遺伝学を用いたセントロメアおよびヘテロクロマチン構造の形成機構の解析

• Project/Area Number: 17687002
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Genetics/Genome dynamics
• Research Institution: National Institute of Genetics
• Principal Investigator: 深川 竜郎 National Institute of Genetics, 分子遺伝研究系, 准教授 (60321600)
• Project Period (FY): 2005 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥30,290,000 (Direct Cost: ¥23,300,000、Indirect Cost: ¥6,990,000); Fiscal Year 2007: ¥8,840,000 (Direct Cost: ¥6,800,000、Indirect Cost: ¥2,040,000); Fiscal Year 2006: ¥9,880,000 (Direct Cost: ¥7,600,000、Indirect Cost: ¥2,280,000); Fiscal Year 2005: ¥11,570,000 (Direct Cost: ¥8,900,000、Indirect Cost: ¥2,670,000)
• Keywords: セントロメア / DT40細胞 / 染色体分配 / キネトコア / RNAiマシーナリー / CENPs / プロテオミクス / 細胞分裂

Research Abstract

染色体複製・分配のような基本的な生体反応に狂いが生じると染色体の異数化、がん化など細胞に対する悪影響が生じる。したがって、染色体複製や分配の正確な分子機構を解明することは、遺伝学における本質的な課題の一つである。本研究では、主にプロテオミクスを用いて新規セントロメアタンパク質を同定してセントロメアを構成する巨大複合体の実体を知ることを目的としている。また、同時にセントロメアやその近傍のヘテロクロマチンの形成に必要な分子機構の解明を試みといる。これまでの研究で、ヒトおよびニワトリ細胞からセントロメアタンパク質CENP-HおよびCENP-Iを含むCENP-H/I複合体に少なくも13種類のタンパク質を含むことが明らかにできた。19年度は、更なるセントロメタンパク質の同定に成功し、CENP-H/I複合体が他のセントロメアタンパク質複合体とどのような関係にあるのかを明らかにできた。また、セントロメアやその近傍のヘテロクロマチンの重要な働きを担う可能性がAgo3遺伝子およびAgo3/Ago4のダブルノックアウト細胞を作成し、ノックアウト細胞において、ヘテロクロマチン形成の必要と思われる遺伝子の発現およびタンパク質量が変化していることを明らかにできた。

Research Products

• [Journal Article] CENP-O-class proteins form a stable complex and are required for proper kinetochore function. (2008)
  • Author(s): Tetsuya Hori
  • Journal Title: Molecular Biology of the Cell 19 Pages: 843-854
  • Peer Reviewed
• [Journal Article] KNL1 and CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates. (2008)
  • Author(s): Iain M. Cheeseman
  • Journal Title: Molecular Biology of the Cell 19 Pages: 587-594
  • Peer Reviewed
• [Journal Article] The kinetochore and spindle checkpoint in vertebrate cells. (2008)
  • Author(s): Tatsuo Fukagawa
  • Journal Title: Frontiers in Bioscience 13 Pages: 2705-2713
  • Peer Reviewed
• [Journal Article] CENP-C is involved in chromosome segregation, mitotic checkpoint function and kinetochore assembly. (2007)
  • Author(s): Mi-Sun Kwon
  • Journal Title: Molecular Biology of the Cell 18 Pages: 2155-2168
  • Peer Reviewed
• [Journal Article] The human Nup107-160 nuclear pore subcomplex contributes to proper kinetochore functions. (2007)
  • Author(s): Michela Zuccolo
  • Journal Title: The EMBO Journal 26 Pages: 1853-1864
  • Peer Reviewed
• [Journal Article] Essential Role of CAF-1-mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells. (2007)
  • Author(s): Yasunari Takami
  • Journal Title: Molecular Biology of the Cell 18 Pages: 129-141
  • Peer Reviewed
• [Journal Article] Purification of a protein complex that associates with chromatin. (2006)
  • Author(s): Masahiro Okada
  • Journal Title: Nature Protocols DOI:10.1038/nprot.2006
• [Journal Article] A novel histone-exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A/H2B. (2006)
  • Author(s): Hiroshi Kimura
  • Journal Title: Journal of Cell Biology 175 Pages: 389-400
• [Journal Article] The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. (2006)
  • Author(s): Masahiro Okada
  • Journal Title: Nature Cell Biology 8 Pages: 446-457
• [Journal Article] The Human Mis12 Complex is Required for Kinetochore Assembly and Proper Chromosome Segregation. (2006)
  • Author(s): Susan Kline
  • Journal Title: Journal of Cell Biology 173 Pages: 9-17
• [Journal Article] Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells. (2006)
  • Author(s): Fumiyuki Sanematsu
  • Journal Title: Journal of Biological Chemistry 281 Pages: 13817-13827
• [Journal Article] The Human Mis12 Complex is Required for Kinetochore Assembly and Proper Chromosome Segregation. (2006)
  • Author(s): Susan Kline-Smith
  • Journal Title: Journal of Cell Biology (in press)
• [Journal Article] Cell cycle dependent pharmacology of methotrexate in HL-60. (2005)
  • Author(s): Atsushi Yamauchi
  • Journal Title: Journal of Pharmacological Sciences 99 Pages: 335-341
• [Journal Article] The Constitutive Centromere Component CENP-50 is Required for Recovery from Spindle damage. (2005)
  • Author(s): Yukinori Minoshima
  • Journal Title: Molecular and Cellular Biology 25 Pages: 10315-10328
• [Journal Article] CENP-A depletion leads to impaired mitotic checkpoint maintenace in vertebrate cells. (2005)
  • Author(s): Vinciane Regnier
  • Journal Title: Molecular and Cellular Biology 25 Pages: 3967-3981
• [Journal Article] Functional region of CENP-H interacts with Nuf2 complex, which functions as a connector between the inner and outer kinetochores. (2005)
  • Author(s): Yoshikazu Mikami
  • Journal Title: Molecular and Cellular Biology 25 Pages: 1958-1970
• [Journal Article] Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. (2005)
  • Author(s): Tomoko Motohashi
  • Journal Title: Biochemical and Biophysical Research Communications 326 Pages: 564-569
• [Presentation] CENP-H/I複合体のサブ複合体であるCENP-Oクラスの性質 (2008)
  • Author(s): 深川竜郎
  • Organizer: 第25回染色体ワークショップ
  • Place of Presentation: 熱海市
  • Year and Date: 2008-02-01
• [Presentation] クロマチン工学への応用に向けたきネトコアタンパク質複合体の再構成 (2007)
  • Author(s): 堀哲也
  • Organizer: BMB2007(第30回日本分子生物学会年会)
  • Place of Presentation: 横浜市
  • Year and Date: 2007-12-15
• [Presentation] Functional role of CENP-H/I complex in the kinetochore (2007)
  • Author(s): Tatsuo Fukagawa
  • Organizer: BMB2007(第30回日本分子生物学会年会)
  • Place of Presentation: 横浜市
  • Year and Date: 2007-12-12
• [Presentation] Functional role of CENP-Q-class proteins in the kinetochore. (2007)
  • Author(s): Tatsuo Fukagawa
  • Organizer: 16th International Chromosome Conference
  • Place of Presentation: Amsterdam
  • Year and Date: 2007-08-28
• [Presentation] CENP-50ノックアウトマウスの染色体動態 (2007)
  • Author(s): 深川竜郎
  • Organizer: 日本遺伝学会第79回大会
  • Place of Presentation: 岡山市