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In-vitro reconstitution of mitotic chromosomes

Research Project

Project/Area Number 18H02381
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Review Section Basic Section 43010:Molecular biology-related
Research InstitutionInstitute of Physical and Chemical Research

Principal Investigator

Shintomi Keishi  国立研究開発法人理化学研究所, 開拓研究本部, 専任研究員 (60462694)

Project Period (FY) 2018-04-01 – 2021-03-31
Project Status Completed (Fiscal Year 2020)
Budget Amount *help
¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000)
Fiscal Year 2020: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2019: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2018: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Keywords染色体 / 再構成 / トポⅡ / コンデンシン / カエル卵抽出液 / トポイソメラーゼ / トポII / トポイソメラーゼⅡ / 分裂期 / トポイソメラーゼII / ヌクレオソーム / DNAトポロジー
Outline of Final Research Achievements

Topoisomerase IIα (topo IIα) is one of the six proteins essential for mitotic chromatid reconstitution in vitro. It is not fully understood how this enzyme regulates chromosome assembly remains elusive. In this project, we found that chromosomal binding of topo IIα is sensitive to buffer conditions and depends on its C-terminal domain (CTD). Enzymological assays using circular DNA substrates supports the idea that topo IIα first resolves inter-chromatid entanglements to drive individualization and then generates intra-chromatid entanglements to promote thickening. Importantly, only the latter process requires the CTD. By using frog egg extracts, we also show that the CTD contributes to proper formation of nucleosome-depleted chromatids by counteracting anomalous aggregated . Our results demonstrate that topo IIα utilizes its CTD to deliver the enzymatic core to crowded environments created during mitotic chromatid assembly, thereby fine-tuning this process.

Academic Significance and Societal Importance of the Research Achievements

本研究成果は、細胞の分裂期におけるトポⅡαの役割を初めて明確に示したものであり、染色体の構造と機能を大きく進展させるものである。さらに、トポⅡαは抗がん剤の標的タンパク質として知られているだけでなく、核移植によってクローン個体を作出する技術との関連も示唆されていることから、実学的研究にも大きなインパクトを与えると期待される。

Report

(4 results)
  • 2020 Annual Research Report   Final Research Report ( PDF )
  • 2019 Annual Research Report
  • 2018 Annual Research Report

Research Products

(15 results)

All 2021 2020 2019 2018 Other

All Int'l Joint Research (1 results) Journal Article (2 results) (of which Peer Reviewed: 2 results,  Open Access: 1 results) Presentation (9 results) (of which Int'l Joint Research: 1 results,  Invited: 6 results) Remarks (3 results)

  • [Int'l Joint Research] カリフォルニア大学バークレー校(米国)

    • Related Report
      2019 Annual Research Report
  • [Journal Article] Guiding functions of the C-terminal domain of topoisomerase IIα advance mitotic chromosome assembly2021

    • Author(s)
      Keishi Shintomi and Tatsuya Hirano
    • Journal Title

      Nature Communications

      Volume: - Pages: 2917-2917

    • DOI

      10.1038/s41467-021-23205-w

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Reconstitution of Mitotic Chromatids In Vitro2018

    • Author(s)
      Shintomi Keishi、Hirano Tatsuya
    • Journal Title

      Current Protocols in Cell Biology

      Volume: 79

    • DOI

      10.1002/cpcb.48

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Presentation] 染色体内の混雑した環境におけるトポイソメラーゼⅡαのはたらき2021

    • Author(s)
      新冨圭史
    • Organizer
      新学術領域「非ゲノム情報複製」主催ウェビナー
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 染色体内の混雑した環境におけるトポイソメラーゼⅡαのはたらき2021

    • Author(s)
      新冨圭史、平野達也
    • Organizer
      第38回 染色体ワークショップ・第19回 核ダイナミクス研究会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Reprogramming somatic cell nuclei in Xenopus egg extracts2020

    • Author(s)
      Keishi Shintomi
    • Organizer
      第93回 日本生化学会大会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] Topoisomerase IIa utilizes its C-terminal domain to work in crowded environments of mitotic chromosome assembly2020

    • Author(s)
      Keishi Shintomi and Tatsuya Hirano
    • Organizer
      第43回 日本分子生物学会年会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] Making a chromosome from scratch: a powerful approach to dissecting mitotic functions of topoisomerase II alpha2019

    • Author(s)
      Keishi Shintomi
    • Organizer
      EMBO Workshop - DNA topology and topoisomerase in genome dynamics
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Making a chromosome from scratch: a powerful approach to dissecting mitotic functions of topoisomerase II alpha2019

    • Author(s)
      Keishi Shintomi
    • Organizer
      第42回日本分子生物学会年
    • Related Report
      2019 Annual Research Report
  • [Presentation] 染色体再構成によって見えてきたトポⅡの役割2019

    • Author(s)
      新冨圭史
    • Organizer
      遺伝研研究会:クロマチン・細胞核の形成とダイナミクスによるゲノム制御
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] 再構成系の洗練化を通じた染色体構築メカニズムの解明2019

    • Author(s)
      新冨圭史, 平野達也
    • Organizer
      第36回染色体ワークショップ・第17回核ダイナミクス研究会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Toward the development of a “next-generation” mitotic chromatid reconstitution assay.2018

    • Author(s)
      Keishi Shintomi, Tatusya Hirano
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Remarks] 理化学研究所 平野染色体ダイナミクス研究室

    • URL

      http://www2.riken.jp/chromdyna/

    • Related Report
      2019 Annual Research Report
  • [Remarks] 所属研究室ウェブサイト

    • URL

      http://www2.riken.jp/chromdyna/

    • Related Report
      2018 Annual Research Report
  • [Remarks] research map

    • URL

      https://researchmap.jp/k_shintomi

    • Related Report
      2018 Annual Research Report

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Published: 2018-04-23   Modified: 2022-01-27  

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