Publications by authors named "Yuki Katou"

66 Publications

Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

Commun Biol 2020 May 4;3(1):208. Epub 2020 May 4.

RIKEN Cluster for Pioneering Research, Tsukuba, Ibaraki, Japan.

Paternal environmental factors can epigenetically influence gene expressions in offspring. We demonstrate that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.
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http://dx.doi.org/10.1038/s42003-020-0935-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198565PMC
May 2020

A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint.

EMBO Rep 2020 06 19;21(6):e50257. Epub 2020 Apr 19.

CRBM, University of Montpellier, CNRS, Montpellier, France.

The Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1-3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways.
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http://dx.doi.org/10.15252/embr.202050257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271318PMC
June 2020

ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet.

Mol Cell 2020 05 19;78(3):445-458.e6. Epub 2020 Mar 19.

RIKEN Cluster for Pioneering Research, Tsukuba, Ibaraki 305-0074, Japan; Department of Functional Genomics, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. Electronic address:

Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7 mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7 mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.
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http://dx.doi.org/10.1016/j.molcel.2020.02.028DOI Listing
May 2020

The Phosphatase PP1 Promotes Mitotic Slippage through Mad3 Dephosphorylation.

Curr Biol 2020 01 9;30(2):335-343.e5. Epub 2020 Jan 9.

CRBM, University of Montpellier, CNRS, 1919 Route de Mende, 34293 Montpellier, France. Electronic address:

Accurate chromosome segregation requires bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the spindle assembly checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays anaphase onset until all chromosomes are bipolarly attached [1]. SAC signaling fires at kinetochores and involves a soluble mitotic checkpoint complex (MCC) that inhibits the anaphase-promoting complex (APC) [2, 3]. The mitotic delay imposed by SAC, however, is not everlasting. If kinetochores fail to establish bipolar connections, cells can escape from the SAC-induced mitotic arrest through a process called mitotic slippage [4]. Mitotic slippage occurs in the presence of SAC signaling at kinetochores [5, 6], but whether and how MCC stability and APC inhibition are actively controlled during slippage is unknown. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached [7, 8]. PP1 turns off SAC signaling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores [9-11]. Here, we show that, in budding yeast, PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilizes the MCC without affecting the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.
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http://dx.doi.org/10.1016/j.cub.2019.11.054DOI Listing
January 2020

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

Epigenetics Chromatin 2019 12 19;12(1):77. Epub 2019 Dec 19.

Japan Agency for Medical Research and Development (AMED-CREST), AMED, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.

Background: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.

Results: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.

Conclusions: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.
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http://dx.doi.org/10.1186/s13072-019-0319-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921469PMC
December 2019

Chromosome-associated RNA-protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe.

Nat Commun 2019 12 6;10(1):5598. Epub 2019 Dec 6.

Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, Kobe, 651-2492, Japan.

Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA-protein complexes is required for robust pairing. Moreover, the lncRNA-protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA-protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.
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http://dx.doi.org/10.1038/s41467-019-13609-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898681PMC
December 2019

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken.

Sci Rep 2019 08 29;9(1):12560. Epub 2019 Aug 29.

Department of Systems BioMedicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9's functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9's regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.
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http://dx.doi.org/10.1038/s41598-019-48979-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715657PMC
August 2019

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

PLoS Genet 2018 11 12;14(11):e1007783. Epub 2018 Nov 12.

Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, United Kingdom.

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.
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http://dx.doi.org/10.1371/journal.pgen.1007783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258251PMC
November 2018

Mapping of histone-binding sites in histone replacement-completed spermatozoa.

Nat Commun 2018 09 24;9(1):3885. Epub 2018 Sep 24.

Cluster for Pioneering Research, CREST Research Project of JST (Japan Science and Technology Agency), RIKEN Tsukuba Institute, Tsukuba, Ibaraki, 305-0074, Japan.

The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Here, we find that the swim-up sperm used in many studies contain about 10% population of sperm which have not yet completed the histone-to-protamine replacement. We develop a method to purify histone replacement-completed sperm (HRCS) and to completely solubilize histones from cross-linked HRCS without MNase digestion. Our results indicate that histones are retained at specific promoter regions in HRCS. This method allows the study of epigenetic status in mature sperm.
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http://dx.doi.org/10.1038/s41467-018-06243-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155156PMC
September 2018

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks.

EMBO Rep 2018 09 13;19(9). Epub 2018 Aug 13.

Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome-wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild-type Rif1 and truncated Rif1 lacking the Rap1-interaction domain, we identify hundreds of Rap1-dependent and Rap1-independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1-independent manner, associating with both early and late-initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.
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http://dx.doi.org/10.15252/embr.201846222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123642PMC
September 2018

Re-evaluating the Localization of Sperm-Retained Histones Revealed the Modification-Dependent Accumulation in Specific Genome Regions.

Cell Rep 2018 06;23(13):3920-3932

Laboratory of Pathology and Development, the Institute for Quantitative Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-0032, Japan. Electronic address:

The question of whether retained histones in the sperm genome localize to gene-coding regions or gene deserts has been debated for years. Previous contradictory observations are likely caused by the non-uniform sensitivity of sperm chromatin to micrococcal nuclease (MNase) digestion. Sperm chromatin has a highly condensed but heterogeneous structure and is composed of 90%∼99% protamines and 1%∼10% histones. In this study, we utilized nucleoplasmin (NPM) to improve the solubility of sperm chromatin by removing protamines in vitro. NPM treatment efficiently solubilized histones while maintaining quality and quantity. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses using NPM-treated sperm demonstrated the predominant localization of H4 to distal intergenic regions, whereas modified histones exhibited a modification-dependent preferential enrichment in specific genomic elements, such as H3K4me3 at CpG-rich promoters and H3K9me3 in satellite repeats, respectively, implying the existence of machinery protecting modified histones from eviction.
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http://dx.doi.org/10.1016/j.celrep.2018.05.094DOI Listing
June 2018

Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics.

Mol Cell 2017 Nov 9;68(4):758-772.e4. Epub 2017 Nov 9.

Instituto de Biología Funcional y Genómica (IBFG-CSIC), Universidad de Salamanca, Calle Zacarías González 2, 37007 Salamanca, Spain; Centro de Investigaciones Biológicas (CIB-CSIC), Calle Ramiro de Maeztu 9, 28040 Madrid, Spain. Electronic address:

Replication fork integrity is challenged in conditions of stress and protected by the Mec1/ATR checkpoint to preserve genome stability. Still poorly understood in fork protection is the role played by the structural maintenance of chromosomes (SMC) cohesin complex. We uncovered a role for the Rsp5 ubiquitin ligase in promoting survival to replication stress by preserving stalled fork integrity. Rsp5 physically interacts with cohesin and the Mec1 kinase, thus promoting checkpoint-dependent cohesin ubiquitylation and cohesin-mediated fork protection. Ubiquitylation mediated by Rsp5 promotes cohesin mobilization from chromatin neighboring stalled forks, likely by stimulating the Cdc48/p97 ubiquitin-selective segregase, and its timely association to nascent chromatids. This Rsp5 fork protection mechanism requires the Wpl1 cohesin mobilizer as well as the function of the Eco1 acetyltransferase securing sister chromatid entrapment. Our data indicate that ubiquitylation facilitates cohesin dynamic interfacing with replication forks within a mechanism preserving stalled-fork functional architecture.
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http://dx.doi.org/10.1016/j.molcel.2017.10.012DOI Listing
November 2017

interleukin-11 induces and maintains progenitors of different cell lineages during Xenopus tadpole tail regeneration.

Nat Commun 2017 09 8;8(1):495. Epub 2017 Sep 8.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Unlike mammals, Xenopus laevis tadpoles possess high ability to regenerate their lost organs. In amphibians, the main source of regenerated tissues is lineage-restricted tissue stem cells, but the mechanisms underlying induction, maintenance and differentiation of these stem/progenitor cells in the regenerating organs are poorly understood. We previously reported that interleukin-11 (il-11) is highly expressed in the proliferating cells of regenerating Xenopus tadpole tails. Here, we show that il-11 knockdown (KD) shortens the regenerated tail length, and the phenotype is rescued by forced-il-11-expression in the KD tadpoles. Moreover, marker genes for undifferentiated notochord, muscle, and sensory neurons are downregulated in the KD tadpoles, and the forced-il-11-expression in intact tadpole tails induces expression of these marker genes. Our findings demonstrate that il-11 is necessary for organ regeneration, and suggest that IL-11 plays a key role in the induction and maintenance of undifferentiated progenitors across cell lineages during Xenopus tail regeneration. Xenopus laevis tadpoles have maintained their ability to regenerate various organs. Here, the authors show that interleukin-11 is necessary for organ regeneration, by inducing and maintaining undifferentiated progenitors across cell lineages during Xenopus tail regeneration.
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http://dx.doi.org/10.1038/s41467-017-00594-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591189PMC
September 2017

An In Vitro Human Liver Model by iPSC-Derived Parenchymal and Non-parenchymal Cells.

Stem Cell Reports 2017 08 27;9(2):490-498. Epub 2017 Jul 27.

Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan. Electronic address:

During liver development, hepatoblasts and liver non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) constitute the liver bud where they proliferate and differentiate. Accordingly, we reasoned that liver NPCs would support the maturation of hepatocytes derived from human induced pluripotent stem cells (hiPSCs), which usually exhibit limited functions. We found that the transforming growth factor β and Rho signaling pathways, respectively, regulated the proliferation and maturation of LSEC and HSC progenitors isolated from mouse fetal livers. Based on these results, we have established culture systems to generate LSECs and HSCs from hiPSCs. These hiPSC-derived NPCs exhibited distinctive phenotypes and promoted self-renewal of hiPSC-derived liver progenitor cells (LPCs) over the long term in the two-dimensional culture system without exogenous cytokines and hepatic maturation of hiPSC-derived LPCs. Thus, a functional human liver model can be constructed in vitro from the LPCs, LSECs, and HSCs derived from hiPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2017.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549957PMC
August 2017

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress.

Proc Natl Acad Sci U S A 2017 07 3;114(29):7671-7676. Epub 2017 Jul 3.

Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, United Kingdom;

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.
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http://dx.doi.org/10.1073/pnas.1620208114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530651PMC
July 2017

Decreased cohesin in the brain leads to defective synapse development and anxiety-related behavior.

J Exp Med 2017 05 13;214(5):1431-1452. Epub 2017 Apr 13.

Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan

Abnormal epigenetic regulation can cause the nervous system to develop abnormally. Here, we sought to understand the mechanism by which this occurs by investigating the protein complex cohesin, which is considered to regulate gene expression and, when defective, is associated with higher-level brain dysfunction and the developmental disorder Cornelia de Lange syndrome (CdLS). We generated conditional -knockout mice and observed greater dendritic complexity and larger numbers of immature synapses in the cerebral cortex of mice. mice also exhibited more anxiety-related behavior, which is a symptom of CdLS. Further, a gene ontology analysis after RNA-sequencing suggested the enrichment of immune processes, particularly the response to interferons, in the mice. Indeed, fewer synapses formed in their cortical neurons, and this phenotype was rescued by STAT1 knockdown. Thus, low levels of cohesin expression in the developing brain lead to changes in gene expression that in turn lead to a specific and abnormal neuronal and behavioral phenotype.
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http://dx.doi.org/10.1084/jem.20161517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413336PMC
May 2017

-TCF3 complex is required for the tumorigenicity of colorectal cancer cells.

Proc Natl Acad Sci U S A 2016 Nov 21;113(45):12739-12744. Epub 2016 Oct 21.

Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan;

Wnt/β-catenin signaling plays a key role in the tumorigenicity of colon cancer. Furthermore, it has been reported that lncRNAs are dysregulated in several steps of cancer development. Here we show that β-catenin directly activates the transcription of the long noncoding RNA (lncRNA) [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3), both of which are required for the survival and tumorigenicity of colorectal cancer cells. interacts with and recruits TCF3 to the activating transcription factor 3 () locus, where it represses the expression of ATF3. Furthermore, we demonstrate that -TCF3-mediated down-regulation of ATF3 expression is required for the proliferation and tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of Our results reveal a pathway involving an lncRNA and two transcription factors that plays a key role in Wnt/β-catenin-mediated tumorigenesis. These results may provide insights into the variety of biological and pathological processes regulated by Wnt/β-catenin signaling.
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http://dx.doi.org/10.1073/pnas.1605938113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111701PMC
November 2016

ARCN1 Mutations Cause a Recognizable Craniofacial Syndrome Due to COPI-Mediated Transport Defects.

Am J Hum Genet 2016 Aug 28;99(2):451-9. Epub 2016 Jul 28.

Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan; CREST, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan.

Cellular homeostasis is maintained by the highly organized cooperation of intracellular trafficking systems, including COPI, COPII, and clathrin complexes. COPI is a coatomer protein complex responsible for intracellular protein transport between the endoplasmic reticulum and the Golgi apparatus. The importance of such intracellular transport mechanisms is underscored by the various disorders, including skeletal disorders such as cranio-lenticulo-sutural dysplasia and osteogenesis imperfect, caused by mutations in the COPII coatomer complex. In this article, we report a clinically recognizable craniofacial disorder characterized by facial dysmorphisms, severe micrognathia, rhizomelic shortening, microcephalic dwarfism, and mild developmental delay due to loss-of-function heterozygous mutations in ARCN1, which encodes the coatomer subunit delta of COPI. ARCN1 mutant cell lines were revealed to have endoplasmic reticulum stress, suggesting the involvement of ER stress response in the pathogenesis of this disorder. Given that ARCN1 deficiency causes defective type I collagen transport, reduction of collagen secretion represents the likely mechanism underlying the skeletal phenotype that characterizes this condition. Our findings demonstrate the importance of COPI-mediated transport in human development, including skeletogenesis and brain growth.
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http://dx.doi.org/10.1016/j.ajhg.2016.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974084PMC
August 2016

Exome Sequencing Identification of EP300 Mutation in a Proband with Coloboma and Imperforate Anus: Possible Expansion of the Phenotypic Spectrum of Rubinstein-Taybi Syndrome.

Mol Syndromol 2015 Jul 3;6(2):99-103. Epub 2015 Mar 3.

Institute for Molecular and Cellular Biosciences, Research Center for Epigenetic Disease, The University of Tokyo, Tokyo, Japan ; Division of Medical Genetics, Nagano Children's Hospital, Azumino, Japan.

Rubinstein-Taybi syndrome (RSTS) is a multisystem developmental disorder characterized by facial dysmorphisms, broad thumbs and halluces, growth retardation, and intellectual disability. In about 8% of RSTS cases, mutations are found in EP300. Previously, the EP300 mutation has been shown to cause the highly variable RSTS phenotype. Using exome sequencing, we identified a de novo EP300 frameshift mutation in a proband with coloboma, facial asymmetry and imperforate anus with minimal RSTS features. Previous molecular studies have demonstrated the importance of EP300 in oculogenesis, supporting the possibility that EP300 mutation may cause ocular coloboma. Since a wide phenotypic spectrum is well known in EP300-associated RSTS cases, the atypical phenotype identified in our proband may be an example of rare manifestations of RSTS.
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http://dx.doi.org/10.1159/000375542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521068PMC
July 2015

Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation.

Cell Rep 2015 Aug 23;12(5):774-87. Epub 2015 Jul 23.

Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK.

The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.
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http://dx.doi.org/10.1016/j.celrep.2015.06.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534484PMC
August 2015

Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells.

Cell Stem Cell 2015 May 19;16(5):517-32. Epub 2015 Mar 19.

Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan; JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan; Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome.
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http://dx.doi.org/10.1016/j.stem.2015.03.002DOI Listing
May 2015

Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis.

PLoS One 2015 16;10(3):e0111655. Epub 2015 Mar 16.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan.

Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in the blastema tissues in various animals, but transcriptome analysis of the isolated proliferating blastema cells has not yet been reported. In the present study, we used RNA-sequencing analysis to analyze the gene expression profile of isolated proliferating blastema cells of regenerating Xenopus laevis tadpole tails. We used flow cytometry to isolate proliferating cells, and non-proliferating blastema cells, from regenerating tadpole tails as well as proliferating tail bud cells from tail bud embryos, the latter two of which were used as control cells, based on their DNA content. Among the 28 candidate genes identified by RNA-sequencing analysis, quantitative reverse transcription-polymerase chain reaction identified 10 genes whose expression was enriched in regenerating tadpole tails compared with non-regenerating tadpole tails or tails from the tail bud embryos. Among them, whole mount in situ hybridization revealed that chromosome segregation 1-like and interleukin 11 were expressed in the broad area of the tail blastema, while brevican, lysyl oxidase, and keratin 18 were mainly expressed in the notochord bud in regenerating tails. We further combined whole mount in situ hybridization with immunohistochemistry for the incorporated 5-bromo-2-deoxyuridine to confirm that keratin 18 and interleukin 11 were expressed in the proliferating tail blastema cells. Based on the proposed functions of their homologs in other animal species, these genes might have roles in the extracellular matrix formation in the notochord bud (brevican and lysyl oxidase), cell proliferation (chromosome segregation 1-like and keratin 18), and in the maintenance of the differentiation ability of proliferating blastema cells (interleukin 11) in regenerating tadpole tails.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111655PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361676PMC
January 2016

Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin.

Nat Genet 2015 Apr 2;47(4):338-44. Epub 2015 Mar 2.

1] Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. [2] The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.
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http://dx.doi.org/10.1038/ng.3229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380798PMC
April 2015

The chromosomal association of the Smc5/6 complex depends on cohesion and predicts the level of sister chromatid entanglement.

PLoS Genet 2014 Oct 16;10(10):e1004680. Epub 2014 Oct 16.

Karolinska Institutet, Department of Cell and Molecular Biology, Stockholm, Sweden.

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.
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http://dx.doi.org/10.1371/journal.pgen.1004680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199498PMC
October 2014

5-Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex.

Cell Rep 2014 Oct 2;9(1):48-60. Epub 2014 Oct 2.

Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address:

The development of cancer is driven not only by genetic mutations but also by epigenetic alterations. Here, we show that TET1-mediated production of 5-hydroxymethylcytosine (5hmC) is required for the tumorigenicity of glioblastoma cells. Furthermore, we demonstrate that chromatin target of PRMT1 (CHTOP) binds to 5hmC. We found that CHTOP is associated with an arginine methyltransferase complex, termed the methylosome, and that this promotes the PRMT1-mediated methylation of arginine 3 of histone H4 (H4R3) in genes involved in glioblastomagenesis, including EGFR, AKT3, CDK6, CCND2, and BRAF. Moreover, we found that CHTOP and PRMT1 are essential for the expression of these genes and that CHTOP is required for the tumorigenicity of glioblastoma cells. These results suggest that 5hmC plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex to selective sites on the chromosome, where it methylates H4R3 and activates the transcription of cancer-related genes.
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http://dx.doi.org/10.1016/j.celrep.2014.08.071DOI Listing
October 2014

Dimeric combinations of MafB, cFos and cJun control the apoptosis-survival balance in limb morphogenesis.

Development 2014 Jul;141(14):2885-94

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-17, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. Here, we show that MafB was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos heterodimers repressed apoptosis, whereas MafB/cJun heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. Our data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73.
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http://dx.doi.org/10.1242/dev.099150DOI Listing
July 2014

Chromatin immunoprecipitation protocol for mammalian cells.

Methods Mol Biol 2014 ;1164:33-8

Laboratory of Genome Structure and Function, Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

The genomic approach (ChIP-seq) we introduce here is now a widely used powerful tool to explore protein-DNA interaction at genome-wide level in high resolution. This technology opens up the way to understand how local event mediated by protein-protein or protein-DNA interactions lead to the dynamic changes of overall chromosome structure and how variety of proteins make a regulatory network for the faithful execution of various chromosomal functions (i.e., transcription, replication, recombination, repair, and partition).
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http://dx.doi.org/10.1007/978-1-4939-0805-9_4DOI Listing
January 2015

Pathological and immunohistochemical findings of natural highly pathogenic avian influenza infection in tufted ducks during 2010-2011 outbreaks in Japan.

J Vet Med Sci 2014 Sep 30;76(9):1285-90. Epub 2014 May 30.

Department of Pathogenetic Veterinary Sciences, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

In the winter of 2010-2011, an outbreak of highly pathogenic avian influenza virus (HPAIV) infection occurred in wild and domestic birds in Japan. Tufted ducks were found dead in an urban area of Toyota City, Koriyama, Fukushima Prefecture. Two tufted ducks were examined histopathologically, immunohistochemically and molecularly. Gross findings included marked dark-red clotted blood in the pectoral muscles and multifocal hemorrhages on the serous membranes. Microscopically, non-suppurative meningoencephalitis, multifocal to coalescing pancreatic necrosis and severe pulmonary congestion were observed. HPAIV antigen was detected in the malacic areas, neuronal, glial and ependymal cells, pulmonary capillary endothelial cells and epithelium of pulmonary bronchioles, necrotic pancreatic acini and degenerated cardiac myocytes. The HPAIV isolate was genetically classified into clade 2.3.2.1 group A. The broad distribution of virus antigen in brain and pulmonary tissues associated with HPAIV spontaneous infection in tufted ducks might be useful in understanding its pathogenesis in nature.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197160PMC
http://dx.doi.org/10.1292/jvms.14-0084DOI Listing
September 2014