1γ, does not easily bridge two nucleosomes in extended conformations because of the absence of the above binding motif and its shorter hinge region. We propose a molecular mechanism for chromatin structural changes caused by HP1.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SN Ar Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-Nucleoside.

In chromatin, 5-methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine-5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2-amino-4-halopyridine-C-nucleosides (dX P) and oligodeoxyribonucleotides (ODNs) containing dX P as a novel mechanism-based inhibitor of DNMTs. The designed ODN containing X PpG forms a complex with DNMTs by covalent bonding through a nucleophilic aromatic substitution (SN Ar) reaction, and its cell proliferation activity is investigated. This study suggests that dX P in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chemical probe for studies of epigenetics. Our molecular design using a SN Ar reaction would be useful for DNMTs and other protein-DNA interactions.

Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation.

Post-translational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitin modification is one such modification. We previously reported on the use of a thiirane linker to introduce a 1,2-aminothiol moiety at a cysteine residue for native chemical ligation with peptide thioesters, which permitted isopeptide mimetics to be produced. In this report, we describe the preparation of the ubiquitylated full length histone H3 at the 18 position and the construction of tetranucleosomes with recombinant histones H2A, H2B, H4, and DNA, which are slightly more stable than those that are prepared without ubiquitin modification. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Nucleosome compaction facilitates HP1γ binding to methylated H3K9.

The α, ß and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1's chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg(2+) or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.

Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants.

Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.

Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases.

In mammals, three DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains, and in this chapter, the structures and functions of these domains are described.

The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA.

Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.

Selective oxidation of 5-hydroxymethylcytosine with micelle incarcerated oxidants to determine it at single base resolution.

5-Methylcytosine (5mC) is oxidized by ten-eleven translocation (TET) enzymes. This process followed by thymine DNA glycosylase is proposed to be the mechanism for methylcytosine demethylation. 5-Hydroxymethylcytosine (5hmC) is one of the most important key oxidative metabolites in the demethylation process, and therefore, simple and accurate method to determine 5hmC at single base resolution is desired. In the present study, we developed a mild catalytic oxidation of 5-hmC using micelle incarcerated oxidants that enables to determine the position of 5hmC at single base resolution.

Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.

The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine.