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挿入的クロマチン免疫沈降法(iChIP)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 25118512
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) 2013-04-01 – 2015-03-31
Project Status Completed (Fiscal Year 2014)
Budget Amount *help
¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
Fiscal Year 2014: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2013: ¥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 / 遺伝子座特異的クロマチン免疫沈降法
Outline of Annual Research Achievements

1. Pax5遺伝子転写調節機構の解明
iChIP-SILAC法を用いて、Pax5遺伝子プロモーター領域にB細胞特異的に結合する蛋白質を同定し、そのうちの一つであるThy28蛋白質がPax5遺伝子の発現に重要な働きをしていることを明らかにした。Thy28結合蛋白質を同定し、Thy28は、そのうちの一つであるmyosin-9 (MYH9) 蛋白質のPax5遺伝子プロモーターへの結合に重要であることを示した。加えて、MYH9もPax5遺伝子の発現に重要であることを明らかにした。こうした成果をPLoS One誌に発表した。また、iChIP-RNA-Seq法を用いて、Pax5遺伝子プロモーター領域にB細胞特異的に結合するRNAを同定した。加えて、iChIP-Seq法を用いて、Pax5遺伝子プロモーター領域にB細胞特異的に結合するゲノム領域を同定した。次に、この領域をCRISPR系を用いて欠損させた細胞株を樹立した。現在、この領域がPax5遺伝子発現制御に果たす役割を解析中である。
2. Foxp3遺伝子転写調節機構の解明
ジーンターゲティング法を用いて、Foxp3遺伝子のTSS近傍にLexA結合配列を挿入したマウスES細胞を作製した。この細胞を用いて、マウス個体を作製し、これと全身の細胞でCreリコンビネースを発現しているマウスとの掛け合わせによって、ES細胞の選択に用いたネオマイシン耐性遺伝子を除去した。このマウス及び対照として野生型C57BL/6マウスから、T-regと対照細胞であるナイーブCD4T細胞を単離した。現在、新たに開発した組換えタグ付きLexA蛋白質を用いたin vitro iChIP法を用いて、Foxp3遺伝子プロモーター領域を単離し、次世代シークエンス法によるT-reg特異的に相互作用しているゲノム領域の同定を進めている。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2014 Annual Research Report
  • 2013 Annual Research Report
  • Research Products

    (26 results)

All 2015 2014 2013 Other

All Journal Article (8 results) (of which Peer Reviewed: 5 results,  Open Access: 3 results,  Acknowledgement Compliant: 3 results) Presentation (15 results) (of which Invited: 4 results) Remarks (2 results) Patent(Industrial Property Rights) (1 results) (of which Overseas: 1 results)

  • [Journal Article] A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells2015

    • Author(s)
      Fujita T, Kitaura F, and Fujii H
    • Journal Title

      PLoS One

      Volume: 10 Issue: 1 Pages: e0116579-e0116579

    • DOI

      10.1371/journal.pone.0116579

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR2014

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

      PLoS One

      Volume: 9 Issue: 7 Pages: e103084-e103084

    • DOI

      10.1371/journal.pone.0103084

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins2014

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

      BMC Mol. Biol.

      Volume: 15 Issue: 1 Pages: 26-26

    • DOI

      10.1186/s12867-014-0026-0

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] TALやCRISPRを用いたenChIP法による特定ゲノム領域の単離と結合分子の同定2014

    • Author(s)
      藤井 穂高
    • Journal Title

      実験医学別冊 「今すぐ始めるゲノム編集」

      Volume: 2014 Pages: 42-43

    • Related Report
      2013 Annual Research Report
  • [Journal Article] Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.2013

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

      Biochem Biophys Res Commun.

      Volume: 439 Issue: 1 Pages: 132-6

    • DOI

      10.1016/j.bbrc.2013.08.013

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).2013

    • Author(s)
      Toshitsugu Fujita, Yoshinori Asano, Junko Ohtsuka, Yoko Takada, Kazunobu Saito, Rieko Ohki, Hodaka Fujii.
    • Journal Title

      Scientific Reports

      Volume: 3 Issue: 1 Pages: 3-3

    • DOI

      10.1038/srep03171

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed
  • [Journal Article] iChIP法による特定ゲノム領域の単離と結合分子の同定2013

    • Author(s)
      藤井 穂高
    • Journal Title

      遺伝子医学Mook「エピジェネティクスと病気」(メディカルドゥ社)

      Volume: 25 Pages: 254-259

    • Related Report
      2013 Annual Research Report
  • [Journal Article] 挿入的クロマチン免疫沈降法 (iChIP) による特定ゲノム領域結合分子の網羅的同定2013

    • Author(s)
      藤田 敏次、藤井 穂高
    • Journal Title

      実験医学(羊土社)

      Volume: 31 Pages: 2629-2636

    • Related Report
      2013 Annual Research Report
  • [Presentation] Capturing chromatin: biochemical analysis of genome functions using the locus-specific chromatin immunoprecipitation technologies2014

    • Author(s)
      Fujita T, Fujii H
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] TALやCRISPR/Cas9を利用したenChIP法による遺伝子座特異的生化学的ゲノム機能解析2014

    • Author(s)
      藤田 敏次、藤井 穂高
    • Organizer
      第37回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2014-11-25 – 2014-11-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) 法による特定ゲノム領域の単離と結合分子の同定2014

    • Author(s)
      藤井 穂高
    • Organizer
      第4回ゲノム編集研究会
    • Place of Presentation
      広島国際会議場
    • Year and Date
      2014-10-06 – 2014-10-07
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] enChIP: a method for locus-specific biochemical analysis of genome functions2014

    • Author(s)
      Fujita T, Fujii H
    • Organizer
      11th EMBL Conference: Transcription and Chromatin
    • Place of Presentation
      Heidelberg, Germany
    • Year and Date
      2014-08-23 – 2014-08-26
    • Related Report
      2014 Annual Research Report
  • [Presentation] enChIP: a method for locus-specific biochemical analysis of genome functions2014

    • Author(s)
      Fujita T, Asano Y, Ohtsuka J, Saito K, Takada Y, Ohki R, Fuji H
    • Organizer
      第8回日本エピジェネティクス研究会年会
    • Place of Presentation
      伊藤国際学術研究センター
    • Year and Date
      2014-05-25 – 2014-05-27
    • Related Report
      2014 Annual Research Report
  • [Presentation] Biochemical analysis of lymphocyte lineage commitment using the locus-specific chromatin immunoprecipitation technology (iChIP and enChIP)2014

    • Author(s)
      Fujii H
    • Organizer
      2nd International Molecular Immunology & Immunogenetics Congress (MIMIC-II)
    • Place of Presentation
      Ramada Plaza Hotel Antalya, Turkey
    • Year and Date
      2014-04-27 – 2014-04-30
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Analysis of chromatin mechanisms by locus-specific chromatin immunoprecipitation technologies2014

    • Author(s)
      Hodaka Fujii, Toshitsugu Fujita
    • Organizer
      Keystone Symposium: Chromatin Mechanisms and Cell Physiology
    • Place of Presentation
      Oberstdorf, Germany
    • Related Report
      2013 Annual Research Report
  • [Presentation] Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immnoprecipitation (iChIP)2013

    • Author(s)
      Toshitsugu Fujita, Hodaka Fujii
    • Organizer
      第7回日本エピジェネティクス研究年会
    • Place of Presentation
      奈良
    • Related Report
      2013 Annual Research Report
  • [Presentation] Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immnoprecipitation (iChIP)2013

    • Author(s)
      Hodaka Fujii
    • Organizer
      Epigenomics & Metabolomics Symposia-2013: Epigenomics, Sequencing & SNiPs-2013
    • Place of Presentation
      Boston, USA
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immnoprecipitation (iChIP)2013

    • Author(s)
      Toshitsugu Fujita, Hodaka Fujii
    • Organizer
      15th International Congress of Immunology
    • Place of Presentation
      Milano, Italy
    • Related Report
      2013 Annual Research Report
  • [Presentation] Identification and characterization of promoter-bound molecules involved in transcriptional regulation by insertional chromatin immunoprecipitation (iChIP)2013

    • Author(s)
      Toshitsugu Fujita, Hodaka Fujii
    • Organizer
      Cold Spring Harbor meeting: Mechanisms of Eukaryotic Transcription
    • Place of Presentation
      Cold Spring Harbor, USA
    • Related Report
      2013 Annual Research Report
  • [Presentation] Locus-specific biochemical analysis of genome functions by iChIP and enChIP2013

    • Author(s)
      Hodaka Fujii
    • Organizer
      Therapeutics Discovery Symposia Asia - 2013, Small RNAs to Stem Cells & Epigenetic Reprogramming - 2013
    • Place of Presentation
      東京
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] enChIP: a method for locus-specific biochemical analysis of genome functions2013

    • Author(s)
      Toshitsugu Fujita, Yoshinori Asano, Junko Ohtsuka, Kazunobu Saito, Yoko Takada, Reiko Ohki, Hodaka Fujii
    • Organizer
      第36回日本分子生物学会年会
    • Place of Presentation
      神戸
    • Related Report
      2013 Annual Research Report
  • [Presentation] 新規テロメア結合タンパク質の同定と機能解析 ~がん化への作用の解明~2013

    • Author(s)
      Takigawa Haruka, Yoshinori Asano, Junko Ohtsuka, Toshitsugu Fujita, Namiki Hideo, Hodaka Fujii, Reiko Ohki
    • Organizer
      第36回日本分子生物学会年会
    • Place of Presentation
      神戸
    • Related Report
      2013 Annual Research Report
  • [Presentation] Identification and characterization of promoter-bound molecules involved in transcriptional regulation of Pax5 by insertional chromatin immunoprecipitation (iChIP)2013

    • Author(s)
      Toshitsugu Fujita, Hodaka Fujii
    • Organizer
      2013 日本免疫学会学術集会
    • Place of Presentation
      千葉
    • Related Report
      2013 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
      2014 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
      2013 Annual Research Report
  • [Patent(Industrial Property Rights)] 内在性DNA配列特異的結合分子を用いる特定ゲノム領域の単離方法2013

    • Inventor(s)
      藤井穂高、藤田敏次
    • Industrial Property Rights Holder
      藤井穂高、藤田敏次
    • Industrial Property Rights Type
      特許
    • Filing Date
      2013-09-06
    • Related Report
      2013 Annual Research Report
    • Overseas

URL: 

Published: 2013-05-15   Modified: 2019-07-29  

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