2021/10/20 更新

写真a

フクダ トモユキ
福田 智行
FUKUDA Tomoyuki
所属
教育研究院 医歯学系 医学系列 准教授
医歯学総合研究科 生体機能調節医学専攻 腎科学 准教授
職名
准教授
外部リンク

学位

  • 博士(生命科学) ( 2005年3月   東京大学 )

研究キーワード

  • 細胞周期

  • 減数分裂

  • 細胞増殖

  • 染色体

  • オートファジー

  • シグナル伝達

  • 細胞分裂

  • 生命科学

  • オルガネラ

  • 細胞分化

研究分野

  • ライフサイエンス / 細胞生物学

  • ライフサイエンス / 機能生物化学

  • ライフサイエンス / 遺伝学

  • ライフサイエンス / 分子生物学

経歴(researchmap)

  • 新潟大学大学院   医歯学総合研究科   准教授

    2016年4月 - 現在

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  • 奈良先端科学技術大学院大学   バイオサイエンス研究科   助教

    2013年3月 - 2016年3月

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  • カロリンスカ研究所   細胞分子生物学部門   シニアラボマネージャー

    2011年4月 - 2013年2月

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  • カロリンスカ研究所   細胞分子生物学部門   博士研究員

    2008年4月 - 2011年3月

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  • 理化学研究所   基礎科学特別研究員

    2005年4月 - 2008年3月

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経歴

  • 新潟大学   医歯学総合研究科 生体機能調節医学専攻 腎科学   准教授

    2016年4月 - 現在

学歴

  • 東京大学大学院   新領域創成科学研究科   先端生命科学専攻

    2000年4月 - 2005年3月

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  • 京都大学   理学部

    1996年4月 - 2000年3月

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所属学協会

 

論文

  • Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis. 国際誌

    Tatsuro Maruyama, Jahangir Md Alam, Tomoyuki Fukuda, Shun Kageyama, Hiromi Kirisako, Yuki Ishii, Ichio Shimada, Yoshinori Ohsumi, Masaaki Komatsu, Tomotake Kanki, Hitoshi Nakatogawa, Nobuo N Noda

    Nature structural & molecular biology   2021年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

    DOI: 10.1038/s41594-021-00614-5

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  • Multiplexed suppression of TOR complex 1 induces autophagy during starvation. 国際誌

    Tomoyuki Fukuda, Kazuhiro Shiozaki

    Autophagy   1 - 2   2021年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Target of rapamycin complex 1 (TORC1) promotes cellular anabolism and suppresses macroautophagy/autophagy. In mammalian cells starved of amino acid, the GATOR1 complex, a negative regulator of TORC1, is released from its inhibitor GATOR2 and inactivates TORC1. We have recently identified the evolutionarily conserved GATOR2 components in fission yeast including Sea3, an ortholog of mammalian WDR59, but, unexpectedly, Sea3 acts as a part of GATOR1 to suppress TORC1. Moreover, fission yeast GATOR1 is not required for the amino-acid starvation-induced TORC1 attenuation, which is instead mediated by the Gcn2 pathway. Conversely, absence of a nitrogen source suppresses TORC1 in a manner dependent on GATOR1 as well as the Tsc1-Tsc2 complex, whose mammalian equivalent functions as a growth-factor sensitive TORC1 inhibitor. Thus, the evolutionarily conserved signaling modules are utilized differently between fission yeast and mammals to control TORC1 activity and autophagy.

    DOI: 10.1080/15548627.2021.1938915

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  • Mitophagy reporter mouse analysis reveals increased mitophagy activity in disuse-induced muscle atrophy. 国際誌

    Shun-Ichi Yamashita, Masanao Kyuuma, Keiichi Inoue, Yuki Hata, Ryu Kawada, Masaki Yamabi, Yasuyuki Fujii, Junko Sakagami, Tomoyuki Fukuda, Kentaro Furukawa, Satoshi Tsukamoto, Tomotake Kanki

    Journal of cellular physiology   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

    DOI: 10.1002/jcp.30404

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  • Atg43, a novel autophagy-related protein, serves as a mitophagy receptor to bridge mitochondria with phagophores in fission yeast. 国際誌

    Tomoyuki Fukuda, Tomotake Kanki

    Autophagy   17 ( 3 )   826 - 827   2021年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Mitophagy is a selective type of autophagy in which damaged or unnecessary mitochondria are sequestered by double-membranous structures called phagophores and delivered to vacuoles/lysosomes for degradation. The molecular mechanisms underlying mitophagy have been studied extensively in budding yeast and mammalian cells. To gain more diverse insights, our recent study identified Atg43 as a mitophagy receptor in the fission yeast Schizosaccharomyces pombe. Atg43 is localized on the mitochondrial outer membrane through the Mim1-Mim2 complex and binds to Atg8, a ubiquitin-like protein conjugated to phagophore membranes. Artificial tethering of Atg8 to mitochondria can bypass the requirement of Atg43 for mitophagy, suggesting that the main role of Atg43 in mitophagy is to stabilize phagophore expansion on mitochondria by interacting with Atg8. Atg43 shares no sequence similarity with mitophagy receptors in other organisms and has a mitophagy-independent function, raising the possibility that Atg43 has acquired the mitophagic function by convergent evolution.

    DOI: 10.1080/15548627.2021.1874662

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  • Tripartite suppression of fission yeast TORC1 signaling by the GATOR1-Sea3 complex, the TSC complex, and Gcn2 kinase. 国際誌

    Tomoyuki Fukuda, Fajar Sofyantoro, Yen Teng Tai, Kim Hou Chia, Takato Matsuda, Takaaki Murase, Yuichi Morozumi, Hisashi Tatebe, Tomotake Kanki, Kazuhiro Shiozaki

    eLife   10   2021年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.

    DOI: 10.7554/eLife.60969

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  • Association and dissociation between the mitochondrial Far complex and Atg32 regulate mitophagy. 国際誌

    Aleksei Innokentev, Kentaro Furukawa, Tomoyuki Fukuda, Tetsu Saigusa, Keiichi Inoue, Shun-Ichi Yamashita, Tomotake Kanki

    eLife   9   2020年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.

    DOI: 10.7554/eLife.63694

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  • Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast. 査読 国際誌

    Tomoyuki Fukuda, Yuki Ebi, Tetsu Saigusa, Kentaro Furukawa, Shun-Ichi Yamashita, Keiichi Inoue, Daiki Kobayashi, Yutaka Yoshida, Tomotake Kanki

    eLife   9   2020年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Degradation of mitochondria through mitophagy contributes to the maintenance of mitochondrial function. In this study, we identified that Atg43, a mitochondrial outer membrane protein, serves as a mitophagy receptor in the model organism Schizosaccharomyces pombe to promote the selective degradation of mitochondria. Atg43 contains an Atg8-family-interacting motif essential for mitophagy. Forced recruitment of Atg8 to mitochondria restores mitophagy in Atg43-deficient cells, suggesting that Atg43 tethers expanding isolation membranes to mitochondria. We found that the mitochondrial import factors, including the Mim1-Mim2 complex and Tom70, are crucial for mitophagy. Artificial mitochondrial loading of Atg43 bypasses the requirement of the import factors, suggesting that they contribute to mitophagy through Atg43. Atg43 not only maintains growth ability during starvation but also facilitates vegetative growth through its mitophagy-independent function. Thus, Atg43 is a useful model to study the mechanism and physiological roles, as well as the origin and evolution, of mitophagy in eukaryotes.

    DOI: 10.7554/eLife.61245

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  • Gemcitabine induces Parkin-independent mitophagy through mitochondrial-resident E3 ligase MUL1-mediated stabilization of PINK1. 査読 国際誌

    Ryoko Igarashi, Shun-Ichi Yamashita, Tomohiro Yamashita, Keiichi Inoue, Tomoyuki Fukuda, Takeo Fukuchi, Tomotake Kanki

    Scientific reports   10 ( 1 )   1465 - 1465   2020年1月

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    記述言語:英語  

    Mitophagy plays an important role in the maintenance of mitochondrial homeostasis. PTEN-induced kinase (PINK1), a key regulator of mitophagy, is degraded constitutively under steady-state conditions. During mitophagy, it becomes stabilized in the outer mitochondrial membrane, particularly under mitochondrial stress conditions, such as in treatment with uncouplers, generation of excessive mitochondrial reactive oxygen species, and formation of protein aggregates in mitochondria. Stabilized PINK1 recruits and activates E3 ligases, such as Parkin and mitochondrial ubiquitin ligase (MUL1), to ubiquitinate mitochondrial proteins and induce ubiquitin-mediated mitophagy. Here, we found that the anticancer drug gemcitabine induces the stabilization of PINK1 and subsequent mitophagy, even in the absence of Parkin. We also found that gemcitabine-induced stabilization of PINK1 was not accompanied by mitochondrial depolarization. Interestingly, the stabilization of PINK1 was mediated by MUL1. These results suggest that gemcitabine induces mitophagy through MUL1-mediated stabilization of PINK1 on the mitochondrial membrane independently of mitochondrial depolarization.

    DOI: 10.1038/s41598-020-58315-w

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  • The PP2A-like Protein Phosphatase Ppg1 and the Far Complex Cooperatively Counteract CK2-Mediated Phosphorylation of Atg32 to Inhibit Mitophagy 査読

    Kentaro Furukawa, Tomoyuki Fukuda, Shun-ichi Yamashita, Tetsu Saigusa, Yusuke Kurihara, Yutaka Yoshida, Hiromi Kirisako, Hitoshi Nakatogawa, Tomotake Kanki

    Cell Reports   23 ( 12 )   3579 - 3590   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier B.V.  

    Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151–200) of Atg32 caused the same phenotypes as in ppg1Δ cells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy. Mitophagy in yeast is initiated by CK2-mediated phosphorylation of the mitophagy receptor Atg32. However, how this phosphorylation is prevented under non-mitophagy-inducing conditions is unclear. Furukawa et al. show that the PP2A-like protein phosphatase Ppg1 and the Far complex negatively regulate mitophagy by counteracting CK2-mediated phosphorylation of Atg32.

    DOI: 10.1016/j.celrep.2018.05.064

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  • H2AFX and MDC1 promote maintenance of genomic integrity in male germ cells 査読

    Erika Testa, Daniela Nardozi, Cristina Antinozzi, Monica Faieta, Stefano Di Cecca, Cinzia Caggiano, Tomoyuki Fukuda, Elena Bonanno, Lou Zhenkun, Andros Maldonado, Ignasi Roig, Monica Di Giacomo, Marco Barchi

    Journal of Cell Science   131 ( 6 )   2018年3月

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    記述言語:英語   出版者・発行元:Company of Biologists Ltd  

    In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx-/- spermatocytes, we found that the synapsis of autosomes and X-Y chromosomes was impaired in a fraction of cells. Such defects correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes and played only a minor role in X-Y synapsis, compared with the action of H2afx. This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we show that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data show that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity.

    DOI: 10.1242/jcs.214411

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  • Mechanisms and Physiological Roles of Mitophagy in Yeast. 査読

    Fukuda T, Kanki T

    Molecules and cells   41 ( 1 )   35 - 44   2018年1月

  • The Rag GTPase-Ragulator complex attenuates TOR complex 1 signaling in fission yeast. 査読

    Fukuda T, Shiozaki K

    Autophagy   14 ( 6 )   1105 - 1106   2018年

  • Ragulator and GATOR1 complexes promote fission yeast growth by attenuating TOR complex 1 through rag GTPases 査読

    Kim Hou Chia, Tomoyuki Fukuda, Fajar Sofyantoro, Takato Matsuda, Takamitsu Amai, Kazuhiro Shiozaki

    eLife   6   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:eLife Sciences Publications Ltd  

    TOR complex 1 (TORC1) is an evolutionarily conserved protein kinase complex that promotes cellular macromolecular synthesis and suppresses autophagy. Amino-acid-induced activation of mammalian TORC1 is initiated by its recruitment to the RagA/B-RagC/D GTPase heterodimer, which is anchored to lysosomal membranes through the Ragulator complex. We have identified in the model organism Schizosaccharomyces pombe a Ragulator-like complex that tethers the Gtr1-Gtr2 Rag heterodimer to the membranes of vacuoles, the lysosome equivalent in yeasts. Unexpectedly, the Ragulator-Rag complex is not required for the vacuolar targeting of TORC1, but the complex plays a crucial role in attenuating TORC1 activity independently of the Tsc1-Tsc2 complex, a known negative regulator of TORC1 signaling. The GATOR1 complex, which functions as Gtr1 GAP, is essential for the TORC1 attenuation by the Ragulator-Rag complex, suggesting that Gtr1GDP-Gtr2 on vacuolar membranes moderates TORC1 signaling for optimal cellular response to nutrients.

    DOI: 10.7554/eLife.30880

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  • The central element of the synaptonemal complex in mice is organized as a bilayered junction structure 査読

    Abrahan Hernandez-Hernandez, Sergej Masich, Tomoyuki Fukuda, Anna Kouznetsova, Sara Sandin, Bertil Daneholt, Christer Hoog

    JOURNAL OF CELL SCIENCE   129 ( 11 )   2239 - 2249   2016年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COMPANY OF BIOLOGISTS LTD  

    The synaptonemal complex transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the synaptonemal complex is mediated through integration of opposing transverse filaments into a central element, a process that is poorly understood. We have, here, analyzed the localization of the transverse filament protein SYCP1 and the central element proteins SYCE1, SYCE2 and SYCE3 within the central region of the synaptonemal complex in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the synaptonemal complex, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure, and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth central element protein) localization to the central element abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four central element proteins, in an interdependent manner, contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered transverse-filament-central-element junction structure that promotes synaptonemal complex formation and synapsis.

    DOI: 10.1242/jcs.182477

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  • Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes 査読

    Rocio Gomez, Natalia Felipe-Medina, Miguel Ruiz-Torres, Ines Berenguer, Alberto Viera, Sara Perez, Jose Luis Barbero, Elena Llano, Tomoyuki Fukuda, Manfred Alsheimer, Alberto M. Pendas, Ana Losada, Jose A. Suja

    EMBO REPORTS   17 ( 5 )   695 - 707   2016年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II. The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.

    DOI: 10.15252/embr.201541060

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  • STAG3-mediated stabilization of REC8 cohesin complexes promotes chromosome synapsis during meiosis 査読

    Tomoyuki Fukuda, Nanaho Fukuda, Ana Agostinho, Abrahan Hernandez-Hernandez, Anna Kouznetsova, Christer Hoog

    EMBO JOURNAL   33 ( 11 )   1243 - 1255   2014年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Cohesion between sister chromatids in mitotic and meiotic cells is promoted by a ring-shaped protein structure, the cohesin complex. The cohesin core complex is composed of four subunits, including two structural maintenance of chromosome (SMC) proteins, one alpha-kleisin protein, and one SA protein. Meiotic cells express both mitotic and meiosis-specific cohesin core subunits, generating cohesin complexes with different subunit composition and possibly separate meiotic functions. Here, we have analyzed the in vivo function of STAG3, a vertebrate meiosis-specific SA protein. Mice with a hypomorphic allele of Stag3, which display a severely reduced level of STAG3, are viable but infertile. We show that meiocytes in homozygous mutant Stag3 mice display chromosome axis compaction, aberrant synapsis, impaired recombination and developmental arrest. We find that the three different alpha- kleisins present in meiotic cells show different dosage-dependent requirements for STAG3 and that STAG3-REC8 cohesin complexes have a critical role in supporting meiotic chromosome structure and functions.

    DOI: 10.1002/embj.201387329

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  • Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling 査読

    Yuval Tabach, Tamar Golan, Abrahan Hernandez-Hernandez, Arielle R. Messer, Tomoyuki Fukuda, Anna Kouznetsova, Jian-Guo Liu, Ingrid Lilienthal, Carmit Levy, Gary Ruvkun

    MOLECULAR SYSTEMS BIOLOGY   9   692   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor.

    DOI: 10.1038/msb.2013.50

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  • The Transacting Factor CBF-A/Hnrnpab Binds to the A2RE/RTS Element of Protamine 2 mRNA and Contributes to Its Translational Regulation during Mouse Spermatogenesis 査読

    Nanaho Fukuda, Tomoyuki Fukuda, John Sinnamon, Abrahan Hernandez-Hernandez, Manizheh Izadi, Chandrasekhar S. Raju, Kevin Czaplinski, Piergiorgio Percipalle

    PLOS GENETICS   9 ( 10 )   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3' UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5' cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs.

    DOI: 10.1371/journal.pgen.1003858

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  • Dynamic localization of SMC5/6 complex proteins during mammalian meiosis and mitosis suggests functions in distinct chromosome processes 査読

    Rocio Gomez, Philip W. Jordan, Alberto Viera, Manfred Alsheimer, Tomoyuki Fukuda, Rolf Jessberger, Elena Llano, Alberto M. Pendas, Mary Ann Handel, Jose A. Suja

    JOURNAL OF CELL SCIENCE   126 ( 18 )   4239 - 4252   2013年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COMPANY OF BIOLOGISTS LTD  

    Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase II alpha, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase II alpha colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase II alpha colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase II alpha, in regulating centromere cohesion during meiosis II.

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  • ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing. 査読

    Royo H, Prosser H, Ruzankina Y, Mahadevaiah SK, Cloutier JM, Baumann M, Fukuda T, Höög C, Tóth A, de Rooij DG, Bradley A, Brown EJ, Turner JM

    Genes & development   27 ( 13 )   1484 - 1494   2013年7月

  • Phosphorylation of Chromosome Core Components May Serve as Axis Marks for the Status of Chromosomal Events during Mammalian Meiosis 査読

    Tomoyuki Fukuda, Florencia Pratto, John C. Schimenti, James M. A. Turner, R. Daniel Camerini-Otero, Christer Hoog

    PLOS GENETICS   8 ( 2 )   e1002485   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin) machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation.

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  • The Mouse Cohesin-Associated Protein PDS5B Is Expressed in Testicular Cells and Is Associated with the Meiotic Chromosome Axes 査読

    Tomoyuki Fukuda, Christer Hoog

    GENES   1 ( 3 )   484 - 494   2010年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    During the first meiotic prophase, the cohesin complex is localized to the chromosome axis and contributes to chromosome organization, pairing, synapsis, and recombination. The PDS5 protein, an accessory factor of the cohesin complex, is known to be a component of meiotic chromosome cores in fungi and to be implicated in meiotic chromosome structure and function. We found by immunoblotting experiments that a mammalian PDS5 protein, PDS5B, is abundantly expressed in mouse testis compared to other tissues. Immunofluorescence labeling experiments revealed that PDS5B is highly expressed in spermatogonia and that most PDS5B is depleted from chromatin as cells enter meiosis. During the first meiotic prophase, PDS5B associates with the axial cores of chromosomes. The axial association of PDS5B was observed also in the absence of synaptonemal complex proteins, such as SYCP1 and SYCP3, suggesting that PDS5B is an integral part of the chromosome axis as defined by the cohesin complex. These results suggest that PDS5B modulates cohesin functions in spermatocytes as well as in spermatogonia, contributing to meiotic chromosome structure and function.

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  • A novel mammalian HORMA domain-containing protein, HORMAD1, preferentially associates with unsynapsed meiotic chromosomes 査読

    Tomoyuki Fukuda, Katrin Daniel, Lukasz Wojtasz, Attila Toth, Christer Hoog

    EXPERIMENTAL CELL RESEARCH   316 ( 2 )   158 - 171   2010年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER INC  

    HORMA domain-containing proteins regulate interactions between homologous chromosomes (homologs) during meiosis in a wide range of eukaryotes. We have identified a mouse HORMA domain-containing protein, HORMAD1, and biochemically and cytologically shown it to be associated with the meiotic chromosome axis. HORMAD1 first accumulates on the chromosomes during the leptotene to zygotene stages of meiotic prophase I. As germ cells progress into the pachytene stage, HORMAD1 disappears from the synapsed chromosomal regions. However, once the chromosomes desynapse during the diplotene stage, HORMAD1 again accumulates on the chromosome axis of the desynapsed homologs. HORMAD1 thus preferentially localizes to unsynapsed or desynapsed chromosomal regions during the prophase I stage of meiosis. Analysis of mutant strains lacking different components of the synaptonemal complex (SC) revealed that establishment of the SC is required for the displacement of HORMAD1 from the chromosome axis. Our results therefore strongly suggest that also mammalian cells use a HORMA domain-containing protein as part of a surveillance system that monitors synapsis or other interactions between homologs. (C) 2009 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.yexcr.2009.08.007

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  • Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes 査読

    Kazuto Kugou, Tomoyuki Fukuda, Shintaro Yamada, Masaru Ito, Hiroyuki Sasanuma, Saori Mori, Yuki Katou, Takehiko Itoh, Kouji Matsumoto, Takehiko Shibata, Katsuhiko Shirahige, Kunihiro Ohta

    MOLECULAR BIOLOGY OF THE CELL   20 ( 13 )   3064 - 3076   2009年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC CELL BIOLOGY  

    Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.

    DOI: 10.1091/mbc.E08-12-1223

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  • Analysis of chromatin structure at meiotic DSB sites in yeasts 査読

    Kouji Hirota, Tomoyuki Fukuda, Takatomi Yamada, Kunihiro Ohta

    Methods in Molecular Biology   557   253 - 266   2009年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion of chromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.

    DOI: 10.1007/978-1-59745-527-5_16

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  • Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination 査読

    Tomoyuki Fukuda, Kazuto Kugou, Hiroyuki Sasanuma, Takehiko Shibata, Kunihiro Ohta

    NUCLEIC ACIDS RESEARCH   36 ( 3 )   984 - 997   2008年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.

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  • Cdc7-dependent phosphorylation of Mer2 facilitates initiation of yeast meiotic recombination 査読

    Hiroyuki Sasanuma, Kouji Hirota, Tomoyuki Fukuda, Naoko Kakusho, Kazuto Kugou, Yasuo Kawasaki, Takehiko Shibata, Hisao Masai, Kunihiro Ohta

    GENES & DEVELOPMENT   22 ( 3 )   398 - 410   2008年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

    Meiosis ensures genetic diversification of gametes and sexual reproduction. For successful meiosis, multiple events such as DNA replication, recombination, and chromosome segregation must occur coordinately in a strict regulated order. We investigated the meiotic roles of Cdc7 kinase in the initiation of meiotic recombination, namely, DNA double-strand breaks (DSBs) mediated by Spo11 and other coactivating proteins. Genetic analysis using bob1-1 cdc7 Delta reveals that Cdc7 is essential for meiotic DSBs and meiosis I progression. We also demonstrate that the N-terminal region of Mer2, a Spo11 ancillary protein required for DSB formation and phosphorylated by cyclin-dependent kinase (CDK), contains two types of Cdc7-dependent phosphorylation sites near the CDK site (Ser30): One (Ser29) is essential for meiotic DSB formation, and the others exhibit a cumulative effect to facilitate DSB formation. Importantly, mutations on these sites confer severe defects in DSB formation even when the CDK phosphorylation is present at Ser30. Diploids of cdc7 Delta display defects in the chromatin binding of not only Spo11 but also Rec114 and Mei4, other meiotic coactivators that may assist Spo11 binding to DSB hot spots. We thus propose that Cdc7, in concert with CDK, regulates Spo11 loading to DSB sites via Mer2 phosphorylation.

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  • Conditional genomic rearrangement by designed meiotic recombination using VDE (PI-SceI) in yeast 査読

    Tomoyuki Fukuda, Yoshikazu Ohya, Kunihiro Ohta

    MOLECULAR GENETICS AND GENOMICS   278 ( 4 )   467 - 478   2007年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Meiotic recombination plays critical roles in the acquisition of genetic diversity and has been utilized for conventional breeding of livestock and crops. The frequency of meiotic recombination is normally low, and is extremely low in regions called "recombination cold domains". Here, we describe a new and highly efficient method to modulate yeast meiotic gene rearrangements using VDE (PI-SceI), an intein-encoded endonuclease that causes an efficient unidirectional meiotic gene conversion at its recognition sequence (VRS). We designed universal targeting vectors, by use of which the strain that inserts the VRS at a desired site is acquired. Meiotic induction of the strains provided unidirectional gene conversions and frequent genetic rearrangements of flanking genes with little impact on cell viability. This system thus opens the way for the designed modulation of meiotic gene rearrangements, regardless of recombinational activity of chromosomal domains. Finally, the VDE-VRS system enabled us to conduct meiosis-specific conditional knockout of genes where VDE-initiated gene conversion disrupts the target gene during meiosis, serving as a novel approach to examine the functions of genes during germination of resultant spores.

    DOI: 10.1007/s00438-007-0264-7

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  • Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114 査読

    Hiroyuki Sasanuma, Hajime Murakami, Tomoyuki Fukuda, Takehiko Shibata, Alain Nicolas, Kunihiro Ohta

    NUCLEIC ACIDS RESEARCH   35 ( 4 )   1119 - 1133   2007年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11 at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.

    DOI: 10.1093/nar/gkl1162

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  • Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site 査読

    T Fukuda, K Ohta, Y Ohya

    EUKARYOTIC CELL   5 ( 6 )   981 - 990   2006年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

    VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.

    DOI: 10.1128/EC.00052-06

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  • Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI). 査読 国際誌

    Tomoyuki Fukuda, Yoshikazu Ohya

    Molecular genetics and genomics : MGG   275 ( 2 )   204 - 14   2006年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

    DOI: 10.1007/s00438-005-0078-4

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  • High-dimensional and large-scale phenotyping of yeast mutants 査読

    Y Ohya, J Sese, M Yukawa, F Sano, Y Nakatani, TL Saito, A Saka, T Fukuda, S Ishihara, S Oka, G Suzuki, M Watanabe, A Hirata, M Ohtani, H Sawai, N Fraysse, JP Latge, JM Francois, M Aebi, S Tanaka, S Muramatsu, H Araki, K Sonoike, S Nogami, S Morishita

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   102 ( 52 )   19015 - 19020   2005年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    One of the most powerful techniques for attributing functions to genes in uni- and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits. Deletion of nearly half of the yeast genes not essential for growth affects these morphological traits. Similar morphological phenotypes are caused by deletions of functionally related genes, enabling a functional assignment of a locus to a specific cellular pathway. The high-dimensional phenotypic analysis of defined yeast mutant strains provides another step toward attributing gene function to all of the genes in the yeast genome.

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  • Molecular mechanism of VDE-initiated intein homing in yeast nuclear genome 査読

    Tomoyuki Fukuda, Yuri Nagai, Yoshikazu Ohya

    Advances in Biophysics   38   215 - 232   2004年

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    記述言語:英語   出版者・発行元:Japan Scientific Societies Press  

    In Saccharomyces cerevisiae, VMA1 intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMA1 intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spo11p-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDE-initiated homing in yeast cells.

    DOI: 10.1016/S0065-227X(04)80181-3

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  • VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner 査読

    T Fukuda, S Nogami, Y Ohya

    GENES TO CELLS   8 ( 7 )   587 - 602   2003年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL PUBLISHING LTD  

    Background: Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae , the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus.
    Results: We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5Delta clb6Delta mutation reduces VDE-mediated DSBs.
    Conclusion: The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.

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  • Homing at an extragenic locus mediated by VDE (PI-Scel) in Saccharomyces cerevisiae 査読

    S Nogami, T Fukuda, Y Nagai, S Yabe, M Sugiura, R Mizutani, Y Satow, Y Anraku, Y Ohya

    YEAST   19 ( 9 )   773 - 782   2002年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JOHN WILEY & SONS LTD  

    PI-SceI (VDE), a homing endonuclease with protein splicing activity, is a genomic parasite in the VMA1 gene of Saccharomyces cerevisiae. In a heterozygous diploid of the VDE-less VMA1 allele and a VDE-containing VMA1 allele, VDE specifically cleaves its recognition sequence (VRS) in the VDE-less VMA1 allele at meiosis, followed by 'homing', i.e. a conversion to a VDE-containing allele. We found that upon VDE expression, homing of a marker gene at an extragenic locus occurs only when a 45 bp element containing the VRS is inserted at its allelic site, while mutants of VDE with no endonuclease activity lack authentic extragenic homing activity. Thus, both the VRS and VDE are required for homing. Insertion of the VRS in a homozygous diploid significantly lowered the spore germination ability, indicating that a template for gene repair at its allelic locus is essential for efficient homing and survival of yeast cells. Copyright (C) 2002 John Wiley Sons, Ltd.

    DOI: 10.1002/yea.872

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  • Viability of Escherichia coli cells under long-term cultivation in a rich nutrient medium 査読

    T Fukuda, K Nakahigashi, H Inokuchi

    GENES & GENETIC SYSTEMS   76 ( 5 )   271 - 278   2001年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:GENETICS SOC JAPAN  

    We investigated the viability of Escherichia coli cells during long-term cultivation in Brain Heart Infusion (BHI) medium and observed that the number of viable cells increased, then decreased, and increased again, in this medium, and finally the cells died out within about 10 days. This cell death may result from an increase in the pH of the medium. After repeated cultivation in BHI, bacterial cells that did not die out even under conditions of further cultivation were obtainable from cultures showing a stabilized viable count. We propose that long-term cultivation in BHI medium is a good system for studying growth phase-specific events in E. coli cells, because the total life-cycle of a population of E. coli, including exponential growth, stationary phase, and extinction, can be seen during a period of only about 10 days. Also, this system clearly allows detection of a phenotype that may not be detectable in other commonly used media. Moreover, in this report, we show that mutants displaying the GASP (growth advantage in stationary phase) phenotype appear at high frequency under long-term cultivation conditions.

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  • 酵母のすべて (系統, 細胞から分子まで)

    福田智行, 太田邦史( 担当: 分担執筆 ,  範囲: 組換え、修復、突然変異)

    シュプリンガー・ジャパン  2007年9月 

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MISC

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受賞

  • 会長賞

    2019年9月   酵母遺伝学フォーラム  

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  • キャッチコピー「無限の可能性、ここが最先端」採用

    2013年11月   奈良先端科学技術大学院大学  

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  • ポスター賞

    2009年10月   Solbacka 2009 (14th Karolinska Institutet CMB/Ludwig Conference)  

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  • 日本学術振興会海外特別研究員

    2009年4月  

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  • 海外留学助成ポストドクトラルフェローシップ

    2008年4月   上原記念生命科学財団  

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  • 博士論文特別賞

    2005年3月   東京大学大学院新領域創成科学研究科先端生命科学専攻  

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  • 日本学術振興会特別研究員(DC1)

    2002年4月  

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  • IB賞(最優秀修士論文)

    2002年3月   東京大学大学院新領域創成科学研究科先端生命科学専攻  

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共同研究・競争的資金等の研究

  • マイトファジーによるミトコンドリア分解の機構と生理機能の解明

    2021年11月 - 2024年5月

    公益財団法人武田科学振興財団  2021年度医学系研究継続助成(基礎) 

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  • 選択的オートファジーによるオルガネラ分解の機構と意義の解明

    研究課題/領域番号:20K06552  2020年4月 - 2023年3月

    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)

    福田 智行

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    配分額:4290000円 ( 直接経費:3300000円 、 間接経費:990000円 )

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  • マイトファジーによるミトコンドリア分解の機構と生理機能の解明

    2018年11月 - 2020年3月

    公益財団法人武田科学振興財団  2018年度医学系研究助成(基礎) 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • TOR複合体が刺激に応答して細胞増殖を制御する仕組みの解明

    2017年4月 - 2020年3月

    日本学術振興会  基盤研究(C) 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • TOR複合体シグナル伝達経路による細胞増殖と代謝の制御機構解明

    2014年11月 - 2015年10月

    公益財団法人鈴木謙三記念医科学応用研究財団  平成26年度調査研究助成金 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • 主要栄養シグナルを感知・統合するTORキナーゼ複合体ネットワーク

    研究課題/領域番号:26291024  2014年4月 - 2018年3月

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

    塩崎 一裕, 建部 恒, 福田 智行, 児嶋 長次郎

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    配分額:16510000円 ( 直接経費:12700000円 、 間接経費:3810000円 )

    TORキナーゼを含むTORC1およびTORC2複合体は、細胞の成長・増殖制御に中心的な役割を果たす。分裂酵母をモデルとして解析を行い、Rab様Gタンパク質が、グルコースに応答してTORC2を活性化することを発見した。また、TORC2のサブユニットの一つであるSin1のCRIMドメインが特異的にTORC2基質に結合することを示し、その立体構造を決定した。さらに、Rag様Gタンパク質のGtr1-Gtr2 ヘテロ二量体が、TORC1の活性を抑制することを発見した。この抑制制御には、Gtr1のGTPase-Activating Protein として働くGATOR1複合体が必須であった。

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  • TOR複合体の活性制御機構の解明

    2014年4月 - 2016年3月

    日本学術振興会  若手研究(B) 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • 哺乳類シナプトネマ複合体の分子ネットワークの解明

    2013年4月 - 2014年3月

    日本学術振興会  研究活動スタート支援 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • 可動性遺伝因子による部位特異的染色体切断を利用した組換えの研究

    2006年4月 - 2008年3月

    日本学術振興会  若手研究(スタートアップ) 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • 利己的遺伝子VDEによる染色体DNA切断を用いた減数分裂期組換えに関する研究

    2005年4月 - 2006年3月

    理化学研究所  平成17年度研究奨励ファンド 

    福田 智行

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    担当区分:研究代表者  資金種別:競争的資金

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  • 複製・分配・クロマチン構造と組換え開始の相互作用

    研究課題/領域番号:17080011  2005年 - 2009年

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    太田 邦史, 廣田 耕志, 福田 智行, ブランゼイ ダーナ, 山田 貴富

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    配分額:64700000円 ( 直接経費:64700000円 )

    組換えは遺伝情報の多様性獲得や子孫への継承に重要な役割を果たす。減数分裂においては、組換え酵素の活性化およびクロマチン構造の変化とともに組換えが顕著に上昇するが、その分子メカニズムに関しては不明な点が多い。本研究では、減数分裂期組換えの開始酵素であるDNA切断酵素Spo11およびその関連因子の役割を、DNA複製サイクルの調節因子との関係や、分子集合過程などの観点から明らかにした。また、組換えが開始される部位の決定に関しては、クロマチン再編成因子・ヒストン修飾・姉妹染色体結合・セントロメアなどとの関係を示した。加えて、ストレス応答遺伝子上流に頻繁に見出される組換えホットスポット配列を介して転写制御を受ける、新たな長鎖ノンコーディングRNAを発見し、そのストレス応答遺伝子発現におけるクロマチン再編成への役割を明らかにした。

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  • 出芽酵母の可動性DNA領域VDEがゲノム中で伝播する分子機構に関する研究

    研究課題/領域番号:02J08383  2002年 - 2004年

    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

    福田 智行

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    配分額:3000000円 ( 直接経費:3000000円 )

    出芽酵母の可動性DNA領域VDEは、コードする部位特異的エンドヌクレアーゼVDEが減数分裂期特異的に染色体の二重鎖切断を誘導し、宿主がこの切断を修復することでゲノム中を動く。これらの反応機構をより詳細に解析するために、1)VDEによって切断されたDNAとDNA修復系タンパク質との結合解析、2)VDEによる切断の減数分裂期特異性を産み出す機構の解析を行った。
    1)VDEによる二重鎖切断末端にはRecAホモログであるDmc1pとRad51pが互いに独立に結合することをクロマチン免疫沈降法により明らかにした。また、RAD52、RAD55、RAD57遺伝子破壊株においてはいずれもRad51pの結合に欠損が見られた。一方、SAE3遺伝子破壊株ではDmc1pの結合が欠損していた。以上の結果からRad51pはRad52p、Rad55p、Rad57pの働きにより、Dmc1pはSae3pの働きによりそれぞれ独立に二重鎖切断部位にリクルートされ、修復反応をおこなうことが示唆された。
    2)昨年度の解析によりVDEの認識配列付近のクロマチン構造は減数分裂期に変化していることを示したが、今年度は、種々の変異株を用いた解析により、このクロマチン構造の変化は減数分裂の進行に依存していることを明らかにした。また、VDEは減数分裂誘導前にも発現しており、エンドヌクレアーゼ活性も保持していることを確認した。一方、抗VDE抗体を用いたクロマチン免疫沈降法により、VDEは減数分裂期に初めて認識配列に結合し得ることを示した。従ってVDEの減数分裂期特異的な切断はaccessibilityにより制御されており、減数分裂期に見られたクロマチン構造の変化はこのaccessibilityの上昇を反映している可能性が示唆された。

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