染色体の核内高次構築を支配する非コード領域とそれを制御するタンパク質の解析

• Project Area: Functions of non-coding DNA region for genome integrity
• Project/Area Number: 26114723
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: 正井 久雄 公益財団法人東京都医学総合研究所, ゲノム医科学研究分野, 副所長 (40229349)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000); Fiscal Year 2015: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000); Fiscal Year 2014: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
• Keywords: Rif1タンパク質 / 染色体 / ゲノム / 核 / 転写 / 複製タイミング / クロマチンループ / グアニン4重鎖 / DNA複製 / 非コードDNA / グアニン４重鎖

Outline of Annual Research Achievements

染色体の核内高次構築を支配する非コード領域に結合するRif1タンパク質による染色体機能の制御機構の解析の結果、下記の成果が得られた。
1 酵母：Rif1 ①Rif1結合配列は、その保存されたG-tract配列に依存してグアニン４重鎖構造を形成する。②Rif1は一本鎖上および二本鎖上に(熱処理により形成される)G4構造に特異的に結合する。③Rif1は多量体を形成し、同時に複数のDNA分子と結合することができる。④Rif1結合部位二本鎖DNAは転写されるとG4様構造を形成し、Rif1はそれに結合する。⑤Rif1結合配列の変異によりその近傍の複製タイミングが約100kbにわたり活性化する。
2 動物細胞：①Rif1 ノックアウトによりES細胞の分化の過程が障害される。②マウスES細胞におけるRif1結合部位をChIPSeqにより決定した。③Rif1ノックアウトにより、活性化される遺伝子クラスターにおいてはDNAメチル化の減少が観察された。④動物細胞Rif1タンパク質もG4構造に結合し、さらに、その効率のよい結合にはN端HEAT RepeatとC端ドメインの両者が必要である。
このようにRif1タンパク質は非コード領域に存在するG4DNA形成配列に、これが転写などによりG4構造を形成すると結合し、同時に複数のクロマチンに結合し束ねることにより複製や転写の機能ドメインの形成に貢献する可能性を提唱した。

Research Progress Status

27年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

27年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex. (2016)
  • Author(s): Zhiying You, Koji L. Ode, Haruhiko Takisawa, and *Hisao Masai
  • Journal Title: Cell Cycle Volume: 3
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Fork restart protein, PriA, binds around oriC after depletion of nucleotide precursors: Replication fork arrest near the replication origin. (2016)
  • Author(s): Tanaka T, Nishito Y, *Masai H.
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 470 Issue: 3 Pages: 546-551
  • DOI: 10.1016/j.bbrc.2016.01.108
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Epigenetic Regulation of the Blimp-1 Gene (Prdm1) in B Cells Involves Bach2 and Histone Deacetylase 3* (2016)
  • Author(s): Tanaka H
  • Journal Title: JBC Volume: 291 Issue: 12 Pages: 6316-6330
  • DOI: 10.1074/jbc.m116.713842
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells. (2016)
  • Author(s): Chi-Chun Yang, Masahiro Suzuki, Shiori Yamakawa, Syuzi Uno, Ai Ishii, Satoshi Yamazaki, Rino Fukatsu, Ryo Fujisawa, Kenji Sakimura3, Toshiki Tsurimoto, *Hisao Masai
  • Journal Title: Nature Communications Volume: TBA
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Rif1はグアニン４重鎖構造を介して染色体に結合し、広範囲に複製を抑制する (2016)
  • Author(s): 加納 豊、松本 清治、正井 久雄
  • Journal Title: 実験医学 Volume: 34-no.3 Pages: 458-461
• [Journal Article] CDC28 phosphorylates Cac1p and regulates the association of Chromatin Assembly Factor I with chromatin. (2015)
  • Author(s): Jeffery, D., Kakusho, N., You, Z., Gharib, M., Wyse, B., Drury, E., Weinreich, M., Thibault, P. Verreault, A., Masai, H. and *Yankulov, K.
  • Journal Title: Cell Cycle Volume: 14 Issue: 1 Pages: 74-85
  • DOI: 10.4161/15384101.2014.973745
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Definition of the transcription factor TdIF1 consensus binding sequence through genome-wide mapping of its binding sites. (2015)
  • Author(s): Kotaro Koiwai, Takashi Kubota, Nobuhisa Watanabe, Katsutoshi Hori, Osamu Koiwai and Hisao Masai
  • Journal Title: Genes to Cells Volume: 20 Issue: 3 Pages: 242-254
  • DOI: 10.1111/gtc.12216
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] The DNA-Binding Domain of S. pombe Mrc1 (Claspin) Acts to Enhance Stalling at Replication Barriers. (2015)
  • Author(s): Zech, J., Godfrey, E.L., Masai, H., Hartsuiker, E. and *Dalgaard, J.Z.
  • Journal Title: PLoS One Volume: 10 Issue: 7 Pages: e0132595-e0132595
  • DOI: 10.1371/journal.pone.0132595
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells. (2015)
  • Author(s): Iguchi T, Aoki K, Ikawa T, Taoka M, Taya C, Yoshitani H, Toma-Hirano M, Koiwai O, Isobe T, Kawamoto H, Masai H, Miyatake S.
  • Journal Title: The Journal of Immunology Volume: 195 Issue: 3 Pages: 982-93
  • DOI: 10.4049/jimmunol.1500602
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Rif1 binds to G-quadruplexes and suppresses replication over long distances. (2015)
  • Author(s): Yutaka Kanoh, Seiji Matsumoto, Rino Fukatsu, Naoko Kakusho, Nobuaki Kono, Claire Renard-Guillet, Koji Masuda, Keisuke Iida, Kazuo Nagasawa, Katsuhiko Shirahige, and *Hisao Masai
  • Journal Title: Nature Struct. Mol. Biol. Volume: 22 Issue: 11 Pages: 889-897
  • DOI: 10.1038/nsmb.3102
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Rif1はグアニン4重鎖構造を形成するDNAを介して染色体へ結合し複製の開始を広範囲にわたり抑制する (2015)
  • Author(s): 加納 豊、松本 清治、正井 久雄
  • Journal Title: ライフサイエンス 新着論文レビュー Volume: -
  • Open Access
• [Journal Article] NCOA4 Transcriptional Coactivator Inhibits Activation of DNA Replication Origins. (2014)
  • Author(s): Bellelli, R., Castellone, M.D., Guida, T., Limongello, R., Dathan, N.A., Merolla, F., Cirafici, A.M., Affuso, A., Masai, H., Costanzo, V., Grieco, D., Fusco, A., Santoro, M., and Carlomagno, F.
  • Journal Title: Mol. Cell Volume: 55 Issue: 1 Pages: 123-137
  • DOI: 10.1016/j.molcel.2014.04.031
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Regulation and roles of Cdc7 kinase under replication stress. (2014)
  • Author(s): Yamada, M., Masai, H., and Bartek, J.
  • Journal Title: Cell Cycle Volume: 13 Issue: 12 Pages: 1859-1866
  • DOI: 10.4161/cc.29251
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Cell cycle synchronization and flow cytometry analysis of mammalian cell. (2014)
  • Author(s): Yoshizawa-Sugata, N. and Masai, H.
  • Journal Title: Methods in Molecular Biology Volume: 1170 Pages: 279-293
  • DOI: 10.1007/978-1-4939-0888-2_13
  • ISBN: 9781493908875, 9781493908882
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] ATM in prevention of genomic instability. (2014)
  • Author(s): Hisao Masai
  • Journal Title: Cell Cycle Volume: 13 Issue: 6 Pages: 882-883
  • DOI: 10.4161/cc.28216
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Temporal and spatial regulation of eukaryotic DNA replication: from regulated initiation to genome-scale timing program. (2014)
  • Author(s): Renard-Guillet, C., Kanoh, Y., Shirahige, K., and Masai, H.
  • Journal Title: Seminars in Cell & Developmental Biology, Volume: 30 Pages: 110-120
  • DOI: 10.1016/j.semcdb.2014.04.014
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Interaction with G-quadruplex structures forms a basis for Rif1-mediated regulation of DNA replication, transcription and chromatin architecture (2015)
  • Author(s): 正井 久雄、加納 豊、松本 清治、覺正 直子、深津 理乃、森山 賢治、河野 暢明、飯田 啓介、長澤 和夫、増田 晃士、Claire Renard-Guillet、白髭 克彦、山崎 聡志、吉沢 直子
  • Organizer: BMB2015
  • Place of Presentation: 神戸国際会議場（兵庫県神戸市）
  • Year and Date: 2015-12-01
  • Int'l Joint Research / Invited
• [Presentation] Roles of G-quadruplex structures in regulation of DNA replication. (2015)
  • Author(s): Hisao Masai, Yutaka Kanoh, Taku Tanaka, Kenji Moriyama, Yumika Seki, Seiji Matsumoto, Rino Fukatsu, Naoko Kakusho, Satoshi Yamazaki, Yumeka Matsushima, Naoko Yoshizawa, Keisuke Iida, Kazuo Nagasawa
  • Organizer: Cold Spring Harbor Meeting EUKARYOTIC DNA REPLICATION & GENOME MAINTENANCE
  • Place of Presentation: Cold Spring Harbor, New York,USA
  • Year and Date: 2015-09-01
  • Int'l Joint Research / Invited
• [Presentation] Interaction of Rif1 and G-quadruplex forms a basis of Rif1-mediated chromatin regulation. (2015)
  • Author(s): Hisao Masai, Rino Fukatsu, Naoko Kakusho, Yutaka Kanoh, Kenji Moriyama
  • Organizer: The 8th international fission yeast meeting (Pombe2015)
  • Place of Presentation: 神戸国際会議場（兵庫県神戸市）
  • Year and Date: 2015-06-21
  • Int'l Joint Research / Invited
• [Presentation] ゲノムDNA複製制御のメカニズム：生物種を超えた統一像と多様性 (2014)
  • Author(s): 正井 久雄
  • Organizer: 第37回日本分子生物学会年会 (パシフィコ横浜)ワークショップ
  • Place of Presentation: パシフィコ横浜（神奈川）
  • Year and Date: 2014-11-25 – 2014-11-27
  • Invited
• [Presentation] Regulation of mammalian Mcm helicase complexes, (2014)
  • Author(s): Hisao Masai, You Zhiying, Koji L. Ode, Naoko Kakusho, Rino Fukatsu, and Haruhiko Takisawa
  • Organizer: 第87回日本生化学会大会シンポジウム「ゲノムを支えるDNAヘリカーゼの動態を探求する新たな研究展開」
  • Place of Presentation: 国立京都国際会館（京都）
  • Year and Date: 2014-10-15 – 2014-10-18
  • Invited
• [Presentation] 染色体DNA複製プログラムの制御機構 (2014)
  • Author(s): 正井 久雄
  • Organizer: 大阪大学蛋白質研究所セミナー「染色体伝承の分子背景：複製から染色体分離まで」
  • Place of Presentation: 大阪大学（大阪）
  • Year and Date: 2014-09-25 – 2014-09-26
  • Invited
• [Presentation] Regulation of DNA replication program in fission yeast and human cells (2014)
  • Author(s): Hisao Masai
  • Organizer: Marco Foiani（Italy, Milan）教授の招聘により IFOM Foundation – F.I.R.C. Institute of Molecular Oncology Foundationで講演
  • Place of Presentation: Italia, Milan
  • Year and Date: 2014-09-16
  • Invited
• [Presentation] Mrc1/Claspinの分子内相互作用による活性制御および新規機能 (2014)
  • Author(s): 正井 久雄
  • Organizer: 九州大学 大学院薬学研究院 招聘研究セミナー
  • Place of Presentation: 九州大学（福岡）
  • Year and Date: 2014-08-29
  • Invited
• [Presentation] Regulation of replication program in fission yeast and human cells. (2014)
  • Author(s): Hisao Masai
  • Organizer: Peking University（特別招聘講演）
  • Place of Presentation: Peking University（中国）
  • Year and Date: 2014-05-16
  • Invited
• [Book] The Initiation of DNA Replication in Eukaryotes "Rif1, a conserved chromatin factor regulating DNA replication, DNA repair and transcription." (2015)
  • Author(s): Naoko Yoshizawa, Satoshi Yamazaki, and *Hisao Masai
  • Total Pages: 623
  • Publisher: Springer
• [Book] DNA Replication, Recombination, and Repair "DNA Replication Timing: Temporal and Spatial Regulation of Eukaryotic DNA Replication." (2015)
  • Author(s): Hayano, M., Matsumoto, S. and *Masai, H.
  • Total Pages: 555
  • Publisher: Springer
• [Book] DNA Replication, Recombination and Repair: Molecular Mechanisms and Pathology (2015)
  • Author(s): Motoshi Hayano, Seiji Matsumoto and Hisao Masai
  • Publisher: Springer