Improvement of the AID technology through chemical biology approaches

• Project/Area Number: 18H02170
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 38060:Applied molecular and cellular biology-related
• Research Institution: National Institute of Genetics
• Principal Investigator: KANEMAKI MASATO 国立遺伝学研究所, 遺伝メカニズム研究系, 教授 (20444507)
• Co-Investigator(Kenkyū-buntansha): 林 謙一郎 岡山理科大学, 理学部, 教授 (30289136)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2020: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2018: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000)
• Keywords: 発現制御 / デグロン / タンパク質分解 / オーキシン / 細胞工学 / ケミカルバイオロジー / 阻害剤

Outline of Final Research Achievements

The auxin-inducible degron (AID) method allows the degradation of degron-fused proteins in cells by expressing the plant-derived ubiquitin ligase subunit TIR1 by the addition of auxin. The problem with the AID method is that degron-fused proteins are weakly degraded even without auxin.

To overcome this problem, we improved AID. By using a mutant TIR1 and a new ligand, we succeeded in developing AID2, which overcomes the problem. Furthermore, we showed that AID2 can be applied not only to cells but also to living mice. We have published the results of this research in a paper.

Academic Significance and Societal Importance of the Research Achievements

AID2を確立できたことにより、タンパク質分解による発現制御を細胞から個体まで応用できることを示した。タンパク質分解に基づく発現制御は、ごく短時間に標的タンパク質の発現を抑制できるために、標的タンパク質除去直後に起きる影響を観察できる。また、この分解は可逆的であるため、再発現も可能である。本技術は細胞から個体を用いた基礎研究の貢献できるのみならず、創薬における実証実験や疾患モデル動物作成に役立つことが期待される。

Research Products

• [Int'l Joint Research] University of Dundee/University of Aberdeen(英国)
• [Int'l Joint Research] Florida State University/San Diego Biomedical Research Institute(米国)
• [Int'l Joint Research] Stony Brook University/Baylor College of Medicine(米国)
• [Int'l Joint Research] University of Aberdeen/University college of London(英国)
• [Int'l Joint Research] University of Aberdeen(英国)
• [Int'l Joint Research] Baylor College of Medicine(米国)
• [Journal Article] Targeted Protein Depletion Using the Auxin‐Inducible Degron 2 (AID2) System (2021)
  • Author(s): Saito Yuichiro、Kanemaki Masato T.
  • Journal Title: Current Protocols Volume: 1 Issue: 8 Pages: 1-21
  • DOI: 10.1002/cpz1.219
  • Peer Reviewed
• [Journal Article] Replication timing maintains the global epigenetic state in human cells (2021)
  • Author(s): Klein Kyle N.、Zhao Peiyao A.、Lyu Xiaowen、Sasaki Takayo、Bartlett Daniel A.、Singh Amar M.、Tasan Ipek、Zhang Meng、Watts Lotte P.、Hiraga Shin-ichiro、Natsume Toyoaki、Zhou Xuemeng、Baslan Timour、Leung Danny、Kanemaki Masato T.、Donaldson Anne D.、Zhao Huimin、Dalton Stephen、Corces Victor G.、Gilbert David M.
  • Journal Title: Science Volume: 372 Issue: 6540 Pages: 371-378
  • DOI: 10.1126/science.aba5545
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice (2020)
  • Author(s): Yesbolatova Aisha、Saito Yuichiro、Kitamoto Naomi、Makino-Itou Hatsune、Ajima Rieko、Nakano Risako、Nakaoka Hirofumi、Fukui Kosuke、Gamo Kanae、Tominari Yusuke、Takeuchi Haruki、Saga Yumiko、Hayashi Ken-ichiro、Kanemaki Masato T.
  • Journal Title: Nature Communications Volume: 11 Issue: 1 Pages: 5701-5701
  • DOI: 10.1038/s41467-020-19532-z
  • Peer Reviewed / Open Access
• [Journal Article] Constructing Auxin-Inducible Degron Mutants Using an All-in-One Vector (2020)
  • Author(s): Yesbolatova Aisha、Saito Yuichiro、Kanemaki Masato T.
  • Journal Title: Pharmaceuticals Volume: 13 Issue: 5 Pages: 103-103
  • DOI: 10.3390/ph13050103
  • Peer Reviewed / Open Access
• [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 Issue: 4 Pages: 101027-101027
  • DOI: 10.1016/j.isci.2020.101027
  • 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] 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] 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] 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 Issue: 13 Pages: 908-913
  • DOI: 10.1097/wnr.0000000000001299
  • Peer Reviewed
• [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 Issue: 19-20 Pages: 1397-1415
  • DOI: 10.1101/gad.329508.119
  • Peer Reviewed / Open Access / Int'l Joint Research
• [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 Issue: 1 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: J. Cell Sci. Volume: 132 Issue: 2
  • 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: Curr Biol. Volume: 28(16) Issue: 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 Issue: 5 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 Issue: 9
  • 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) Issue: 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: Journal of Cell Science 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 Issue: 4
  • DOI: 10.1242/jcs.210187
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Publisher Correction: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells (2018)
  • Author(s): Takahashi Akiko、Okada Ryo、Nagao Koji、Kawamata Yuka、Hanyu Aki、Yoshimoto Shin、Takasugi Masaki、Watanabe Sugiko、Kanemaki Masato T.、Obuse Chikashi、Hara Eiji
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 06613-3
  • DOI: 10.1038/s41467-018-06613-3
  • Peer Reviewed / Open Access
• [Journal Article] Endosomal Rab cycles regulate Parkin-mediated mitophagy. (2018)
  • Author(s): Yamano K, Wang C, Sarraf SA, Münch C, Kikuchi R, Noda NN, Hizukuri Y, Kanemaki MT, Harper W, Tanaka K, Matsuda N, Youle RJ.
  • 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] AID2技術を使ってゲノムと非ゲノム情報複製の関係を探る (2020)
  • Author(s): 鐘巻将人
  • Organizer: 日本遺伝学会
  • Invited
• [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] オーキシンデグロン技術を標的タンパク質誘導分解薬開発に利用する (2019)
  • Author(s): 鐘巻将人
  • Organizer: 日本分子生物学会 ワークショップ3AW-19
  • Invited
• [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
  • ISBN: 9784860435530
• [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
  • ISBN: 9784758125086
• [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
  • ISBN: 9784758125147
• [Remarks] オーキシンデグロン(AID)法のためのCRISPR/Cas9を利用した新規タグ導入と分解阻害剤の開発
  • URL: https://www.nig.ac.jp/nig/ja/2019/05/research-highlights_ja/rh20190510.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
• [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
• [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・システム研究機構、学校法人加計学園
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2019-131464
  • Filing Date: 2019