Updated on 2024/03/19

写真a

 
NISHIMURA Kohei
 
Organization
Graduate School of Science Lecturer
Graduate School
Graduate School of Science
Undergraduate School
School of Science Department of Biological Science
Title
Lecturer

Degree 1

  1. PhD ( 2010.3   Osaka University ) 

Research Interests 5

  1. 染色体分配

  2. degron

  3. セントロメア

  4. auxin

  5. DNA replication

Research Areas 3

  1. Life Science / Genome biology

  2. Life Science / Genetics

  3. Life Science / Molecular biology

Research History 8

  1. Nagoya University   Lecturer

    2023.4

  2. Nagoya University   Assistant Professor

    2019.10 - 2023.3

  3. Osaka University   Designated assistant professor

    2015.10 - 2019.9

  4. Medical Research Institute   Protein Phosphorylation and Ubiquitination Unit   Oversea Research Fellowships from JSPS

    2014.4 - 2015.9

  5. National Institute of Genetics   Center of Frontier Research   Postdoctoral Fellowship for young Scientists from JSPS

    2011.4 - 2014.3

  6. National Institute of Genetics   Center of Frontier Research

    2011.2 - 2011.3

  7. Osaka University   Graduate School of Science Department of Biological Sciences

    2010.4 - 2011.2

  8. Osaka University   Graduate School of Science Department of Biological Sciences   Doctoral Fellowship for young Scientists

    2007.4 - 2010.3

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Education 1

  1. Osaka University   Graduate School of Science

    2007.4 - 2010.3

Professional Memberships 3

  1. The Genetics Society of Japan

  2. THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

  3. 日本植物生理学会

Awards 3

  1. 内藤記念海外研究留学助成金

    2014.4   内藤記念科学振興財団   植物ホルモンを用いたタンパク質分解系AID法による新たな動物細胞遺伝学の構築

    西村 浩平

  2. 海外特別研究員

    2014.4   日本学術振興会   植物ホルモンを用いたタンパク質分解系AID法による新たな動物細胞遺伝学の構築

    西村 浩平

  3. 特別研究員(PD)

    2011.4   日本学術振興会   動物細胞におけるオーキシン誘導デグロン法を応用した、合成生物学的遺伝学の創出

    西村 浩平

 

Papers 18

  1. Development of AlissAID system targeting GFP or mCherry fusion protein. Reviewed International journal

    Yoshitaka Ogawa, Kohei Nishimura, Keisuke Obara, Takumi Kamura

    PLoS genetics   Vol. 19 ( 6 ) page: e1010731   2023.6

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Conditional control of target proteins using the auxin-inducible degron (AID) system provides a powerful tool for investigating protein function in eukaryotes. Here, we established an Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody). In this system, target proteins fused with GFP or mCherry were degraded depending on a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In AlissAID system, nanomolar concentration of 5-Ad-IAA induces target degradation, thus minimizing the side effects from chemical compounds. In addition, in AlissAID system, we observed few basal degradations which was observed in other AID systems including ssAID system. Furthermore, AlissAID based conditional knockdown cell lines are easily generated by using budding yeast GFP Clone Collection. Target protein, which has antigen recognition sites exposed in cytosol or nucleus, can be degraded by the AlissAID system. From these advantages, the AlissAID system would be an ideal protein-knockdown system in budding yeast cells.

    DOI: 10.1371/journal.pgen.1010731

    PubMed

  2. Proteolysis of adaptor protein Mmr1 during budding is necessary for mitochondrial homeostasis in Saccharomyces cerevisiae. Reviewed International journal

    Keisuke Obara, Taku Yoshikawa, Ryu Yamaguchi, Keiko Kuwata, Kunio Nakatsukasa, Kohei Nishimura, Takumi Kamura

    Nature communications   Vol. 13 ( 1 ) page: 2005 - 2005   2022.4

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    Language:English   Publishing type:Research paper (scientific journal)  

    In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae. Redundant ubiquitin ligases Dma1 and Dma2 are responsible for Mmr1 ubiquitination. Dma1/2-mediated Mmr1 ubiquitination requires phosphorylation, most likely at S414 residue by Ste20 and Cla4. These kinases are mostly localized to the growing bud and nearly absent from mother cells, ensuring phosphorylation and ubiquitination of Mmr1 after the mitochondria enter the growing bud. In dma1Δ dma2Δ cells, transported mitochondria are first stacked at the bud-tip and then pulled back to the bud-neck. Stacked mitochondria in dma1Δ dma2Δ cells exhibit abnormal morphology, elevated respiratory activity, and increased level of reactive oxygen species, along with hypersensitivity to oxidative stresses. Collectively, spatiotemporally-regulated Mmr1 turnover guarantees mitochondrial homeostasis.

    DOI: 10.1038/s41467-022-29704-8

    PubMed

  3. Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase. Reviewed International journal

    Tokuko Haraguchi, Takako Koujin, Tomoko Shindo, Şükriye Bilir, Hiroko Osakada, Kohei Nishimura, Yasuhiro Hirano, Haruhiko Asakawa, Chie Mori, Shouhei Kobayashi, Yasushi Okada, Yuji Chikashige, Tatsuo Fukagawa, Shinsuke Shibata, Yasushi Hiraoka

    Communications biology   Vol. 5 ( 1 ) page: 78 - 78   2022.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

    DOI: 10.1038/s42003-022-03021-8

    PubMed

  4. A Simple Method that Combines CRISPR and AID to Quickly Generate Conditional Knockouts for Essential Genes in Various Vertebrate Cell Lines. Invited Reviewed International journal

    Kohei Nishimura, Tatsuo Fukagawa

    Methods in molecular biology (Clifton, N.J.)   Vol. 2377   page: 109 - 122   2022

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Cells with a loss-of-function mutation in a gene (knockout cells) are powerful tools for characterizing the function of such gene product. However, for essential genes, conditional knockout cell lines must be generated. The auxin-inducible degron (AID) technique enables us to conditionally and rapidly deplete a target protein from various eukaryotic cell lines. A combination of CRISPR-/Cas9-based gene editing and AID technique allows us to generate AID-based conditional knockout cell lines. Using these two techniques, we recently proposed a simple and quick way to generate conditional knockout cells for essential genes. In this chapter, we introduce the reader to the experimental procedures to generate these AID-based conditional knockout cell lines.

    DOI: 10.1007/978-1-0716-1720-5_6

    PubMed

  5. A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines. Invited Reviewed International journal

    Kohei Nishimura, Tatsuo Fukagawa

    Bio-protocol   Vol. 11 ( 14 ) page: e4092   2021.7

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to essential genes, which require a conditional knockout system. The auxin-inducible degron (AID) system enables rapid depletion of the target protein in an auxin-dependent manner and has been used to generate conditional mutants in various eukaryotic cell lines. One problem with the AID system is the use of high auxin concentrations for protein degradation, which can cause cytotoxicity. Recently, we established a super-sensitive AID (ssAID) system that allowed a reduction in the amount of auxin required by more than 1,000-fold. We also utilized a single-step method to generate AID-based conditional knockout cells with a ssAID system in various cell lines. In this protocol, we introduce our improved method, which provides a powerful tool for the investigation of the roles of essential genes.

    DOI: 10.21769/BioProtoc.4092

    PubMed

  6. CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle. Reviewed International journal

    Fabrizio Villa, Ryo Fujisawa, Johanna Ainsworth, Kohei Nishimura, Michael Lie-A-Ling, Georges Lacaud, Karim Pm Labib

    EMBO reports     page: e52164   2021.2

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    Language:English   Publishing type:Research paper (scientific journal)  

    The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.

    DOI: 10.15252/embr.202052164

    PubMed

  7. Essentiality of CENP-A Depends on Its Binding Mode to HJURP. Reviewed International journal

    Tetsuya Hori, JingHui Cao, Kohei Nishimura, Mariko Ariyoshi, Yasuhiro Arimura, Hitoshi Kurumizaka, Tatsuo Fukagawa

    Cell reports   Vol. 33 ( 7 ) page: 108388 - 108388   2020.11

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    Language:English   Publishing type:Research paper (scientific journal)  

    CENP-A incorporation is critical for centromere specification and is mediated by the chaperone HJURP. The CENP-A-targeting domain (CATD) of CENP-A specifically binds to HJURP, and this binding is conserved. However, the binding interface of CENP-A-HJURP is yet to be understood. Here, we identify the critical residues for chicken CENP-A or HJURP. The A59Q mutation in the α1-helix of chicken CENP-A causes CENP-A mis-incorporation and subsequent cell death, whereas the corresponding mutation in human CENP-A does not. We also find that W53 of HJURP, which is a contact site of A59 in CENP-A, is also essential in chicken cells. Our comprehensive analyses reveal that the affinities of HJURP to CATD differ between chickens and humans. However, the introduction of two arginine residues to the chicken HJURP αA-helix suppresses CENP-A mis-incorporation in chicken cells expressing CENP-AA59Q. Our data explain the mechanisms and evolution of CENP-A essentiality by the CENP-A-HJURP interaction.

    DOI: 10.1016/j.celrep.2020.108388

    PubMed

  8. A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair. Reviewed International journal

    Kohei Nishimura, Ryotaro Yamada, Shinya Hagihara, Rie Iwasaki, Naoyuki Uchida, Takumi Kamura, Koji Takahashi, Keiko U Torii, Tatsuo Fukagawa

    Nucleic acids research   Vol. 48 ( 18 ) page: e108   2020.10

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

    DOI: 10.1093/nar/gkaa748

    PubMed

  9. Rapid turnover of transcription factor Rim101 confirms a flexible adaptation mechanism against environmental stress in Saccharomyces cerevisiae. Reviewed International journal

    Keisuke Obara, Mai Higuchi, Yuki Ogura, Kohei Nishimura, Takumi Kamura

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 25 ( 10 ) page: 651 - 662   2020.10

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    Language:English   Publishing type:Research paper (scientific journal)  

    Saccharomyces cerevisiae cells activate the Rim101 pathway to adapt to alkaline and salt stresses. On activation of this pathway, the transcription factor Rim101 undergoes proteolytic activation and regulates the expression of responsive genes. We found Rim101 to be a short-lived protein with a half-life of approximately 15 min. Its rapid turnover was supposedly mediated by the ubiquitin-proteasome system. Excess accumulation of the processed active Rim101 through its over-expression conferred tolerance to both alkaline and salt stresses in yeast cells; in contrast, it had detrimental effects under cadmium stress condition. Cadmium ion inhibited proteolytic activation of Rim101, implying reciprocal interaction between the Rim101 pathway and cadmium stress. Our results showed yeast cells to be equipped with two protective systems to prevent overaccumulation of the processed active Rim101; Rim101 processing is inhibited when Rim101 level is high, and turnover of processed Rim101 is accelerated when it is abundant. Collectively, the results confirmed the flexible aspect of stress response in yeast cell; the cells not only prevent excess activation of one stress-responsive pathway but also facilitate its attenuation to cope with other environmental stresses.

    DOI: 10.1111/gtc.12801

    PubMed

  10. 3D genomic architecture reveals that neocentromeres associate with heterochromatin regions Reviewed

    Kohei Nishimura, Masataka Komiya, Tetsuya Hori, Takehiko Itoh, Tatsuo Fukagawa

    Journal of Cell Biology   Vol. 218 ( 1 ) page: 134 - 149   2019.1

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)  

    © 2018 Nishimura et al. The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere–heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres.

    DOI: 10.1083/jcb.201805003

    Scopus

    PubMed

  11. An efficient method to generate conditional knockout cell lines for essential genes by combination of auxin-inducible degron tag and CRISPR/Cas9 Reviewed

    Kohei Nishimura, Tatsuo Fukagawa

    CHROMOSOME RESEARCH   Vol. 25 ( 3-4 ) page: 253 - 260   2017.10

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER  

    Generation of cells with a loss-of-function mutation in a gene (knockout cells) is a valuable technique for studying the function of a given gene product. However, if the product of the target gene is essential for cell viability, conditional knockout cell lines must be generated. Recently, as gene editing technology using CRISPR/Cas9 has developed, it has become possible to produce conditional knockout cell lines using this technique. However, to obtain final conditional knockout cell lines, it is necessary to perform several experiments with multiple complicated steps. In this paper, we introduce an easy and efficient method to generate conditional knockout cell lines based on combining auxin-inducible degron (AID) technology with CRISPR/Cas9 gene editing. Our method only requires performing a single transfection and is therefore an easy and rapid method to obtain a conditional knockout cell line.

    DOI: 10.1007/s10577-017-9559-7

    Web of Science

    PubMed

  12. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis Reviewed

    Toyoaki Natsume, Kohei Nishimura, Sheroy Minocherhomji, Rahul Bhowmick, Ian D. Hickson, Masato T. Kanemaki

    GENES & DEVELOPMENT   Vol. 31 ( 8 ) page: 816 - 829   2017.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

    DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8-9 complex, a paralog of the MCM2-7 replicative helicase. We show that MCM8-9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase.

    DOI: 10.1101/gad.297663.117

    Web of Science

    PubMed

  13. Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex Reviewed

    Ryosuke Imai, Seiji Komeda, Mari Shimura, Sachiko Tamura, Satoshi Matsuyama, Kohei Nishimura, Ryan Rogge, Akihiro Matsunaga, Ichiro Hiratani, Hideaki Takata, Masako Uemura, Yutaka Iida, Yuko Yoshikawa, Jeffrey C. Hansen, Kazuto Yamauchi, Masato T. Kanemaki, Kazuhiro Maeshima

    SCIENTIFIC REPORTS   Vol. 6   page: 24712   2016.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers.

    DOI: 10.1038/srep24712

    Web of Science

    PubMed

  14. Rapid depletion of budding yeast proteins via the fusion of an auxin-inducible degron (AID) Reviewed

    Kohei Nishimura, Masato T. Kanemaki

    Current Protocols in Cell Biology   Vol. 2014   page: 20.9.1 - 20.9.16   2014

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:John Wiley and Sons Inc.  

    The auxin-inducible degron (AID) system allows the rapid and reversible proteolysis of proteins of interest, and enables the generation of conditional mutants of budding yeast. The construction of budding yeast AID mutants is simple, and the effect of depletion of essential proteins on proliferation can be confirmed by analyzing their phenotype. In this protocol, we describe a procedure to generate AID mutants of budding yeast via a simple transformation using PCR-amplified DNA. We also describe methods to confirm the depletion of proteins of interest that are required for proliferation by serial-dilution and liquid-culture assays.

    DOI: 10.1002/0471143030.cb2009s64

    Scopus

    PubMed

  15. The Elg1 Replication Factor C-like Complex Functions in PCNA Unloading during DNA Replication Reviewed

    Takashi Kubota, Kohei Nishimura, Masato T. Kanemaki, Anne D. Donaldson

    MOLECULAR CELL   Vol. 50 ( 2 ) page: 273 - 280   2013.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    The ring-shaped complex PCNA coordinates DNA replication, encircling DNA to act as a polymerase clamp and a sliding platform to recruit other replication proteins. PCNA is loaded onto DNA by replication factor C, but it has been unknown how PCNA is removed from DNA when Okazaki fragments are completed or the replication fork terminates. Here we show that the Elg1 replication factor C-like complex (Elg1-RLC) functions in PCNA unloading. Using an improved degron system we show that without Elg1, PCNA accumulates on Saccharomyces cerevisiae chromatin during replication. The accumulated PCNA can be removed from chromatin in vivo by switching on Elg1 expression. We find moreover that treating chromatin with purified Elg1-RLC causes PCNA unloading in vitro. Our results demonstrate that Elg1-RLC functions in unloading of both unmodified and SUMOylated PCNA during DNA replication, while the genome instability of an elg1 Delta mutant suggests timely PCNA unloading is critical for chromosome maintenance.

    DOI: 10.1016/j.molcel.2013.02.012

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  16. Mcm8 and Mcm9 Form a Complex that Functions in Homologous Recombination Repair Induced by DNA Interstrand Cross links Reviewed

    Kohei Nishimura, Masamichi Ishiai, Kazuki Horikawa, Tatsuo Fukagawa, Minoru Takata, Haruhiko Takisawa, Masato T. Kanemaki

    MOLECULAR CELL   Vol. 47 ( 4 ) page: 511 - 522   2012.8

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    DNA interstrand crosslinks (ICLs) are highly toxic lesions that stall the replication fork to initiate the repair process during the S phase of vertebrates. Proteins involved in Fanconi anemia (FA), nucleotide excision repair (NER), and translesion synthesis (TS) collaboratively lead to homologous recombination (HR) repair. However, it is not understood how ICL-induced HR repair is carried out and completed. Here, we showed that the replicative helicase-related Mm family of proteins, Mcm8 and Mcm9, forms a complex required for HR repair induced by ICLs. Chicken DT40 cells lacking MCM8 or MCM9 are viable but highly sensitive to ICL-inducing agents, and exhibit more chromosome aberrations in the presence of mitomycin C compared with wild-type cells. During ICL repair, Mcm8 and Mcm9 form nuclear foci that partly colocalize with Rad51. Mcm8-9 works downstream of the FA and BRCA2/Rad51 pathways, and is required for HR that promotes sister chromatid exchanges, probably as a hexameric ATPase/helicase.

    DOI: 10.1016/j.molcel.2012.05.047

    Web of Science

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  17. Auxin-inducible protein depletion system in fission yeast Reviewed

    Mai Kanke, Kohei Nishimura, Masato Kanemaki, Tatsuo Kakimoto, Tatsuro S. Takahashi, Takuro Nakagawa, Hisao Masukata

    BMC CELL BIOLOGY   Vol. 12   page: 8   2011.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BIOMED CENTRAL LTD  

    Background: Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe.
    Results: We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly-ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (improved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest.
    Conclusion: Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.

    DOI: 10.1186/1471-2121-12-8

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  18. An auxin-based degron system for the rapid depletion of proteins in nonplant cells Reviewed

    Kohei Nishimura, Tatsuo Fukagawa, Haruhiko Takisawa, Tatsuo Kakimoto, Masato Kanemaki

    NATURE METHODS   Vol. 6 ( 12 ) page: 917 - U78   2009.12

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase. Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability. The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.

    DOI: 10.1038/nmeth.1401

    Web of Science

    PubMed

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Books 2

  1. First Author's Mcm8とMcm9は複合体を形成しDNA二本鎖間架橋のひき起こす相同組換え修復において機能する

    西村浩平, 鐘巻将人( Role: Sole author)

    2012 

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    Language:Japanese

  2. 動物の培養細胞における迅速なタンパク質発現制御法(AID法)

    西村浩平, 鐘巻将人( Role: Joint author ,  実験医学 2010年 Vol. 28 No.14, 2279-2284)

    羊土社  2010.9 

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    Language:Japanese

MISC 18

  1. 植物ホルモン・オーキシンによるタンパク質分解系を利用した核内セントロメア構造の解析

    西村浩平, 嘉村巧, 深川竜郎

    アグリバイオ   Vol. 6 ( 13 ) page: 77 - 80   2022.12

  2. Centromere structure analysis by AID system and 4C method

    西村浩平, 深川竜郎, 嘉村巧

    月刊細胞   Vol. 54 ( 11 )   2022

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  3. 植物ホルモン応答の自在操作ツールの開発と動物細胞での標的タンパク質分解系への応用

    打田直行, 打田直行, 西村浩平

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

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  4. 人工オーキシンとTIR1のペアを用いた高感受性AID法

    西村浩平, 西村浩平, 山田遼太郎, 山田遼太郎, 萩原伸也, 萩原伸也, 岩崎理恵, 打田直行, 嘉村巧, 高橋宏二, 鳥居啓子, 深川竜郎

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

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  5. 脊椎動物細胞におけるセントロメアの3次元構造解析

    西村浩平, 堀哲也, 豊田敦, 古宮正隆, 伊藤武彦, 深川竜郎

    日本遺伝学会大会プログラム・予稿集   Vol. 91st   page: 148   2019.8

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  6. ヒト染色体維持における二本鎖切断誘導DNA複製の役割

    夏目豊彰, 夏目豊彰, 西村浩平, HICKSON Ian, 鐘巻将人, 鐘巻将人

    日本遺伝学会大会プログラム・予稿集   Vol. 90th   page: 96   2018.8

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  7. ゲノムの安定性を維持するためのヒトMCM8‐9依存的な複製フォークの再生機構

    夏目豊彰, 西村浩平, HICKSON Ian D, 鐘巻将人

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 41st   page: ROMBUNNO.1PW1‐07‐5 (WEB ONLY)   2018

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  8. ネオセントロメアから迫るセントロメア領域における染色体構造の解析

    西村浩平, 堀哲也, 古宮正隆, 伊藤武彦, 深川竜郎

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 41st   page: ROMBUNNO.1P‐0213 (WEB ONLY)   2018

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  9. セントロメア機能を制御するエピジェネティックメカニズム

    堀哲也, 曹静暉, 西村浩平, 有村泰宏, 有吉眞理子, 豊田敦, 三須定彦, 池尾一穂, 胡桃坂仁志, 深川竜郎

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 41st   page: ROMBUNNO.2P‐0234 (WEB ONLY)   2018

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  10. 4C‐Seqを用いたネオセントロメア形成に伴う染色体高次構造変化の解析

    古宮正隆, 西村浩平, 堀哲也, 深川竜郎, 伊藤武彦

    日本生化学会大会(Web)   Vol. 2017年度   page: [1P - 0621]   2017.12

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  11. ネオセントロメアの解析により明らかになったセントロメア領域のゲノム3D構造の実体

    西村浩平, 堀哲也, 古宮正隆, 伊藤武彦, 深川竜郎

    日本生化学会大会(Web)   Vol. 2017年度   page: [4P2T18 - 0530)]   2017.12

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  12. ネオセントロメア形成領域における染色体3D構造

    西村浩平, 堀哲也, 古宮正隆, 伊藤武彦, 深川竜郎

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 39th   page: ROMBUNNO.1P‐0022 (WEB ONLY)   2016

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  13. ヒト細胞における人為的複製フォーク破壊システムから見えてきたMcm8‐9依存的な複製フォークの再生メカニズム

    夏目豊彰, 夏目豊彰, 西村浩平, 鐘巻将人, 鐘巻将人

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 39th   page: ROMBUNNO.1AS10‐9(3P‐0108) (WEB ONLY)   2016

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    J-GLOBAL

  14. A hidden DNA synthesis that contributes integrity of genomic DNA

    Toyoaki Natsume, Kohei Nishimura, Yoshiki Kanehara, Masato Kanemaki

    DNA REPAIR   Vol. 28   page: 142 - 142   2015.4

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:ELSEVIER SCIENCE BV  

    Web of Science

  15. Mcm8‐9複合体はRad51依存的鎖潜り込み反応後のDNA伸長反応に関わる

    西村浩平, 夏目豊彰, 石合正道, 深川竜郎, 高田穣, 鐘巻将人

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 36th   page: 1PW8-5 (WEB ONLY)   2013

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  16. Mcm8とMcm9は複合体を形成し,DNA二本鎖架橋修復時に引き起こされる相同組換え修復において働く

    西村浩平, 石合正道, 堀川一樹, 深川竜郎, 高田穣, 滝澤温彦, 鐘巻将人

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 35th   page: 2W5II-5 (WEB ONLY)   2012

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  17. Mcm8 and Mcm9 from a novel complex involved in resistance to DNA crosslinking agents. Reviewed

    Kohei Nishimura, Masamichi Ishiai, Tatsuo Fukagawa, Minoru Takata, Haruhiko Takisawa, Masato Kanemaki

    日本分子生物学会第 34 回年会 2011 年12 月 横浜     2011

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  18. クローズアップ実験法 動物の培養細胞における迅速なタンパク質発現制御法(AID法)

    西村浩平, 鐘巻将人

    実験医学   Vol. 28 ( 14 ) page: 2279 - 2284   2010.9

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    J-GLOBAL

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Presentations 18

  1. Auxin-Inducible Degron (AID)法を⽤いた細胞内タンパク質分解コントロール Invited

    西村浩平

    日本プロテオーム学会2023年大会  2023.7.26 

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    Event date: 2023.7

    Language:English   Presentation type:Oral presentation (general)  

  2. 3D structural analysis of neo-centromere region in vertebrate cell

    西村 浩平

    EMBO workshop "Dynamic kinetochore"  2017.6.8 

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  3. 植物ホルモン・オーキシン依存的タンパク質分解系を応用した迅速なタンパク質除去法による出芽酵母と動物細胞コンディショナル変異株の作成 - オーキシン誘導デグロン法 –

    西村 浩平

    第32回日本分子生物学会年会  2009.12.11 

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    Language:English   Presentation type:Oral presentation (general)  

  4. 植物ホルモン・オーキシン依存的タンパク質分解系を応用した迅速なタンパク質除去法による出芽酵母と動物細胞における条件特異的変異株の作成-オーキシン誘導デグロン法-

    西村 浩平

    第20回DNA複製・組換え・修復ワークショップ  2009.11.2 

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  5. 植物ホルモン・オーキシンを利用したユビキチン依存的タンパク質分解系とその利用 Invited

    西村 浩平

    名古屋大学GTRセミナー  2019.4.16 

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  6. 新規 Mcm8-9 複合体の機能解析

    西村 浩平

    第21回 複製・組換え・ゲノム安定性制御ワークショップ  2011.10.25 

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  7. ネオセントロメア形成領域における染色体構造の解析

    西村 浩平

    第39回 日本分子生物学会年会  2016.12.1 

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  8. ネオセントロメアの解析により明らかになったセントロメア領域のゲノム3D構造の実態

    西村 浩平

    生命科学系学会合同年次学会 (第40回 日本分子生物学会年会)  2017.12.9 

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  9. The Mcm8-9 complex promotes DNA synthesis after strand invasion in recombination repair

    西村 浩平

    第36回 日本分子生物学会年会  2013.12.3 

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  10. The auxin dependent degradation pathway can be transplanted to non-plant cells for the construction of a novel degron system. Invited

    西村 浩平

    Perspective of Plant Science  2010.3.27 

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  11. Mcm8-9複合体はICL修復における 相同組換え鎖潜り込み反応後の DNA合成反応を促進する

    西村 浩平

    第22回 DNA複製・組換え・修復ワークショップ  2013.11.21 

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  12. Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand-crosslinks

    西村 浩平

    第35回 日本分子生物学会年会  2012.12.12 

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  13. Exploring factors involving maintenance and re-start of the stalled fork by using a novel method to induce rapid degradation in budding yeast.

    西村 浩平

    Cold Spring Harbor Meeting ‘Eukaryotic DNA Replication & Genome Maintenance’  2009.9.2 

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  14. Easy construction of Auxin Inducible Degron mutants in vertebrate cells

    西村 浩平

    Genome Editing and Functional Genomics 2018  2018.7.4 

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  15. A novel method to regulate protein function in living cells using a phytohormone Invited

    西村 浩平

    Minisymposium of Tsing-Hua University-Osaka University Life Science Student Academic Activity  2009.5.13 

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  16. 4C解析による核内セントロメア構造 分子基盤とその役割の解明

    西村 浩平

    第36回 染色体ワークショップ  2019.1.24 

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  17. 3D structural analysis of neo-centromere region in vertebrate cells. International conference

    Kohei Nishimura

    ASCB/EMBO 2018 meeting, San Diego  2018.12.11 

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  18. A super-sensitive Auxin-Inducible Degron (ssAID) system for conditional knockdown in various vertebrate cell lines Invited

    Kohei Nishimura

    2nd Webinar on Nucleic Acid and CRISPR  2021.3.10 

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KAKENHI (Grants-in-Aid for Scientific Research) 11

  1. 小分子抗体を活用した細胞内悪性タンパク質の除去法の構築

    2023.12 - 2024.12

    公益財団法人 持田記念医学薬学振興財団  研究助成 

    西村浩平

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  2. 小分子抗体を利用したオーキシン依存的タンパク質分解システムの開発

    2023.4 - 2025.3

    IFO 公益財団法人 発酵研究所  一般研究助成 

    西村浩平

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    Authorship:Principal investigator 

  3. タンパク質工学およびケミカルバイオロジーを駆使したタンパク質分解系の開発

    Grant number:22K05558  2022.4 - 2025.3

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    西村 浩平

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    Grant amount:\4160000 ( Direct Cost: \3200000 、 Indirect Cost:\960000 )

    細胞内のタンパク質分解システムを利用した標的タンパク質分解法は基礎的な研究、応用研究から医薬品の開発に至るまで、幅広い領域で研究、開発が進んでいる。MGDやPROTACのように直接的に医薬品の開発に向かう研究が必要であることは明らかではあるが、一方で、違った方向からの研究、開発も必要である。申請者が開発したAID法は全ての細胞内に存在するユビキチンプロテアソーム系というタンパク質分解システムを利用した分解系であるという点で前述のMGDやPROTACと同様である。そのため、本システムの開発がもたらすユビキチンプロテアソーム系に対する理解の向上がどの分解系においても有益な情報となると考えた。
    申請者が確立したAID法は標的とするタンパク質をオーキシン依存的に分解することが可能であり、様々な真核生物種において、標的とするタンパク質の迅速なノックダウン系として用いられている(Nishimura et al., Nature Methods, 2009)。しかしながら、TIR1の導入や標的タンパク質へのAIDタグの付加など、実験に先立ち行わなければならないことも多い。また、過剰量のオーキシンがもたらす細胞毒性の問題も、避けては通れない問題であった。このような状況の中、申請者は動物細胞で簡便にAID細胞株を作製する方法を確立し(Nishimura and Fukagawa, Chromosome Res, 2017)、また、勘案事項の一つであった過剰量のオーキシン問題も、OsTIR1F74A変異体と人工合成オーキシンである5-Ad-IAAとの組み合わせにより1/1000にまで減少させることに成功した(Nishimura et al., Nucleic Acids res, 2020)。本年はナノボディと呼ばれる小分子抗体をこのssAID法に組み込み、ナノボディが認識したタンパク質を分解するAlissAID systemの構築を行った。その結果、真核生物のモデル生物である出芽酵母細胞において、GFPやmCherryなどの蛍光タンパク質を標的としたAlissAID systemの開発に成功した。出芽酵母細胞においてはGFPタグのクローンコレクションが存在しているため、これらの酵母株を利用することで簡便にAlissAID株を作製することができるため、このAlissAID systemは出芽酵母の分子遺伝学にとって重要なツールとなると考えられる。
    既存のオーキシンデグロン法で標的タンパク質の分解を誘導するためには分解用のタグであるAIDタグを標的とするタンパク質に付加する必要がある。出芽酵母や一部の培養細胞では相同組換えにより、簡便にAIDタグを付加することが可能である。一方で、他の動物培養細胞や生物個体ではこのようなタンパク質へのタグの付加は難しいものも多い。そのため、AIDタグの付加を必要としないssAID法、すなわちAlissAID法の確立を試みた。この目的のために小分子抗体であるナノボディを利用した。ナノボディとAIDタグとを融合させ、この融合タンパク質の中からユビキチン化の標的となるリシン残基を取り除くことによって、このタンパク質はユビキチン化されず、ナノボディ によって認識された標的タンパク質がユビキチン化され、分解されるようになると考えられた。本研究ではまず、既知のナノボディであるGFPナノボディとGFP融合タンパク質を用いて、GFP融合タンパク質の分解が可能であるかを真核生物のモデル生物である出芽酵母細胞を用いて検証を行った。すると、細胞質や核に局在するGFP融合タンパク質を5-Ad-IAA依存的に分解することが可能であった。次に赤色蛍光タンパク質としてGFPとともに多用されるmCherryに対するナノボディを用いてmCherry融合タンパク質に対するAlissAID法の構築を行った。得られたデータをもとに論文を作成し、プレプリントサーバーであるBioRxivへとデポジットを行った。
    本年、確立した出芽酵母によるAlissAID法が動物の細胞においても機能するか検証を行う。また、上記の研究により、構築したAlissAID法により、内在性のタンパク質を標的とした分解系を構築するために、内在性の因子に対するナノボディの探索を行う。現在、ナノボディ を探索する方法としては、これを保有するリャマなどへの免疫による通常の抗体取得以外に、ファージや酵母の表層に抗体を提示させるディスプレイ法などが知られている。またこの他にもペプチドバーコード法によるナノボディ の探索法などが開発されている。そのため、これらの方法を用いることにより標的とするタンパク質に対するナノボディ の探索を行い、上記の分解誘導法に用いる。分解の標的としては核や細胞膜など多様な局在を持つタンパク質を標的とすることとで本分解システムの汎用性を確かめていく。本システムの特徴として、分解標的の認識を小分子抗体により担わせる点にある。既存のタンパク質分解法であるAID法、MGD、PROTACではタンパク質の修飾状況にかかわらず、全てのタンパク質が分解の標的となってしまう。しかし、本システムでは修飾がなされたタンパク質のみに対する小分子抗体を取得することができれば、修飾されたタンパク質のみを標的として分解誘導することが可能となる。

  4. B型肝炎ウィルスの全長POL発現技術を基盤としたPOL機能の解明と新規薬剤開発

    2022.4 - 2025.3

    日本医療研究開発機構(AMED)  肝炎等克服実用化研究事業 B型肝炎創薬実用化等研究事業 

    杉山真也, 金井雅武, 西村浩平, 竹田浩平

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    Authorship:Coinvestigator(s) 

  5. 内在性タンパク質を標的とした超高感度オーキシン依存的タンパク質分解系の開発

    2022.4 - 2024.3

    加藤記念バイオサイエンス振興財団  研究助成「バイオテクノロジー分野」 

    西村浩平

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    Authorship:Principal investigator 

  6. Artificial cell cycle control using the AID method, a low-toxicity and rapid proteolytic system

    Grant number:20K21423  2020.7 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    Kamura Takumi

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    The AID method, a low-toxicity and rapid proteolytic system, was used to artificially control the cell cycle in cultured cells. Since cell cycle progression is positively regulated by CDK complexes and negatively regulated by CKI, the expression of these factors was controlled by the AID method and their effects on the cell cycle were examined. We found that in the Cdk1 AID cell line, the cell cycle is arrested in the G2 phase due to Cdk1 degradation. Similar results were obtained not only in the chicken DT40 cell line but also in mouse ES cells, suggesting that artificial cell cycle control can be achieved by regulating Cdk1 in a variety of cells.

  7. 変異型オーキシン・システムによる高効率なタンパク質分解系の構築

    2020.4 - 2021.3

    堀科学芸術振興財団  研究助成 

    西村浩平

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  8. 3D structural analysis of centromere in vertebrate cells

    Grant number:19K06611  2019.4 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Nishimura Kohei

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    We successfully detected genome regions that specifically interact with centromeres in interphase nuclei using centromeres without repetitive sequences and the chicken DT40 cell line. This success served as a starting point for this research project. The aim of this study is to elucidate the molecular basis and role of the specific three-dimensional genome organization of centromeres in the nuclei of vertebrates during interphase. The applicants combined their previously achieved results with a protein removal technique they developed, the Auxin-Inducible Degron system (AID), with high-precision genome three-dimensional structural analysis methods (4C and Hi-C) to find that centromeres function as boundaries for binding locks in interphase nuclei.

  9. 3D structural analysis of centromere in vertebrate cells

    Grant number:17K15041  2017.4 - 2019.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)  Grant-in-Aid for Young Scientists (B)

    NISHIMURA Kohei

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    We carried out 4C-seq analysis of chicken DT40 cells. 4C-seq analysis showed some features of centromere region in interphase nuclei. In addition, we identified some factors that are involved in the centromere structure by using neocentromere forming cells and Auxin inducible Degron system in chicken DT40 cells.

  10. 動物細胞におけるオーキシン誘導デグロン法を応用した、合成生物学的遺伝学の創出

    Grant number:11J02503  2011 - 2013

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    西村 浩平

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    申請者は昨年DNA複製因子として考えられていたMcm8そしてMcm9が複合体を形成し、DNA二本鎖架橋のダメージの際の相同組換え修復において機能することを見いだし、論文として発表した。本年はこのニワトリDT40細胞におけるMcm8-9の機能をさらに多面的に解析するために、Mcm8-9複合体の精製を行い、ニワトリDT40細胞からMcm8-9複合体を精製することに成功した。この精製した複合体について生化学的また構造学的な解析を進めるための準備を現在行っている。またMcm8,9の遺伝学的な解析をさらに行う上でMcm8, およびMcm9のコンディショナルミュータントを当研究室で開発されたAID法を用いてMcm8-aid, Mcm9-aid株を作成し、表現型を確認したところ、いずれの場合もノックアウト細胞と比べて表現型が弱くなっていた。この結果を受けて当初考えていたマウスES細胞におけるAIDミュータントの作成に先立ち、AID法における標的タンパク質の分解の更なる効率化を行うこととした。出芽酵母において研究を行い、分解誘導に必要なタグの大きさを従来の3分の1にまで縮小することに成功した。さらにこのタグを3つ連続してつなげることによって従来よりも高効率の分解を誘導することに成功した。この分解の効率化によって、解析の困難であったElg1の機能解析が可能となり、分解効率の効率化とElg1の解析結果を併せて、論文に発表した。

  11. 高等真核生物における蛋白質分解系の開発

    Grant number:08J01719  2008 - 2010

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    西村 浩平

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    AID法は植物におけるオーキシン受容体TIR1を出芽酵母細胞内で発現させることでSCF^<TIR1>と呼ばれるE3ユビキチンライゲースを構築し、分解標識となるaidを付加したタンパク質をオーキシン依存的に速やかにポリユビキチン化し、分解する系である。私は昨年度この方法を動物の培養細胞に導入することに成功した。そのため、動物の培養細胞においてAID法によって必須因子を細胞内から除去した際に、細胞がその因子の細胞内機能を反映した表現型を示すかを確認するため、細胞分裂に必須なセントロメアタンパク質であるCENP-Hの発現をAID法によって制御することのできるDT40細胞(CENP-H-Caid)を作製した。この株においてaidを付加したCENP-Hはオーキシン添加によって細胞内で速やかに分解され、蛍光抗体法によりセントロメア領域に局在しているCENP-Hもオーキシン添加後30分でほぼ完全に除去されていることが明らかとなった。また、このCENP-H-Caid株はオーキシン添加直後から細胞周期がM期へ蓄積しはじめ、12時間後にはほぼすべての細胞がM期に蓄積している様子が観察された。これらの結果はAID法が動物細胞で標的タンパク質の急激な除去により、表現型を速やかに誘導することが可能であることを示している。既存のテトラサイクリンによるCENP-H転写制御株ではセントロメア領域からCENP-Hを完全に除去し、表現型が現れるまで24時間以上かかることを考慮すると、AID法は既存の方法と比べて、表現型を得るまでにかかる時間を大幅に短縮することができ、動物の培養細胞においても有用な遺伝学的解析ツールとなることが期待される

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Industrial property rights 5

  1. 真核細胞におけるタンパク質分解誘導方法

    鐘巻 将人, 西村 浩平, 滝澤 温彦, 三村 覚, 小畑 有以

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    Applicant:大学共同利用機関法人情報・システム研究機構, 国立大学法人大阪大学

    Application no:JP2012079744  Date applied:2012.11

    Announcement no:WO2013-073653  Date announced:2013.5

    J-GLOBAL

  2. 真核細胞におけるタンパク質分解誘導方法

    鐘巻 将人, 西村 浩平, 滝澤 温彦, 三村 覚, 小畑 有以

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    Applicant:大学共同利用機関法人情報・システム研究機構, 国立大学法人大阪大学

    Application no:特願2013-544335  Date applied:2012.11

    Patent/Registration no:特許第6021116号  Date issued:2016.10

    J-GLOBAL

  3. 哺乳類細胞におけるタンパク質分解誘導方法

    鐘巻 将人, 柿本 辰男, 西村 浩平, 滝澤 温彦, 深川 竜郎

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    Applicant:国立大学法人大阪大学, 大学共同利用機関法人情報・システム研究機構

    Application no:JP2009005863  Date applied:2009.11

    Announcement no:WO2010-125620  Date announced:2010.11

    J-GLOBAL

  4. 哺乳類細胞におけるタンパク質分解誘導方法

    鐘巻 将人, 柿本 辰男, 西村 浩平, 滝澤 温彦, 深川 竜郎

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    Applicant:国立大学法人大阪大学, 大学共同利用機関法人情報・システム研究機構

    Application no:特願2011-511193  Date applied:2009.11

    Patent/Registration no:特許第5605658号  Date issued:2014.9

    J-GLOBAL

  5. タンパク質分解誘導性細胞、その製造方法、および、タンパク質分解の制御方法

    鐘巻 将人, 柿本 辰男, 西村 浩平, 滝澤 温彦

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    Applicant:国立大学法人大阪大学

    Application no:特願2007-026163  Date applied:2007.2

    Announcement no:特開2008-187958  Date announced:2008.8

    Patent/Registration no:特許第5250811号  Date issued:2013.4

    J-GLOBAL

 

Teaching Experience (Off-campus) 4

  1. Special lecture of biology II

    2023.10 Nagoya University)

  2. Basic biochemistry Ib

    2023.4 Nagoya University)

  3. 最先端生命科学持論

    2020.11 Chiba Institute of Technology)

  4. Genetics Ia

    2020.4 Nagoya University)