Science & Technologies

DNAは、周囲のさまざまなタンパク質やRNA等と複合体であるクロマチンを形成し、エピジェネティック制御や転写制御に深く関わっています。クロマチンとその遺伝子制御メカニズムを正しく理解するためには、注目する遺伝子座領域に結合する多くの分子を同定し機能を解明する必要があります。ところが、特定の遺伝子座領域に結合している分子を同定する方法は、従来存在しませんでした。

Wisteriagenが保有する遺伝子座特異的ChIP法は、着目する遺伝子座を任意に決定したのち、この遺伝子座に結合する分子を網羅的に捕捉し同定することができる技術です。遺伝子座特異的ChIP法には、insertional ChIP (iChIP) 法と、engineered DNA-binding molecule-mediated ChIP (enChiP) 法の2種類があります。

Wisteriagenは、遺伝子座特異的ChIP法による創薬ターゲット探索のほか、がん領域と中枢神経領域の自社開発パイプラインを保有し、研究開発を推進しています。

遺伝子座特異的ChIP法のメカニズムは以下のとおりです。

  • 任意の標的ゲノム領域を、人工DNA結合分子を用いてタグ付けした細胞を樹立する。
  • ホルムアルデヒド等の架橋剤でクロスリンクする。
  • 超音波処理により細胞を破砕しゲノムDNAを断片化する。
  • 適当な方法で、人工DNA結合分子が結合したゲノム領域をアフィニティー精製する。
  • クロスリンクをはずし、複合体中のタンパク質・DNA・RNAを同定し、これらの分子のキャラクタリゼーションを行う。

原著論文

  • Fujii H, Fujita T: An enChIP system for the analysis of genome functions in budding yeast. Biol. Methods Protoc. (2022) 7, bpac025.

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  • Sarudate A, Fujita T, Nakayama T, Fujii H: enChIP-Seq analyzer: a software program to analyze and interpret enChIP-Seq data for the detection of physical interactions between genomic regions. Genes (2022) 13, 472.

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  • Takeda T, Yokoyama Y, Takahashi H, Okuzaki D, Asai K, Itakura H, Miyoshi N, Kobayashi S, Uemura M, Fujita T, Ueno H, Mori M, Doki Y, Fujii H, Eguchi H, Yamamoto H: A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells. Br. J. Cancer (2021) 126, 109-119.

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  • Yuno M, Nagata S, Fujita T, Fujii H: MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis. Biol. Methods Protoc. (2021) 6, bpab013.

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  • Husain A, Xu J, Fujii H, Nakata M, Kobayashi M, Wang JY, Rehwinkel J, Honjo T, Begum NA: SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination. EMBO J. (2020) 39, e102931.

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  • Fujita T, Yuno M, Fujii H: An enChIP system for the analysis of bacterial genome functions. BMC Res. Notes (2018) 11, 387.

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  • Hamidian A, Vaapil M, von Stedingk K, Fujita T, Persson CU, Eriksson P, Veerla S, De Preter K, Speleman F, Fujii H, Påhlman S, Mohlin S: Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator. Biochem. Biophys. Res. Commun. (2018) 499, 291-298.

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  • Fujita T, Yuno M, Fujii H: enChIP systems using different CRISPR orthologues and epitope tags. BMC Res. Notes (2018) 11, 154.

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  • Fujita T, Kitaura F, Oji A, Tanigawa N, Yuno M, Ikawa M, Taniuchi I, Fujii H: Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis. Genes Cells (2018) 23, 318-325.

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  • Fujita T, Kitaura F, Yuno M, Suzuki Y, Sugano S, Fujii H: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Res. (2017) 24, 537–548.

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  • Fujita T, Yuno M, Suzuki Y, Sugano S, Fujii H: Identification of physical interactions between genomic regions by enChIP-Seq. Genes Cells (2017) 22, 506-520.

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  • Fujita T, Yuno M, Fujii H: Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus. Sci. Rep. (2016) 6, 30485.

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  • Fujita T, Yuno M, Fujii H: Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells (2016) 21, 370-377.

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  • Fujita T, Yuno M, Okuzaki D, Ohki R, Fujii H: Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing. PLoS One (2015) 10, e0123387.

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  • Fujita T, Kitaura F, and Fujii H: A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells. PLoS One (2015) 10, e0116579.

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  • Fujita T, and Fujii H: Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins. BMC Mol. Biol. (2014) 15, 26.

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  • Fujita T, and Fujii H: Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR. PLoS One (2014) 9, e103084.

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  • Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, and Fujii H: Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci. Rep. (2013) 3, 3171.

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  • Fujita T, and Fujii H: Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. (2013) 439, 132-136.

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  • Fujita T, and Fujii H: Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Adv. Biosci. Biotechnol. (2012) 3, 626-629.

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  • Fujita T, and Fujii H: Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One (2011) 6, e26109.

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  • Hoshino A, and Fujii H: Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J. Biosci. Bioeng. (2009), 108, 446-449.

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総説

  • Fujita H, Fujita T, and Fujii H: Locus-specific genomic DNA purification using the CRISPR system: methods and applications. CRISPR J. (2021) 4 (2), 290-300.

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  • Fujita T, and Fujii H: Purification of specific DNA species using the CRISPR system. Biol. Methods Protoc. (2019) 4 (1), bpz008.

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  • 藤井　穂高：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調節機構の生化学的解析. 弘前医学 (2019) 69 (1-4): 10-18.

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  • Fujita T, and Fujii H: in vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (in vitro enChIP) using CRISPR ribonucleoproteins in combination with next-generation sequencing (in vitro enChIP-Seq) for the identification of chromosomal interactions. Bio-protocol (2017) 7 (22), doi: 10.21769/BioProtoc.2612.

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  • 藤井　穂高、藤田敏次：遺伝子座特異的クロマチン免疫沈降法による特定ゲノム領域結合分子の同定とゲノム高次構造解析. 生体の科学（医学書院）(2017) 6月号、68 (3): 189-193.

  • Fujita T, and Fujii H: Isolation of specific genomic regions and identification of associated molecules by enChIP. JoVE (2016) issue 107, doi: 10.3791/53478.

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  • Fujita T, and Fujii H: Biochemical analysis of genome functions using locus-specific chromatin immunoprecipitation technologies. Gene Regul. Syst. Bio. (2016) Suppl. 1, 1-9.

  • 藤井　穂高: 遺伝子座特異的クロマチン免疫沈降法によるエピジェネティック創薬. 化学工業（化学工業社）(2015) 12月号、66 (12):28-34.

  • Fujita T, and Fujii H: Applications of engineered DNA-binding molecules such as TAL proteins and the CRISPR/Cas system in biology research. Int. J. Mol. Sci. (2015) 16, 23143-23164.

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  • Fujii H, and Fujita T: 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. Int. J. Mol. Sci. (2015) 16, 21802-21812.

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  • 藤田　敏次、藤井　穂高: CRISPR/Casのゲノム編集以外への応用. 実験医学（羊土社）(2014) 7月号、32 (11):1732-1736.

  • Fujita T, and Fujii H: Identification of proteins interacting with genomic regions of interest in vivo using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Bio-protocol (2014) 4 (10), doi: 10.21769/BioProtoc.1124.

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  • 藤田　敏次、藤井　穂高: 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. 実験医学（羊土社）(2013) 10月号、31 (16):2629-2636.

  • Fujita T, and Fujii H: Locus-specific biochemical epigenetics/chromatin biochemistry by insertional chromatin immunoprecipitation. ISRN Biochem. (2013) 2013, 913273.

著書

  • Fujita T, and Fujii H: New directions for epigenetics: application of engineered DNA-binding molecules to locus-specific epigenetic research. Handbook of Epigenetics, Third Edition, Academic Press (Elsevier), Editor: Trygve O. Tollefsbol, (2023) 843-868.

  • 藤田　敏次、藤井　穂高：CRISPRを利用した配列特異的なDNAの単離　実験医学別冊「完全版　ゲノム編集実験スタンダード」（羊土社）　編集：山本　卓，佐久間　哲史　(2019) 309-316.

  • 藤田　敏次、藤井　穂高：ゲノム編集技術の応用：enChIP法とその創薬への応用　DOJIN BIOSCIENCE 29「医療応用を目指すゲノム編集」（化学同人）　編集：真下　知士、金田　安史　(2018) 53-64.

  • Fujita T, and Fujii H: New directions for epigenetics: application of engineered DNA-binding molecules to locus-specific epigenetic research. Handbook of Epigenetics, Second Edition, Academic Press (Elsevier), Editor: Trygve O. Tollefsbol, (2017) 635-652.

  • 藤井　穂高：遺伝子座特異的クロマチン免疫沈降法を用いたエピジェネティック作動薬・抗感染症薬の開発 - バイオベンチャー「Epigeneron」の取り組み　実験医学増刊「All About ゲノム編集」（羊土社）　編集：真下　知士、山本　卓　(2016) 34 (20): 185-191.

  • 藤田　敏次、藤井　穂高：CRISPR/Cas9を用いたenChIP法による遺伝子座特異的ゲノム機能解析　「進化するゲノム編集技術」（株式会社エヌ・ティー・エス）　(2015) 第1編　第2章　第3節

  • Fujita T, and Fujii H: Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Methods Mol. Biol. (2015) 1288:43-52. doi: 10.1007/978-1-4939-2474-5_4.

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  • 藤井　穂高: 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. 実験医学別冊「今すぐ始めるゲノム編集」（羊土社）　編集：山本　卓(2014) 42-43.

  • 藤井　穂高: iChIP法による特定ゲノム領域の単離と結合分子の同定. 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）監修：佐々木　裕之、編集：中尾　光善、中島　欽一　(2013) 25:254-259.

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