Publications by authors named "Kyohei Arita"

25 Publications

  • Page 1 of 1

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza.

Life Sci Alliance 2021 06 5;4(6). Epub 2021 Apr 5.

Department of Nutritional Physiology, Institute of Medical Nutrition, Tokushima University Graduate School, Tokushima, Japan.

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.
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http://dx.doi.org/10.26508/lsa.202000849DOI Listing
June 2021

Structural dynamics of double-stranded DNA with epigenome modification.

Nucleic Acids Res 2021 01;49(2):1152-1162

Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1 Seikadai, Seika, Soraku, Kyoto 619-0284, Japan.

Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson-Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson-Crick base pair to allow base-flipping in living cells.
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http://dx.doi.org/10.1093/nar/gkaa1210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826269PMC
January 2021

Serine 298 Phosphorylation in Linker 2 of UHRF1 Regulates Ligand-Binding Property of Its Tandem Tudor Domain.

J Mol Biol 2020 06 16;432(14):4061-4075. Epub 2020 May 16.

Structure Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address:

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential factor for the maintenance of mammalian DNA methylation and harbors several reader modules for recognizing epigenetic marks. The tandem Tudor domain (TTD) of UHRF1 has a peptide-binding groove that functions as a binding platform for intra- or intermolecular interactions. Besides the groove interacting with unphosphorylated linker 2 and spacer of UHRF1, it also interacts with di/tri-methylated histone H3 at Lys9 and DNA ligase 1 (LIG1) at Lys126. Here we focus on the phosphorylation of Ser298 in linker 2, which was implied to regulate the ligand-binding property of the TTD. Although the protein expression level of UHRF1 is unchanged throughout the cell cycle, Ser298 phosphorylated form of UHRF1 is notably increased in the G2/M phase, which is revealed by immunoprecipitation followed by Western blotting. Molecularly, while unphosphorylated linker 2 covers the peptide-binding groove to prevent access of other interactors, small-angle X-ray scattering, thermal stability assay and molecular dynamics simulation revealed that the phosphate group of Ser298 dissociates linker 2 from the peptide-binding groove of the TTD to permit the other interactors to access to the groove. Our data reveal a mechanism in which Ser298 phosphorylation in linker 2 triggers a change of the TTD's structure and may affect multiple functions of UHRF1 by facilitating associations with LIG1 at DNA replication sites and histone H3K9me2/me3 at heterochromatic regions.
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http://dx.doi.org/10.1016/j.jmb.2020.05.006DOI Listing
June 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

Genes Cells 2020 Jan 3;25(1):22-32. Epub 2019 Dec 3.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Japan.

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.
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http://dx.doi.org/10.1111/gtc.12732DOI Listing
January 2020

Structure of PCNA in complex with DNMT1 PIP box reveals the basis for the molecular mechanism of the interaction.

Biochem Biophys Res Commun 2019 08 22;516(2):578-583. Epub 2019 Jun 22.

Structural Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, 230-0045, Japan; JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. Electronic address:

DNMT1 is a C5-DNA methyltransferase that plays a pivotal role in DNA methylation maintenance. During early and mid S-phase, DNMT1 accumulates at DNA replication sites by binding to proliferating cell nuclear antigen (PCNA), an essential factor for DNA replication, through a PIP box motif. However, the molecular mechanism by which the DNMT1 PIP box motif binds to PCNA remains unclear. Here, we report the crystal structure of PCNA bound to DNMT1 PIP box peptide. The structure reveals the detailed interaction between PCNA and DNMT1 PIP box; conserved glutamine and hydrophobic/aromatic residues in the PIP box are recognized by the Q- and hydrophobic pockets of PCNA, respectively. The structure also shows novel intramolecular interactions within the PIP box motif, which stabilize the helix conformation in the PIP box. Our data provide structural insight into the recruitment of DNMT1 to replication sites by PCNA.
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http://dx.doi.org/10.1016/j.bbrc.2019.06.060DOI Listing
August 2019

Structure of the UHRF1 Tandem Tudor Domain Bound to a Methylated Non-histone Protein, LIG1, Reveals Rules for Binding and Regulation.

Structure 2019 03 10;27(3):485-496.e7. Epub 2019 Jan 10.

Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan; JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address:

The protein UHRF1 is crucial for DNA methylation maintenance. The tandem Tudor domain (TTD) of UHRF1 binds histone H3K9me2/3 with micromolar affinity, as well as unmethylated linker regions within UHRF1 itself, causing auto-inhibition. Recently, we showed that a methylated histone-like region of DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1 TTD with nanomolar affinity, permitting UHRF1 recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a LIG1K126me3 peptide. The data explain the basis for the high TTD-binding affinity of LIG1K126me3 and reveal that the interaction may be regulated by phosphorylation. Binding of LIG1K126me3 switches the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition is relieved. Our results provide structural insight into how UHRF1 performs its key function in epigenetic maintenance.
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http://dx.doi.org/10.1016/j.str.2018.11.012DOI Listing
March 2019

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

Mol Cell 2017 Oct;68(2):350-360.e7

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2017.09.037DOI Listing
October 2017

RFTS-dependent negative regulation of Dnmt1 by nucleosome structure and histone tails.

FEBS J 2017 10 11;284(20):3455-3469. Epub 2017 Sep 11.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Japan.

DNA methylation in promoter regions represses gene expression and is copied over mitotic divisions by Dnmt1. Dnmt1 activity is regulated by its replication foci targeting sequence (RFTS) domain which masks the catalytic pocket. It has been shown that Dnmt1 activity on unmethylated DNA is inhibited in nucleosome cores. In the present study, we aimed to assess the effect of nuclesome formation on maintenance methylation at single CpG resolution. We show that Dnmt1 fully methylates naked linker DNA in dinucleosomes, whereas maintenance methylation was repressed at all CpG sites in nucleosome core particles. Deletion of RFTS partly released obstruction of Dnmt1 activity in core particles. Histone H3 tail peptides inhibited Dnmt1 in an RFTS-dependent manner and repression was modulated by acetylation or methylation. We propose a novel function of RFTS to regulate Dnmt1 activity in nucleosomes.
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http://dx.doi.org/10.1111/febs.14205DOI Listing
October 2017

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

Mol Cell 2017 Aug 10;67(4):550-565.e5. Epub 2017 Aug 10.

Epigenetics and Cell Fate, University Paris Diderot, Sorbonne Paris Cité, UMR 7216 CNRS, 75013 Paris, France. Electronic address:

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.
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http://dx.doi.org/10.1016/j.molcel.2017.07.012DOI Listing
August 2017

Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.

Sci Rep 2017 03 3;7(1):55. Epub 2017 Mar 3.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1, Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601, Japan.

Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.
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http://dx.doi.org/10.1038/s41598-017-00136-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427934PMC
March 2017

The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates.

Nat Commun 2015 Jan 20;6:6116. Epub 2015 Jan 20.

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan.

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.
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http://dx.doi.org/10.1038/ncomms7116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309437PMC
January 2015

The structural basis for receptor recognition of human interleukin-18.

Nat Commun 2014 Dec 15;5:5340. Epub 2014 Dec 15.

1] Department of Pediatrics, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1194, Japan [2] Biomedical Informatics, Medical Information Sciences Division, The United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1194, Japan.

Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors' recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.
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http://dx.doi.org/10.1038/ncomms6340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275594PMC
December 2014

Purification, crystallization and preliminary X-ray crystallographic analysis of human IL-18 and its extracellular complexes.

Acta Crystallogr F Struct Biol Commun 2014 Oct 25;70(Pt 10):1351-6. Epub 2014 Sep 25.

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the interleukin-1 (IL-1) family, is involved in the pathogenesis of autoimmune/autoinflammatory and allergic diseases such as juvenile idiopathic arthritis and bronchial asthma. IL-18 forms a signalling complex with the IL-18 receptor α (IL-18Rα) and β (IL-18Rβ) chains; however, the detailed activation mechanism remains unclear. Here, the IL-18-IL-18Rα binary and IL-18-IL-18Rα-IL-18Rβ ternary complexes were purified and crystallized as well as IL-18 alone. An X-ray diffraction data set for IL-18 was collected to 2.33 Å resolution from a crystal belonging to space group P21, with unit-cell parameters a = 68.15, b = 79.51, c = 73.46 Å, β = 100.97°. Crystals of both the IL-18 binary and ternary complexes belonging to the orthorhombic space groups P21212 and P212121, respectively, diffracted to 3.10 Å resolution. Unit-cell parameters were determined as a = 135.49, b = 174.81, c = 183.40 Å for the binary complex and a = 72.56, b = 111.56, c = 134.57 Å for the ternary complex.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188078PMC
http://dx.doi.org/10.1107/S2053230X14016926DOI Listing
October 2014

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.

J Biol Chem 2014 Jan 19;289(1):379-86. Epub 2013 Nov 19.

From the Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 565-0871, Japan.

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.
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http://dx.doi.org/10.1074/jbc.M113.523209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879560PMC
January 2014

Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication.

Nature 2013 Oct 8;502(7470):249-53. Epub 2013 Sep 8.

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan.

Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.
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http://dx.doi.org/10.1038/nature12488DOI Listing
October 2013

Development of fluorogenic probes for quick no-wash live-cell imaging of intracellular proteins.

J Am Chem Soc 2013 Aug 8;135(33):12360-5. Epub 2013 Aug 8.

Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

We developed novel fluorogenic probes for no-wash live-cell imaging of proteins fused to PYP-tag, which is a small protein tag recently reported by our group. Through the design of a new PYP-tag ligand, specific intracellular protein labeling with rapid kinetics and fluorogenic response was accomplished. The probes crossed the cell membrane, and cytosolic and nuclear localizations of PYP-tagged proteins without cell washing were visualized within a 6-min reaction time. The fluorogenic response was due to the environmental effect of fluorophore upon binding to PYP-tag. Furthermore, the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization, movement, and function of cellular proteins.
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http://dx.doi.org/10.1021/ja405745vDOI Listing
August 2013

Quantitative comparison of protein dynamics in live cells and in vitro by in-cell (19)F-NMR.

Chem Commun (Camb) 2013 Apr;49(27):2801-3

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.

Here we describe how a (19)F-probe incorporated into an endogenous protein by a chemical biology method revealed protein dynamics. By explicit determination of ligand-bound and unbound structures with X-ray crystallography, the quantitative comparison of the protein's dynamics in live cells and in vitro is presented. These results clearly demonstrated the greater conformational fluctuations of the intracellular protein, partially due to macromolecular crowding effects.
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http://dx.doi.org/10.1039/c3cc39205hDOI Listing
April 2013

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

J Biol Chem 2013 Mar 10;288(9):6351-62. Epub 2013 Jan 10.

Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.

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.
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http://dx.doi.org/10.1074/jbc.M112.431098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585070PMC
March 2013

Lysyl 5-hydroxylation, a novel histone modification, by Jumonji domain containing 6 (JMJD6).

J Biol Chem 2013 Mar 9;288(9):6053-62. Epub 2013 Jan 9.

Division of Epigenetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.

JMJD6 is reported to hydroxylate lysyl residues of a splicing factor, U2AF65. In this study, we found that JMJD6 hydroxylates histone lysyl residues. In vitro experiments showed that JMJD6 has a binding affinity to histone proteins and hydroxylates multiple lysyl residues of histone H3 and H4 tails. Using JMJD6 knock-out mouse embryos, we revealed that JMJD6 hydroxylates lysyl residues of histones H2A/H2B and H3/H4 in vivo by amino acid composition analysis. 5-Hydroxylysine was detected at the highest level in histones purified from murine testis, which expressed JMJD6 at a significantly high level among various tissues examined, and JMJD6 overexpression increased the amount of 5-hydroxylysine in histones in human embryonic kidney 293 cells. These results indicate that histones are additional substrates of JMJD6 in vivo. Because 5-hydroxylation of lysyl residues inhibited N-acetylation and N-methylation by an acetyltransferase and a methyltransferase, respectively, in vitro, histone 5-hydroxylation may have important roles in epigenetic regulation of gene transcription or chromosomal rearrangement.
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http://dx.doi.org/10.1074/jbc.M112.433284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585044PMC
March 2013

Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1.

Proc Natl Acad Sci U S A 2012 Aug 25;109(32):12950-5. Epub 2012 Jul 25.

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.

Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3-binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3-binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.
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http://dx.doi.org/10.1073/pnas.1203701109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420164PMC
August 2012

Crystal structure of the ubiquitin-associated (UBA) domain of p62 and its interaction with ubiquitin.

J Biol Chem 2011 Sep 29;286(36):31864-74. Epub 2011 Jun 29.

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Kyoto 615-8510, Japan.

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.
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http://dx.doi.org/10.1074/jbc.M111.259630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173063PMC
September 2011

Structural basis for regulation of poly-SUMO chain by a SUMO-like domain of Nip45.

Proteins 2010 May;78(6):1491-502

Kyoto University, Japan.

Post-translational modification by small ubiquitin-like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin-proteasome degradation system by the action of SUMO-targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly-SUMO chain through their SUMO-interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO-like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a beta-grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO-binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly-SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9-mediated poly-SUMO formation.
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http://dx.doi.org/10.1002/prot.22667DOI Listing
May 2010

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain.

EMBO Rep 2009 Nov 16;10(11):1235-41. Epub 2009 Oct 16.

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan.

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.
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http://dx.doi.org/10.1038/embor.2009.218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775176PMC
November 2009

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism.

Nature 2008 Oct 3;455(7214):818-21. Epub 2008 Sep 3.

Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.
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http://dx.doi.org/10.1038/nature07249DOI Listing
October 2008