Alterations of chromatin loop structures thorough manipulation of G-quadruplex and its binding protein, Rif1

• Project/Area Number: 16K14675
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Molecular biology
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: MASAI Hisao 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, 所長 (40229349)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: Rif1タンパク質 / クロマチンループ / 核内染色体構造 / 複製タイミング / グアニン４重鎖構造 / 核膜 / 染色体ループ構造 / ゲノム改変 / グアニン4重鎖 / Rif1 / 染色体核内配置 / DNA結合 / クロマチン動態 / 核機能 / グアニン４重鎖

Outline of Final Research Achievements

In order to manipulate chromatin loop formations, we used G-quadruplex binding protein, Rif1, which is known to regulate chromatin loops. We first identified L848S mutant, which cannot bind to chromatin. Overexpression of Rif1 resulted in inhibition of S phase progression and aberrant chromosome structures, as well as mitotic arrest with short spindles. The effects can be observed with Rif1 incapable of binding to phosphatase, but not with L848S deficient in chromatin binding. Thus, chromatin binding of Rif1 is responsible for these effects. By using the L848S mutant, we tried to tether Rif1 at a specific location on the genome. We used Gal4DBD-Gal4BS, and replaced a cellular Rif1BS with Gal4BS, and expressed Rif1L848S-Gal4BD at the endogenous locus. However, significant effects on the replication timing near the binding site were not observed, presumably due to low expression of the Gal4DBD fusion protein

Academic Significance and Societal Importance of the Research Achievements

Rif1はTAD(Topologically Associated Domain)等に関連したクロマチン高次構造を、G4結合を介して形成する。このクロマチンドメインは、複製タイミングや転写等の制御ユニットとなる。ゲノム操作が容易な分裂酵母を用いて、今回、それ自身ではクロマチンに結合しない変異Rif1を同定した。これを用いて、別のDNA結合ドメインを介して、任意の場所にRif1を局在化し、近傍のクロマチン高次構造を変化させうる可能性が生まれた。この方法は、特定の遺伝子のみを標的とするのではなく、クロマチンドメイン全体を制御可能であり、従来のゲノム操作技術とは異なる、新しい細胞改変技術をもたらす。

Research Products

• [Journal Article] Both a Unique Motif at the C Terminus and an N-Terminal HEAT Repeat Contribute to G-Quadruplex Binding and Origin Regulation by the Rif1 Protein (2018)
  • Author(s): Kobayashi Shunsuke、Fukatsu Rino、Kanoh Yutaka、Kakusho Naoko、Matsumoto Seiji、Chaen Shigeru、Masai Hisao
  • Journal Title: Molecular and Cellular Biology Volume: 39 Issue: 4
  • DOI: 10.1128/mcb.00364-18
  • Peer Reviewed / Open Access
• [Journal Article] Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture (2018)
  • Author(s): Moriyama Kenji、Yoshizawa-Sugata Naoko、Masai Hisao
  • Journal Title: Journal of Biological Chemistry Volume: 293 Issue: 10 Pages: 3607-3624
  • DOI: 10.1074/jbc.ra117.000446
  • Peer Reviewed / Open Access
• [Journal Article] A BTB-ZF protein, ZNF131, is required for early B cell development (2018)
  • Author(s): Iguchi T, Miyauchi E, Watanabe S, Masai H, Miyatake S
  • Journal Title: Biochem Biophys Res Commun. Volume: 501 Issue: 2 Pages: 570-575
  • DOI: 10.1016/j.bbrc.2018.05.044
  • Peer Reviewed / Open Access
• [Journal Article] Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences (2018)
  • Author(s): Masai Hisao、Kakusho Naoko、Fukatsu Rino、Ma Yue、Iida Keisuke、Kanoh Yutaka、Nagasawa Kazuo
  • Journal Title: Journal of Biological Chemistry Volume: 293 Issue: 44 Pages: 17033-17049
  • DOI: 10.1074/jbc.ra118.005240
  • Peer Reviewed / Open Access
• [Journal Article] Checkpoint-independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 kinase. (2017)
  • Author(s): Matsumoto, S., Kanoh, Y., Shimmoto, M., Hayano, M., Ueda, K., Fukatsu, R., Kakusho, N., and Masai, H.
  • Journal Title: Mol. Cell. Biol. Volume: MCB.00355-16. Issue: 7
  • DOI: 10.1128/mcb.00355-16
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast. (2017)
  • Author(s): Toteva, T., Mason, B., Kanoh, Y. Brogger, P. Green, D., Verhein-Hansen, J. Masai, H. and Thon, G.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 114 Issue: 5 Pages: 1093-1098
  • DOI: 10.1073/pnas.1614837114
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Potent DNA strand annealing activity associated with mouse Mcm2?7 heterohexameric complex (2017)
  • Author(s): You Zhiying、Masai Hisao
  • Journal Title: Nucleic Acids Research Volume: 45 Issue: 11 Pages: 6494-6506
  • DOI: 10.1093/nar/gkx269
  • Peer Reviewed / Open Access
• [Journal Article] Discovery of novel furanone derivatives as potent Cdc7 kinase inhibitors (2017)
  • Author(s): Irie Takayuki、Asami Tokiko、Sawa Ayako、Uno Yuko、Hanada Mitsuharu、Taniyama Chika、Funakoshi Yoko、Masai Hisao、Sawa Masaaki
  • Journal Title: European Journal of Medicinal Chemistry Volume: 130 Pages: 406-418
  • DOI: 10.1016/j.ejmech.2017.02.030
  • Peer Reviewed / Open Access
• [Journal Article] Telomere-binding factors in the regulation of DNA replication (2017)
  • Author(s): Masai, H., Kanoh, Y., Moriyama, K., Yamazaki, S., Yoshizawa, N., and Matsumoto, S.
  • Journal Title: Genes & Genetic Systems Volume: 92 Issue: 3 Pages: 119-125
  • DOI: 10.1266/ggs.17-00008
  • NAID: 130006316033
  • ISSN: 1341-7568, 1880-5779
  • Peer Reviewed / Open Access
• [Journal Article] A novel p53-Cdc7 link induced by genotoix stress. (2017)
  • Author(s): Masai, H.
  • Journal Title: Cell Cycle Volume: 印刷中 Issue: 8 Pages: 735-736
  • DOI: 10.1080/15384101.2017.1304746
  • Peer Reviewed / Open Access
• [Journal Article] Mrc1/Claspin: a new role for regulation of origin firing. (2017)
  • Author(s): Masai, H., Yang, C-C., and Matsumoto, S.
  • Journal Title: Current Genetics Volume: 印刷中 Issue: 5 Pages: 813-818
  • DOI: 10.1007/s00294-017-0690-y
  • Peer Reviewed / Open Access
• [Journal Article] Interaction of Rif1 Protein with G-Quadruplex in Control of Chromosome Transactions (2017)
  • Author(s): Moriyama Kenji、Lai Mong Sing、Masai Hisao
  • Journal Title: Adv Exp Med Biol. Volume: - Pages: 287-310
  • DOI: 10.1007/978-981-10-6955-0_14
  • ISBN: 9789811069543, 9789811069550
  • Peer Reviewed / Open Access
• [Journal Article] 多様な染色体ダイナミクスのmodulator kinase, Cdc7 (2017)
  • Author(s): 正井久雄
  • Journal Title: 生化学 Volume: 5 Pages: 719-730
  • NAID: 40021377418
  • Peer Reviewed
• [Journal Article] Potent DNA strand annealing activity associated with mouse Mcm2~7 heterohexamer complex. (2017)
  • Author(s): You, Z. and Masai, H.
  • Journal Title: Nucleic Acids Res. Volume: 印刷中
  • Peer Reviewed / Open Access
• [Journal Article] Telomere binding factors in regulation of DNA replication. (2017)
  • Author(s): Masai, H., Kanoh, Y., Moriyama, K., Yamazaki, S., Yoshizawa, N., and Matsumoto, S.
  • Journal Title: Genes & Genetic Systems Volume: 印刷中
  • NAID: 130006316033
  • Peer Reviewed
• [Journal Article] Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex. (2016)
  • Author(s): You Z, Ode KL, Shindo M, Takisawa H, Masai H.
  • Journal Title: Cell Cycle. Volume: 15 Issue: 9 Pages: 1213-1226
  • DOI: 10.1080/15384101.2015.1106652
  • Peer Reviewed / Open Access
• [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] Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43. (2016)
  • Author(s): Nonaka T, Suzuki G, Tanaka Y, Kametani F, Hirai S, Okado H, Miyashita T, Saitoe M, Akiyama H, Masai H, Hasegawa M.
  • Journal Title: The journal of Biological Chemistry Volume: 291 Issue: 11 Pages: 5473-83
  • DOI: 10.1074/jbc.m115.695379
  • Peer Reviewed / Open Access
• [Journal Article] Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation. (2016)
  • Author(s): Kozel, C., (他19名), Masai, H., Wagner, G., Beeser, A., Kikkawa, U., Fleming, S.D., and Asano, K.
  • Journal Title: Nucleic Acids Res. Volume: 44 Pages: 8704-8713
  • 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): Yang CC, Suzuki M, Yamakawa S, Uno S, Ishii A, Yamazaki S, Fukatsu R, Fujisawa R, Sakimura K, Tsurimoto T, Masai H.
  • Journal Title: Nat Commun. Volume: 7 Issue: 1 Pages: 12135-12135
  • DOI: 10.1038/ncomms12135
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 生体内でのグアニン４重鎖構造の検出・生物学的意義の解明を目指して (2018)
  • Author(s): 正井 久雄
  • Organizer: 非ワトソン・クリック型核酸に関する九州地区セミナー(日本核酸化学会共催)
  • Invited
• [Presentation] G-quadruplexes: in vivo regulations and biological functions (2018)
  • Author(s): Hisao Masai
  • Organizer: THE 3RD TRANSMED-VN CONFERENCE 2018 University of Medicine & Pharmacy (UMP)
  • Int'l Joint Research / Invited
• [Presentation] 大腸菌染色体のoriC-DnaA非依存性複製開始に必要とされるゲノム配列とその機能 (2018)
  • Author(s): 田中 卓、関 由美香、鷺 朋子、西籐 泰昌、正井 久雄
  • Organizer: 日本遺伝学会ワークショップ
  • Invited
• [Presentation] グアニン４重鎖構造の細胞内動態と生物学的意義の解明にむけて:DNA複製制御における機能 (2018)
  • Author(s): 正井 久雄
  • Organizer: 第91回日本生化学会大会 シンポジウム
  • Invited
• [Presentation] Biochemistry of Mcm, the central factor for DNA replication: a hetelro-hexameric helicase core complex that does not show DNA helicase activity. (2018)
  • Author(s): You, Z.
  • Organizer: 首都大学東京バイオコンファレンス2018
  • Invited
• [Presentation] Roles of Claspin-Cdc7 in initiation and checkpoint activation: differential functions of Cdc7 in cancer and normal cells (2018)
  • Author(s): 正井 久雄
  • Organizer: 3R&3C Symposium
  • Int'l Joint Research / Invited
• [Presentation] RNA-DNA hybrid and G-quadruplex: exploring their biological significance as new genome signature (2018)
  • Author(s): Hisao Masai
  • Organizer: 第41回日本分子生物学会年会 ワークショップ
  • Invited
• [Presentation] DNA複製研究からひもとくゲノムの謎～恩師 新井賢一先生に導かれて～ (2018)
  • Author(s): 正井 久雄
  • Organizer: 第41回日本分子生物学会年会 ランチョンセミナー
  • Invited
• [Presentation] From mechanisms of primer RNA synthesis to revelation of hidden messages of genome (2018)
  • Author(s): Hisao Masai
  • Organizer: OKAZAKI Fragment Memorial Symposium
  • Int'l Joint Research / Invited
• [Presentation] グアニン４重鎖構造：諸刃の剣のゲノム情報 (2017)
  • Author(s): 正井 久雄、(他13名)
  • Organizer: 生命科学系学会合同年次大会（ConBio2017）
  • Invited
• [Presentation] 細胞内におけるグアニン４重鎖DNA構造の存在を証明する新規技法の開発 (2017)
  • Author(s): 正井 久雄、加納 豊、覺正 直子
  • Organizer: 第24回DNA複製・組換え・修復ワークショップ
• [Presentation] グアニン４重鎖結合タンパク質Rif1の構造・機能解析 (2017)
  • Author(s): 小林 駿介、深津 理乃、加納 豊、正井 久雄
  • Organizer: 第24回DNA複製・組換え・修復ワークショップ
• [Presentation] The intramolecular interaction mechanism of human Claspin activation (2017)
  • Author(s): Zhiying You、Chi-Chun Yang、正井 久雄
  • Organizer: 第24回DNA複製・組換え・修復ワークショップ
• [Presentation] Rif1が結合するグアニン４重鎖構造の細胞内存在と形成のメカニズムの解明 (2017)
  • Author(s): 加納 豊、松本 清、深津 理乃、覚正 直子、正井久雄
  • Organizer: 生命科学系学会合同年次大会（ConBio2017）
• [Presentation] 大腸菌染色体のoriC-DnaA非依存性複製開始に必要とされる新たなゲノム領域とその機能 (2017)
  • Author(s): 田中 卓、関 由美香、鷺 朋子、西藤 泰昌、正井 久雄
  • Organizer: 生命科学系学会合同年次大会（ConBio2017）
• [Presentation] マウスES細胞におけるZscan4の脱抑制とRif1依存的なクロマチン構造変化の解析 (2017)
  • Author(s): 吉沢 直子、正井 久雄
  • Organizer: 生命科学系学会合同年次大会（ConBio2017）
• [Presentation] マウスRif1タンパク質のグアニン４重鎖DNA結合活性の生化学的解析 (2017)
  • Author(s): 森山 賢治、吉沢 直子、正井 久雄
  • Organizer: 生命科学系学会合同年次大会（ConBio2017）
• [Presentation] Rif1タンパク質の標的であるグアニン４重鎖DNA構造の分裂酵母細胞内での存在を証明するための新しい方法の開発 (2017)
  • Author(s): 正井 久雄、加納 豊、覺正 直子
  • Organizer: 第35回染色体ワークショップ・第16回核ダイナミクス研究会
• [Presentation] Rif1によるグアニン４重鎖構造を介した DNA複製制御 (2016)
  • Author(s): 加納 豊
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-11-30
• [Presentation] Rif1過剰発現は染色体分配の障害を誘導しM期進行を阻害する (2016)
  • Author(s): 松本 清治
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-11-30
• [Presentation] Novel higher-order DNA structures containing G-quadruplex structures on both strands of Rif1 binding region play a crucial role in determination of replication timing domain (2016)
  • Author(s): Masai, H.
  • Organizer: 第10回3R国際シンポジウム
  • Place of Presentation: ホテル一畑(島根県松江市)
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] Rif1 interacts with chromatin through G-quadruplex and regulates DNA replication (2016)
  • Author(s): Kanoh, Y.
  • Organizer: 第10回3R国際シンポジウム
  • Place of Presentation: ホテル一畑(島根県松江市)
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] G4 binding domain of fission yeast Rif1 protein (2016)
  • Author(s): Kobayashi, S.
  • Organizer: 第10回3R国際シンポジウム
  • Place of Presentation: ホテル一畑(島根県松江市)
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] Multimer formation and G-quadruplex-binding by purified murine Rif1, a key organizer of higher-order chromatin architecture (2016)
  • Author(s): Moriyama, K.
  • Organizer: 第10回3R国際シンポジウム
  • Place of Presentation: ホテル一畑(島根県松江市)
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] Cellular dynamics of G-quadruplex structures and RNA-DNA hybrid in human cells (2016)
  • Author(s): Nishitomi, T.
  • Organizer: 第10回3R国際シンポジウム
  • Place of Presentation: ホテル一畑(島根県松江市)
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] Genome-wide distribution of G-quadruplex structures and RNADNA hybrids on the E. coli genome (2016)
  • Author(s): Seki, Y.
  • Organizer: 第10回3R国際シンポジウム
  • Place of Presentation: ホテル一畑(島根県松江市)
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] Biological roles of G-quadruplex structures (2016)
  • Author(s): Masai, H.
  • Organizer: 14th IGAKUKEN International Symposium IGAKUKEN Summit for Japan and Korea Science Leaders 2016
  • Place of Presentation: 東京都医学総合研究所（東京都世田谷区）
  • Year and Date: 2016-06-30
  • Int'l Joint Research / Invited
• [Presentation] Interaction with G-quadruplex structures forms a basis for Rif1-mediated regulation of DNA replication, transcription and chromatin architecture (2016)
  • Author(s): Masai, H.
  • Organizer: Cold Spring Harbor Asia conference on DNA Metabolism, Genomic Stability and Human Disease
  • Place of Presentation: Suzhou(China)
  • Year and Date: 2016-06-13
  • Int'l Joint Research / Invited
• [Presentation] グアニン４重鎖構造の生物学的意義Molecular basis of chromosome and genome instability in cancer (2016)
  • Author(s): Masai, H.
  • Organizer: HiHA 第 6 回 Workshop Hiroshima Research Center for Healthy Aging
  • Place of Presentation: 広島大学（広島県東広島市）
  • Invited
• [Presentation] 大腸菌の第二の複製システムから考える複製システムの進化 (2016)
  • Author(s): 正井 久雄
  • Organizer: 日本進化学会 第18回東京大会
  • Place of Presentation: 東京工業大学大岡山キャンパス（東京都目黒区）
  • Invited
• [Presentation] 大腸菌染色体の第二の複製システム (2016)
  • Author(s): 正井 久雄
  • Organizer: 第88回日本遺伝学会大会
  • Place of Presentation: 日本大学（静岡県三島市）
  • Invited
• [Presentation] グアニン４重鎖の形成と、その認識タンパク質との相互作用 (2016)
  • Author(s): 正井 久雄
  • Organizer: 第89回 日本生化学会大会
  • Place of Presentation: 仙台国際センター/東北大学(宮城県仙台市)
  • Invited
• [Presentation] グアニン４重鎖構造の生物学的意義 Biological Roles of G-quadruplex (2016)
  • Author(s): 正井 久雄
  • Organizer: 第75回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Invited
• [Presentation] グアニン４重鎖構造の普遍的生物学的意義の解明に向けて (2016)
  • Author(s): 正井 久雄
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Invited
• [Book] Encyclopedia of Signaling Molecules, 2nd Edition: Dbf4 (2017)
  • Author(s): Sangdun Choi (編集)、Masai Hisao
  • Total Pages: 6330
  • Publisher: Springer
  • ISBN: 9783319671987
• [Remarks] ゲノム動態プロジェクト
  • URL: http://www.igakuken.or.jp/genome/
• [Remarks] 公益財団法人東京都医学総合研究所ゲノム動態プロジェクト
  • URL: http://www.igakuken.or.jp/genome/