AID技術を利用したヒト染色体維持機構と染色体構造の関係性の解明

• Project Area: Chromosome Orchestration System
• Project/Area Number: 18H04719
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Research Institution: National Institute of Genetics
• Principal Investigator: 鐘巻 将人 国立遺伝学研究所, 遺伝メカニズム研究系, 教授 (20444507)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000); Fiscal Year 2019: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2018: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
• Keywords: ゲノム維持 / DNA複製 / デグロン / 染色体 / オーキシンデグロン / 細胞工学 / 染色体不安定性 / 染色体構造 / SMCタンパク質 / DNA複製タイミング / 複製フォーク

Outline of Annual Research Achievements

本研究はオーキシンデグロン技術をヒト培養細胞に応用し、ヒト染色体構造と機能を解明することを目標としている。

SMC5-6複合体は、そのリング状の構造からDNAをトポロジカルに捉えることで機能を果たしていると考えられるが、その明確な機能は明らかになっていない。そこで、SMC5-6複合体に対するオーキシンデグロン変異細胞を作成し詳細な解析を行った。その結果、SMC5-6複合体の除去は染色体不安定生と分配異常を引き起こし、DNAダメージやp53の活性化を引き起こすことをしめした。さらに、染色体部分配異常は、直前のS期とG2期にSMC5-6が欠損していると、引き起こされることをみいだした。これら結果をまとめて論文発表した（Bueno Venegas et al. Cell Reports, 2020）。

並行して、複製関連因子MCM8-9の機能解析を継続している。複製ヘリカーゼMCM2にオーキシンデグロンタグを導入し、S期にMCM2を分解すると、MCM8-9に依存したDNA合成が観察される。このDNA合成には相同組換えが関与しているが、そこに新規因子HROBが必要であることを見出した。HROBとMCM8-9は同一の経路で、DNA未複製領域を複製するのに機能すると考えられる。またHROBとMCM8-9は減数分裂組換えにも関与することを見出した。これら結果を論文発表した（Hudest et al., Genes and Development, 2019）。

Research Progress Status

令和元年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和元年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] University of Aberdeen/University college of London(英国)
• [Int'l Joint Research] Stony Brook University/Baylor College of Medicine(米国)
• [Int'l Joint Research] University of Denmark(デンマーク)
• [Int'l Joint Research] University of Toronto(カナダ)
• [Int'l Joint Research] Imperial College of London(英国)
• [Int'l Joint Research] Baylor College of Medicine(米国)
• [Int'l Joint Research] University of Copenhagen(デンマーク)
• [Journal Article] Inducible Degradation of the Human SMC5/6 Complex Reveals an Essential Role Only during Interphase (2020)
  • Author(s): Venegas Andres Bueno、Natsume Toyoaki、Kanemaki Masato、Hickson Ian D.
  • Journal Title: Cell Reports Volume: 31 Pages: 107533-107533
  • DOI: 10.1016/j.celrep.2020.107533
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
  • Author(s): Hegarat Nadia、Crncec Adrijana、Suarez Peredo Rodriguez Maria F、Echegaray Iturra Fabio、Gu Yan、Busby Oliver、Lang Paul F、Barr Alexis R、Bakal Chris、Kanemaki Masato T、Lamond Angus I、Novak Bela、Ly Tony、Hochegger Helfrid
  • Journal Title: The EMBO Journal Volume: e104419
  • DOI: 10.15252/embj.2020104419
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks (2020)
  • Author(s): Akagawa Remi、Trinh Hai Thanh、Saha Liton Kumar、Tsuda Masataka、Hirota Kouji、Yamada Shintaro、Shibata Atsushi、Kanemaki Masato T.、Nakada Shinichiro、Takeda Shunichi、Sasanuma Hiroyuki
  • Journal Title: iScience Volume: 23 Pages: 101027-101027
  • DOI: 10.1016/j.isci.2020.101027
  • Peer Reviewed / Open Access
• [Journal Article] Control of homologous recombination by the HROB-MCM8-MCM9 pathway (2019)
  • Author(s): Hustedt Nicole、Saito Yuichiro、Zimmermann Michal、Alvarez-Quilon Alejandro、Setiaputra Dheva、Adam Salom?、McEwan Andrea、Yuan Jing Yi、Olivieri Michele、Zhao Yichao、Kanemaki Masato T.、Jurisicova Andrea、Durocher Daniel
  • Journal Title: Genes & Development Volume: 33 Pages: 1397-1415
  • DOI: 10.1101/gad.329508.119
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole (2019)
  • Author(s): Yesbolatova A, Natsume T, Hayashi K, Kanemaki MT
  • Journal Title: Methods Volume: 印刷中 Pages: 73-80
  • DOI: 10.1016/j.ymeth.2019.04.010
  • Peer Reviewed / Open Access
• [Journal Article] Auxin-mediated rapid degradation of target proteins in hippocampal neurons. (2019)
  • Author(s): Nakano Risako、Ihara Naoki、Morikawa Shota、Nakashima Ai、Kanemaki Masato T.、Ikegaya Yuji、Takeuchi Haruki
  • Journal Title: Neuroreport Volume: 30 Pages: 908-913
  • DOI: 10.1097/wnr.0000000000001299
  • Peer Reviewed
• [Journal Article] HsSAS-6-dependent cartwheel assembly ensures stabilization of centriole intermediates. (2019)
  • Author(s): Yoshiba Satoko、Tsuchiya Yuki、Ohta Midori、Gupta Akshari、Shiratsuchi Gen、Nozaki Yuka、Ashikawa Tomoko、Fujiwara Takahiro、Natsume Toyoaki、Kanemaki Masato T.、Kitagawa Daiju
  • Journal Title: Journal of Cell Science Volume: 132 Pages: 217521-217521
  • DOI: 10.1242/jcs.217521
  • Peer Reviewed / Open Access
• [Journal Article] Ligand-induced genetic degradation as a tool for target validation (2019)
  • Author(s): Yesbolatova Aisha、Tominari Yusuke、Kanemaki Masato T.
  • Journal Title: Drug Discovery Today: Technologies Volume: 31 Pages: 91-98
  • DOI: 10.1016/j.ddtec.2018.11.001
  • Peer Reviewed
• [Journal Article] Temporal and Spatial Epigenome Editing Allows Precise Gene Regulation in Mammalian Cells (2019)
  • Author(s): Kuscu Cem、Mammadov Rashad、Czikora Agnes、Unlu Hayrunnisa、Tufan Turan、Fischer Natasha Lopes、Arslan Sevki、Bekiranov Stefan、Kanemaki Masato、Adli Mazhar
  • Journal Title: Journal of Molecular Biology Volume: 431 Pages: 111-121
  • DOI: 10.1016/j.jmb.2018.08.001
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Chk1-mediated Cdc25A degradation as a critical mechanism for normal cell-cycle progression (2019)
  • Author(s): Goto H., Natsume T., Kanemaki M.T., Kaito A., Wang S., Gabazza E.C., Inagaki M., Mizoguchi A.
  • Journal Title: Journal of Cell Science Volume: 132
  • DOI: 10.1242/jcs.223123
  • Peer Reviewed / Open Access
• [Journal Article] Temporal Regulation of ESCO2 Degradation by the MCM Complex, the CUL4-DDB1-VPRBP Complex, and the Anaphase-Promoting Complex (2018)
  • Author(s): Minamino M, Tei S, Negishi L, Kanemaki MT, Yoshimura A, Sutani T, Bando M, Shirahige K.
  • Journal Title: Current Biology Volume: 28(16) Pages: 2665-2672
  • DOI: 10.1016/j.cub.2018.06.037
  • Peer Reviewed / Open Access
• [Journal Article] TAGing for destruction (2018)
  • Author(s): Yesbolatova Aisha、Kanemaki Masato T.
  • Journal Title: Nature Chemical Biology Volume: 14 Pages: 414-415
  • DOI: 10.1038/s41589-018-0024-5
  • Open Access
• [Journal Article] Seh1 targets GATOR2 and Nup153 to mitotic chromosomes (2018)
  • Author(s): Platani Melpomeni、Samejima Itaru、Samejima Kumiko、Kanemaki Masato T.、Earnshaw William C.
  • Journal Title: Journal of Cell Science Volume: 131
  • DOI: 10.1242/jcs.213140
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Replication stress induces accumulation of FANCD2 at central region of large fragile genes. (2018)
  • Author(s): Okamoto Y, Iwasaki WM, Kugou K, Takahashi KK, Oda A, Sato K, Kobayashi W, Kawai H, Sakasai R, Takaori-Kondo A, Yamamoto T, Kanemaki MT, Taoka M, Isobe T, Kurumizaka H, Innan H, Ohta K, Ishiai M, Takata M.
  • Journal Title: Nucleic Acids Res Volume: 46(6) Pages: 2932-2944
  • DOI: 10.1093/nar/gky058
  • NAID: 120006488604
  • Peer Reviewed / Open Access
• [Journal Article] Ki-67 and condensins support the integrity of mitotic chromosomes through distinct mechanisms (2018)
  • Author(s): Takagi Masatoshi、Ono Takao、Natsume Toyoaki、Sakamoto Chiyomi、Nakao Mitsuyoshi、Saitoh Noriko、Kanemaki Masato T.、Hirano Tatsuya、Imamoto Naoko
  • Journal Title: J. Cell Sci. Volume: 131 Pages: 212092-212092
  • DOI: 10.1242/jcs.212092
  • Peer Reviewed / Open Access
• [Journal Article] Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis (2018)
  • Author(s): Samejima Kumiko、Booth Daniel G.、Ogawa Hiromi、Paulson James R.、Xie Linfeng、Watson Cara A.、Platani Melpomeni、Kanemaki Masato T.、Earnshaw William C.
  • Journal Title: Journal of Cell Science Volume: 131
  • DOI: 10.1242/jcs.210187
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Endosomal Rab cycles regulate Parkin-mediated mitophagy. (2018)
  • Author(s): Yamano, K., Wang, C., Sarraf, S. A., Munch, C., Kikuchi, R., Noda, N. N., Hizukuri, Y., Kanemaki, M. T., Harper, W., Tanaka, K., Matsuda, N., and Youle, R. J.
  • Journal Title: Elife Volume: 7
  • DOI: 10.7554/elife.31326
  • NAID: 120006457172
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble. (2018)
  • Author(s): Okumura Masako、Natsume Toyoaki、Kanemaki Masato T、Kiyomitsu Tomomi
  • Journal Title: eLife Volume: 7 Pages: 1-24
  • DOI: 10.7554/elife.36559
  • Peer Reviewed / Open Access
• [Presentation] The Auxin-Inducible Degron (AID) system as a tool for target validation (2019)
  • Author(s): Masato Kanemaki
  • Organizer: TPD Forum Japan
  • Int'l Joint Research / Invited
• [Presentation] MCMBP is an MCM chaperon protecting the MCM subunits from degradation (2019)
  • Author(s): Masato Kanemaki
  • Organizer: CSHL meeting on eukaryotic DNA replication and genome maintenance
  • Int'l Joint Research / Invited
• [Presentation] Auxin-inducible degron (AID) technology for rapid protein depletion in human cells (2019)
  • Author(s): Masato Kanemaki
  • Organizer: 日本遺伝学会 国際シンポジウム
  • Int'l Joint Research / Invited
• [Presentation] The Auxin-Inducible Degron (AID) system as a tool for target validation (2019)
  • Author(s): Masato Kanemaki
  • Organizer: TPD summit in Boston
  • Int'l Joint Research / Invited
• [Presentation] MCMBP is an MCM chaperon protecting the MCM subunits from degradation (2019)
  • Author(s): 鐘巻将人
  • Organizer: 3Rワークショップ
• [Presentation] Rapid protein depletion in human cells by the auxin-inducible degron technology (2018)
  • Author(s): Masato Kanemaki
  • Organizer: NIG International Symposium
  • Int'l Joint Research / Invited
• [Presentation] A non-canonical DNA replication important for genome maintenance (2018)
  • Author(s): Masato Kanemaki
  • Organizer: Okazaki Fragment Memorial Symposium
  • Int'l Joint Research / Invited
• [Book] 生物の科学遺伝 ヒト疾患とモデル生物：ヒト培養細胞を用いた解析 (2018)
  • Author(s): 鐘巻将人
  • Total Pages: 5
  • Publisher: エヌ・ティー・エス
• [Book] 実験医学 2018年 6月号：抗体による内在性タンパク質分解除去 (2018)
  • Author(s): 鐘巻将人
  • Total Pages: 2
  • Publisher: 羊土社
• [Book] 実験医学 2018年 12月号：複製フォークの異常が自己炎症性疾患を引き起こす (2018)
  • Author(s): 鐘巻将人
  • Total Pages: 2
  • Publisher: 羊土社
• [Remarks] MCM8–9経路の相同組換えを制御する新規因子HROB
  • URL: https://www.nig.ac.jp/nig/ja/2019/09/research-highlights_ja/rh20190911.html
• [Remarks] 世界初！光で細胞分裂装置の操作に成功 ～体作りに重要な細胞分裂の仕組みの理解促進に期待～
  • URL: https://www.nig.ac.jp/nig/ja/2018/07/research-highlights_ja/20180709.html
• [Remarks] 遺伝子が転写される際のDNAの「動き」を生きた細胞の中で捉えた!
  • URL: https://www.nig.ac.jp/nig/ja/2019/03/research-highlights_ja/20190305.html
• [Patent(Industrial Property Rights)] オーキシンデグロンシステムのキット、およびその使用 (2019)
  • Inventor(s): 鐘巻将人、林謙一郎
  • Industrial Property Rights Holder: 大学共同利用機 関法人情報・シ ステム研究機
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2019-131464
  • Filing Date: 2019