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遺伝子座特異的クロマチン免疫沈降法による細胞分化制御因子の転写機構の解明

Publicly Offered Research

Project AreaIntegral understanding of the mechanism of transcription cycle through quantitative, high-resolution approaches
Project/Area Number 15H01354
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionOsaka University

Principal Investigator

藤井 穂高  大阪大学, 微生物病研究所, 准教授 (30302665)

Project Period (FY) 2015-04-01 – 2017-03-31
Project Status Completed (Fiscal Year 2016)
Budget Amount *help
¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
Fiscal Year 2016: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2015: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Keywords転写 / 分子生物学 / ゲノム生化学 / エピジェネティクス / クロマチン
Outline of Annual Research Achievements

転写制御の分子機構の解明には、転写制御を受けているゲノム領域の生化学的・分子生物学的解析が欠かせない。そのためには当該ゲノム領域の生化学的単離が必要である。申請者らは、これを実現するため、生体内のクロマチン構造を保持したまま特定のゲノム領域を単離する新規な方法として、insertional chromatin immunoprecipitation (iChIP) とengineered DNA-binding molecule-mediated ChIP (enChIP)からなる遺伝子座特異的ChIP法を世界に先駆けて開発した。本研究提案では、遺伝子座特異的ChIP法を用いて、転写制御を受けている特定ゲノム領域を単離して分子生物学・生化学的解析を行う際の方法論を確立するとともに、これを用いて転写制御機構の解明を目指す。
当該年度には以下の研究を行った。
1. Pax5遺伝子転写調節機構の解明:ニワトリの成熟B細胞株であるDT40を用いて、B細胞特異的にPax5遺伝子プロモーター領域と相互作用しているゲノム領域を同定した。その領域を、CRISPR系を用いて欠失させたところ、Pax5遺伝子の発現が減少した。また、その領域では、エンハンサー特異的なヒストン修飾が存在していた。この結果から、この領域がPax5遺伝子の発現を調節している可能性が示唆される。
2. Foxp3遺伝子転写調節機構の解明:iChIP-Seq法を用いて、マウス個体から単離したT細胞で、T-reg特異的にFoxp3遺伝子TSS近傍に結合するゲノム領域を同定した。それらの領域はエンハンサー等Foxp3遺伝子の転写制御領域である可能性がある。現在、in vitro enChIP-Seq法を用いて、iChIP-Seq解析の結果を確認している。
3. 3xFLAG-dCas9 Tgマウスを用いたenChIP法による転写機構の解析:CAGプロモーターを用いて、全身の細胞で3xFLAG-dCas9 Tgがゲノム中に挿入されているTgマウスを作製し、ライン化した。

Research Progress Status

28年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

28年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2016 Annual Research Report
  • 2015 Annual Research Report
  • Research Products

    (27 results)

All 2017 2016 2015 Other

All Int'l Joint Research (6 results) Journal Article (8 results) (of which Peer Reviewed: 8 results,  Open Access: 8 results,  Acknowledgement Compliant: 8 results) Presentation (9 results) (of which Int'l Joint Research: 5 results,  Invited: 2 results) Book (2 results) Remarks (2 results)

  • [Int'l Joint Research] Lund University(スウェーデン)

    • Related Report
      2016 Annual Research Report
  • [Int'l Joint Research] University of Alabama at Birmingham/University of California San Diego(米国)

    • Related Report
      2016 Annual Research Report
  • [Int'l Joint Research] Shaare Zedek Medical Center/Technion, Israel Institute of Technology(イスラエル)

    • Related Report
      2016 Annual Research Report
  • [Int'l Joint Research] l'institut du thorax/Lab Genome et Developpement des Plantes(フランス)

    • Related Report
      2016 Annual Research Report
  • [Int'l Joint Research] Institute for Basic Science(韓国)

    • Related Report
      2016 Annual Research Report
  • [Int'l Joint Research]

    • Related Report
      2016 Annual Research Report
  • [Journal Article] Identification of physical interactions between genomic regions by enChIP-Seq2017

    • Author(s)
      Fujita T, Yuno M, Suzuki Y, Sugano S, Fujii H
    • Journal Title

      Genes Cells

      Volume: in press

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus2016

    • Author(s)
      Fujita T, Yuno M, Fujii H
    • Journal Title

      Sci Rep

      Volume: 6 Issue: 1 Pages: 30485-30485

    • DOI

      10.1038/srep30485

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies.2016

    • Author(s)
      Fujita T, Fujii H.
    • Journal Title

      Gene Regul Syst Bio

      Volume: 10 Pages: 1-9

    • DOI

      10.4137/grsb.s32520

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.2016

    • Author(s)
      Fujita T, Yuno M, Fujii H.
    • Journal Title

      Genes Cells

      Volume: 21 Issue: 4 Pages: 370-377

    • DOI

      10.1111/gtc.12341

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP.2016

    • Author(s)
      Fujita T, Fujii H.
    • Journal Title

      J Vis Exp

      Volume: 107 Issue: 107

    • DOI

      10.3791/53478

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Identification of Non-Coding RNAs Associated with Telomeres Using a Combination of enChIP and RNA Sequencing2015

    • Author(s)
      Toshitsugu Fujita, Miyuki Yuno, Daisuke Okuzaki, Rieko Ohki, Hodaka Fujii
    • Journal Title

      PLOS ONE

      Volume: 10 Issue: 4 Pages: e0123387-e0123387

    • DOI

      10.1371/journal.pone.0123387

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins.2015

    • Author(s)
      Fujita T, Fujii H.
    • Journal Title

      Int J Mol Sci

      Volume: 16 Issue: 9 Pages: 21802-21812

    • DOI

      10.3390/ijms160921802

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research.2015

    • Author(s)
      Fujita T, Fujii H.
    • Journal Title

      Int J Mol Sci

      Volume: 16 Issue: 10 Pages: 23143-23164

    • DOI

      10.3390/ijms161023143

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Presentation] p16INK4a遺伝子座を標的としたCRISPRによるアレル特異的なゲノム結合およびゲノム編集2016

    • Author(s)
      藤田敏次、藤井穂高
    • 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
    • Year and Date
      2016-11-30
    • Related Report
      2016 Annual Research Report
  • [Presentation] Development of enChIP and its applications to locus-specific biochemical analysis of genome functions2016

    • Author(s)
      Fujita T, Fujii H
    • Organizer
      12th EMBL Conference Transcription and Chromatin
    • Place of Presentation
      EMBL Advanced Training Centre, Heidelberg, Germany
    • Year and Date
      2016-08-27
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research
  • [Presentation] エピジェネティック制御機構の生化学的解析に向けたin vitro enChIP法の開発2016

    • Author(s)
      藤田敏次、藤井穂高
    • Organizer
      第10回日本エピジェネティクス研究会年会
    • Place of Presentation
      千里ライフサイエンスセンター、大阪府
    • Year and Date
      2016-05-19
    • Related Report
      2016 Annual Research Report
  • [Presentation] 遺伝子座特異的クロマチン免疫沈降法によるゲノム機能発現機構の解析:核内三次元構造解析への応用を中心として2016

    • Author(s)
      藤田 敏次、藤井 穂高
    • Organizer
      第33回染色体ワークショップ・第14回核ダイナミクス研究会
    • Place of Presentation
      松島一の坊
    • Year and Date
      2016-01-12
    • Related Report
      2015 Annual Research Report
  • [Presentation] Biochemical analysis of genome functions using locus-specific chromatin immunoprecipitation technologies: iChIP and enChIP2016

    • Author(s)
      Fujii H
    • Organizer
      The International Conference on Transcription Cycle International 2016
    • Place of Presentation
      東京大学理学部小柴ホール、東京都
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 組換え蛋白質を利用したin vitro遺伝子座特異的クロマチン免疫沈降法による転写・エピジェネティック制御機構の解析2015

    • Author(s)
      藤井 穂高
    • Organizer
      BMB2015(第38回日本分子生物学会年会・第88回日本生化学会 合同大会)
    • Place of Presentation
      神戸ポートアイランド
    • Year and Date
      2015-12-01
    • Related Report
      2015 Annual Research Report
    • Invited
  • [Presentation] Locus-specific biochemical analysis of genome functions using enChIP: an application of CRISPR/Cas and TAL to purification of specific genomic regions2015

    • Author(s)
      Fujii H
    • Organizer
      TGE2015 Conference on Transposition and Genome Engineering 2015
    • Place of Presentation
      奈良春日野国際フォーラム
    • Year and Date
      2015-11-17
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Locus-specific biochemical analysis of genome functions using enChIP: an application of CRISPR/Cas to purification of specific genomic regions2015

    • Author(s)
      Fujita T, and Fujii H
    • Organizer
      Cold Spring Harbor meeting: Genome Engineering: The CRISPR/Cas Revolution
    • Place of Presentation
      Cold Spring Harbor Laboratory, Cold Spring Harbor, USA
    • Year and Date
      2015-09-24
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Locus-specific biochemical analysis of genome functions using enChIP: an application of CRISPR/Cas and TAL to purification of specific genomic regions2015

    • Author(s)
      Fujita T, and Fujii H
    • Organizer
      FASEB Science Research Conference "Transcription, Chromatin, and Epigenetics"
    • Place of Presentation
      West Palm Beach Marriott, Palm Beach, USA
    • Year and Date
      2015-06-28
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Book] Handbook of Epigenetics 2nd edition2017

    • Author(s)
      Fujita T, Fujii H
    • Publisher
      Elsevier
    • Related Report
      2016 Annual Research Report
  • [Book] Chromatin Protocols2015

    • Author(s)
      Fujita T, Fujii H
    • Total Pages
      492
    • Publisher
      Springer
    • Related Report
      2015 Annual Research Report
  • [Remarks] 藤井研究室ホームページ(大阪大学)

    • URL

      http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/index.html

    • Related Report
      2016 Annual Research Report
  • [Remarks] 藤井研究室ホームページ

    • URL

      http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/index.html

    • Related Report
      2015 Annual Research Report

URL: 

Published: 2015-04-16   Modified: 2018-03-28  

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