Murano, Kensaku

写真a

Affiliation

School of Medicine, Department of Molecular Biology Department of Molecular Biology （Shinanomachi）

Position

Assistant Professor/Senior Assistant Professor

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Research Areas 【 Display / hide 】

  • Life Science / Genome biology

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Books 【 Display / hide 】

  • Generation of Stable Drosophila Ovarian Somatic Cell Lines Using the piggyBac System

    Chikara Takeuchi, Kensaku Murano, Mitsuru Ishikawa, Hideyuki Okano, Yuka W. Iwasaki, Springer US, 2022

    　View Summary

    Transposable elements (TEs) constitute a large proportion of the genome in multiple organisms. Therefore, anti-transposable element machineries are essential to maintain genomic integrity. PIWI-interacting RNAs (piRNAs) are a major force to repress TEs in Drosophila ovaries. Ovarian somatic cells (OSC), in which nuclear piRNA regulation is functional, have been used for research on piRNA pathway as a cell culture system to elucidate the molecular mechanisms underlying the piRNA pathway. Analysis of piRNA pathway using a reporter system to monitor the gene regulation or overexpression of specific genes would be a powerful approach. Here, we present the technical protocol to establish stable cell lines using the piggyBac system, adopted for OSCs. This easy, consistent, and timesaving protocol may accelerate research on the piRNA pathway.

  • タンパク質発現プロトコール

    Muarano Kensaku, 羊土社, 2010

    Scope: 哺乳動物細胞

  • Recent DNA Structure, Chromatin and Gene Expression

    Muarano Kensaku, Transworld Research Network, 2006

    Scope: Molecular and physiological roles of histone chaperones

  • クロマチンと遺伝子機能制御

    Muarano Kensaku, シュプリンガー・フェアラーク東京, 2003

    Scope: ヒトモデルとしてのマウスとウイルスからみたクロマチン制御

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Papers 【 Display / hide 】

  • Transcription of MERVL retrotransposons is required for preimplantation embryo development

    Akihiko Sakashita, Tomohiro Kitano, Hirotsugu Ishizu, Youjia Guo, Harumi Masuda, Masaru Ariura, Kensaku Murano, Haruhiko Siomi

    Nature Genetics （Springer Science and Business Media LLC)   2023.03

    ISSN  1061-4036

    　View Summary

    Abstract

    Zygotic genome activation (ZGA) is a critical postfertilization step that promotes totipotency and allows different cell fates to emerge in the developing embryo. MERVL (murine endogenous retrovirus-L) is transiently upregulated at the two-cell stage during ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we show that full-length MERVL transcripts, but not encoded retroviral proteins, are essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. Both knockdown and CRISPRi-based repression of MERVL result in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that loss of MERVL transcripts led to retention of an accessible chromatin state at, and aberrant expression of, a subset of two-cell-specific genes. Taken together, our results suggest a model in which an endogenous retrovirus plays a key role in regulating host cell fate potential.

  • Memory B Cells and Memory T Cells Induced by SARS-CoV-2 Booster Vaccination or Infection Show Different Dynamics and Responsiveness to the Omicron Variant

    Mise-Omata S., Ikeda M., Takeshita M., Uwamino Y., Wakui M., Arai T., Yoshifuji A., Murano K., Siomi H., Nakagawara K., Ohyagi M., Ando M., Hasegawa N., Saya H., Murata M., Fukunaga K., Namkoong H., Lu X., Yamasaki S., Yoshimura A.

    Journal of Immunology （Journal of Immunology)  209 （ 11 ） 2104 - 2113 2022.12

    ISSN  00221767

    　View Summary

    Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR62 circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

  • TDP-43 safeguards the embryo genome from L1 retrotransposition.

    Ten D Li, Kensaku Murano, Tomohiro Kitano, Youjia Guo, Lumi Negishi, Haruhiko Siomi

    Science advances （Science Advances)  8 （ 47 ） eabq3806 2022.11

    　View Summary

    Transposable elements (TEs) are genomic parasites that propagate within the host genome and introduce mutations. Long interspersed nuclear element-1 (LINE-1 or L1) is the major TE class, which occupies nearly 20% of the mouse genome. L1 is highly active in mammalian preimplantation embryos, posing a major threat to genome integrity, but the mechanism of stage-specific protection against L1 retrotransposition is unknown. Here, we show that TAR DNA-binding protein 43 (TDP-43), mutations in which constitute a major risk factor for amyotrophic lateral sclerosis, inhibits L1 retrotransposition in mouse embryonic stem cells (mESCs) and preimplantation embryos. Knockdown of TDP-43 resulted in massive genomic L1 expansion and impaired cell growth in preimplantation embryos and ESCs. Functional analysis demonstrated that TDP-43 interacts with L1 open reading frame 1 protein (L1 ORF1p) to mediate genomic protection, and loss of this interaction led to derepression of L1 retrotransposition. Our results identify TDP-43 as a guardian of the embryonic genome.

  • The emergence of SARS-CoV-2 variants threatens to decrease the efficacy of neutralizing antibodies and vaccines

    Kensaku Murano, Youjia Guo, Haruhiko Siomi

    Biochemical Society Transactions （Portland Press Ltd.)  49 （ 6 ） 2879 - 2890 2021.12

    ISSN  03005127

    　View Summary

    The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease (COVID-19) pandemic. As of August 2021, more than 200 million people have been infected with the virus and 4.3 million have lost their lives. Various monoclonal antibodies of human origin that neutralize the SARS-CoV-2 infection have been isolated from convalescent patients for therapeutic and prophylactic purposes. Several vaccines have been developed to restrict the spread of the virus and have been rapidly administered. However, the rollout of vaccines has coincided with the spread of variants of concern. Emerging variants of SARS-CoV-2 present new challenges for therapeutic antibodies and threaten the efficacy of current vaccines. Here, we review the problems faced by neutralizing antibodies and vaccines in the midst of the increasing spread of mutant viruses.

  • Potent mouse monoclonal antibodies that block SARS-CoV-2 infection.

    Youjia Guo, Atsushi Kawaguchi, Masaru Takeshita, Takeshi Sekiya, Mikako Hirohama, Akio Yamashita, Haruhiko Siomi, Kensaku Murano

    The Journal of biological chemistry （Journal of Biological Chemistry)  296   100346 - 100346 2021.01

    ISSN  00219258

    　View Summary

    Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that exhibit not only robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies.

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Papers, etc., Registered in KOARA 【 Display / hide 】

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Reviews, Commentaries, etc. 【 Display / hide 】

  • SARS-CoV-2感染を阻害する強力なマウスモノクローナル抗体

    村野健作, GUO Youjia, 川口敦史, 竹下勝, 山下暁朗, 塩見春彦

    日本分子生物学会年会プログラム・要旨集(Web) 43rd 2020

  • PiwiはMaelstromを介してBrm依存的なトランスポゾンを抑制する

    大西遼, 佐藤薫, 村野健作, 根岸瑠美, 塩見春彦, 塩見美喜子

    日本分子生物学会年会プログラム・要旨集(Web) 43rd 2020

  • piRNAを介したトランスポゾン転写抑制機構におけるMaelとBrmの機能

    大西遼, 佐藤薫, 村野健作, 高橋亜紀子, 根岸瑠美, 塩見春彦, 塩見美喜子

    日本RNA学会年会要旨集 21st 2019

  • 転写因子Sp1によるサイレンと遺伝子の活性化

    浅賀正充N, 村野健作, 加藤広介, 永田恭介

    生化学  2007

    ISSN  0037-1017

  • Functional specificity of histone chaperones

    Kyosuke Nagata, Kensaku Murano, Kohsuke Kato, Mitsuru Okuwaki

    GENES & GENETIC SYSTEMS (GENETICS SOC JAPAN)  81 ( 6 ) 456 - 456 2006.12

    ISSN  1341-7568

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Research Projects of Competitive Funds, etc. 【 Display / hide 】

  • Role of retrotransposons in early embryonic development and totipotency control

    2020.07
    -
    2022.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, MURANO Kensaku, Grant-in-Aid for Challenging Research (Exploratory), Principal investigator

    　View Summary

    The host defense mechanism against LINE-1, a retrotransposon highly expressed during early embryogenesis, remains elusive. We identified TDP-43 as a protein interacting with LINE-1 ORF1 protein from cell extract of a two-cell stage-like cell derived from mouse ES cells. We tried to knock down TDP-43 expression by injecting siRNA into fertilized mouse eggs. TDP-43 knockdown significantly increased LINE-1 copy number on the mouse genome. These results indicate that TDP-43 contributes to genome integrity by suppressing LINE-1, which is de-repressed during early embryogenesis.

  • 小分子RNAによるトランスポゾン転写抑制機構の解析

    2020.04
    -
    2023.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Principal investigator

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    生殖細胞系列で転移と増殖を繰り返すトランスポゾンは、生命の次世代継承にとって脅威である。非コード小分子RNA（piRNA）とPiwiタンパク質の複合体は、配列情報と相補的なトランスポゾンを標的とし、周辺にヘテロクロマチンを形成することで抑制している。我々はPiwi-piRNA複合体と相互作用し、トランスポゾンを抑制する因子としてNxf2 (Nuclear RNA export factor 2)を同定した。Nxf2はmRNAの核外輸送に関与はせず、ヘテロクロマチンを形成する前にトランスポゾンの転写反応を抑制することを明らかにした (Murano K et al., EMBO J 2019)。しかし、その転写抑制メカニズムは不明である。本研究では、転写と共役させた系を用いてNxf2の相互作用因子を探索し、Piwi-piRNA経路によるトランスポゾンの転写抑制機構を解明する。
    本研究では、Nxf2-p15-Panx複合体によるトランスポゾンの転写反応抑制機構の解明を通して、Piwi-piRNA経路による生殖ゲノム恒常性維持の全体像を理解する。これまでに、Piwi-piRNA経路によるトランスポゾンの転写抑制は、少なくとも二段階に分けられることが分かった。特に初期段階での転写抑制機構に焦点をあて、転写と共役させた誘導係留レポーターシステムを用いて新生mRNA上におけるNxf2-p15-Panx複合体の相互作用因子の探索を行なった。新生mRNA上へ係留されたラムダNタンパク質を融合したNxf2を免疫沈降法により精製し、相互作用する因子を質量分析法により多数同定した。今後、同定した因子のトランスポゾン 抑制活性を評価し、Piwi-piRNA経路による転写反応と共役した転写抑制機構の解明を目指す。

  • Maintenance of the germ cell genome by non-coding RNA

    2017.04
    -
    2020.03

    MEXT,JSPS, Grant-in-Aid for Scientific Research, MURANO Kensaku, Grant-in-Aid for Scientific Research (C), Principal investigator

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    Retrotransposons, which repeatedly proliferate in the germline genome, are a threat to the next generation of inheritance of life. A complex of non-coding small molecule RNAs (piRNAs) and PIWI proteins represses retrotransposons to maintain genomic homeostasis. This study has shown that the newly identified nuclear export factor 2 (Nxf2) represses the transcription of transposons through its interaction with Piwi-piRNA complex.

  • 新規高感度レポーター系を用いたrRNA遺伝子の種特異的転写開始機構の解析

    2013.06
    -
    2015.03

    University of Tsukuba, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), No Setting

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    rRNA遺伝子の転写反応は厳格な種特異性を示す。ヒトrRNA遺伝子はマウス細胞内で転写されず、またマウスrRNA遺伝子はヒト細胞内で転写されない。この種特異的転写は、マルチサブユニット因子SL1（ヒト）およびTIF-IB（マウス）に起因する。SL1/TIF-IBはrRNA遺伝子のコアプロモーターを認識し、転写開始点を規定する。rRNA遺伝子の転写開始反応を支配する複合体であるにもかかわらず、これまで構成因子によるSL1/TIF-IB活性の再構成はなされてこなかった。我々は、マウス細胞内においてヒトrRNA遺伝子転写を再構成し、マウス細胞内におけるSL1活性にはヒト由来の4種類のTAFI（TBP-associate factor I）が必要かつ十分であることを示してきた。一方で、マウス由来の4種類のTAFIを発現させても、ヒト細胞内においてマウスrRNA遺伝子転写を再構成することができなかった。試験管内転写反応系においても、HeLa細胞核抽出液はマウスF9細胞核抽出液を用いたマウスrRNA遺伝子転写を阻害した。また、マウスF9細胞核抽出液はHeLa細胞核抽出液を用いたヒトrRNA遺伝子の転写反応を阻害することが明らかとなった。そこで、SL1とTIF-IBはそれぞれマウスとヒトのrRNA遺伝子転写に干渉する可能性を検討した。マウス細胞内においてSL1構成因子の過剰発現は、マウスrRNA遺伝子の転写反応を強く抑制した。また、siRNAを用いてSL1構成因子の発現抑制を試みたところ、HeLa細胞内においてTIF-IB構成因子の過剰発現によりマウスrRNA遺伝子の転写に成功した。以上の結果から、HeLa細胞内におけるマウスrRNA遺伝子転写では、SL1がTIF-IB活性に干渉することが明らかとなった。
    26年度が最終年度であるため、記入しない。
    26年度が最終年度であるため、記入しない。

  • Dynamics of sperm chromatin

    2012.04
    -
    2015.03

    University of Tsukuba, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), MURANO Kensaku, NAGATA Kyosuke, Grant-in-Aid for Scientific Research (C), No Setting

    　View Summary

    Sperm chromosome forms highly condensed chromatin structure during spermatogenesis to send their information to next generation. We focus on a biological similarity between adenovirus infection cycle and spermatogenesis/fertilization. A histone chaperone, TAF-I, is not only able to remodel adenoviral chromatin structure, but also to make sperm chromatin decondensed in vitro. A series of experiments suggests that TAF-I is involved in cell growth during differentiation and development through its histone chaperone activity.

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Courses Taught 【 Display / hide 】

  • MOLECULAR BIOLOGY 2

    2023

  • MOLECULAR BIOLOGY 2

    2022

  • MOLECULAR BIOLOGY 2

    2021

  • MOLECULAR BIOLOGY 2

    2020

  • MOLECULAR BIOLOGY 2

    2019

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