Development of New methodology for Genome-wide Detection of Multi-contact Interaction between Chromatin Regions in Single Molecular Resolution

• Project/Area Number: 20K21384
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: Fujiki Katsunori 東京大学, 定量生命科学研究所, 助教 (10646730)
• Project Period (FY): 2020-07-30 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2021: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2020: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: ゲノム / クロマチン高次構造 / 次世代シーケンサー / ロングリードシーケンサー / Hi-C / Pore-C / 多点間相互作用 / HiChIP / ChIA-PET / ChIA-drop / クロマチン構造 / 多領域間クロマチン相互作用 / ゲノム高次構造 / クロマチン相互作用

Outline of Research at the Start

ゲノムの高次構造を理解することはゲノム情報の制御を考える上で非常に重要だが、Hi-Cなどの2点間DNA接合を技術基盤とする既存の解析手法では、昨今注目を集めている相分離によるクロマチン構造制御やsuper enhancerによる転写制御など、[細胞間での変動]がある場合や[多数の領域間]での相互作用等の現象を解析することは難しい。そこで我々は、クロマチンの相互作用を[多点間]で[１分子の解像度]で検出できるChIA-drop法を開発したが、その効率に未だ改良の余地がある。そこで本研究では多点間相互作用検出法の効率を改善し、次世代のゲノム構造解析の主流となり得る新たな研究手法を開発する。

Outline of Final Research Achievements

Through this project, I developed a new method, "targeted Pore-C," which enables the multi-contact analysis in genome-wide with a single molecular resolution. The original Pore-C method is a derivative version of the genome-wide contact detection method "Hi-C." It can detect multi-contact using a long-read sequencer and unfragmented library. I improved this Pore-C to focus on multi-contacts mediated by a specific factor of interest, which was achieved by introducing a chemical modification to DNA nearby the factor. Development of the new method in this project gained a sure success and remaining some challenges for practical use, such as improvement of modification efficiency.

Academic Significance and Societal Importance of the Research Achievements

クロマチン領域の相互の物理的位置関係はゲノム情報の発現に非常に重要な役割を果たしている。これらを解析する手法として現在はHi-CやHiChIPなどの方法が用いられてきたが、これら既存法は原理的にクロマチン高次構造を２領域間の点と点の相互作用の積算としてしか検出できない。今回Pore-C法をもとに開発したtargeted Pore-Cは、標的分子周辺に形成された多点間の相互作用を１分子の解像度で検出することを可能にし、近接領域を点と点ではなく塊で捉え、また細胞間のバリエーションを損なうことなく、既存技術では観察し得なかった核内のクロマチン高次構造を記述できる。

Research Products

• [Int'l Joint Research] Children's Hospital of Philadelphia(米国)
• [Int'l Joint Research] Karolinska Institutet(スウェーデン)
• [Int'l Joint Research] Margarita Salas Centre for Biological Re(スペイン)
• [Int'l Joint Research] カロリンスカ研究所(スウェーデン)
• [Int'l Joint Research] フィラデルフィア小児病院(米国)
• [Int'l Joint Research] Spanish National Research Council(スペイン)
• [Int'l Joint Research] アバディーン大学(英国)
• [Journal Article] Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells (2022)
  • Author(s): Hada M, Miura H, Tanigawa A, Matoba S, Inoue K, Ogonuki N, Hirose M, Watanabe N, Nakato R, Fujiki K, Hasegawa A, Sakashita A, Okae H, Miura K, Shikata D, Arima T, Shirahige K, Hiratani I, Ogura A.
  • Journal Title: Genes Dev Volume: 36 Issue: 1-2 Pages: 84-102
  • DOI: 10.1101/gad.348782.121
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] MAB21L1 modulates gene expression and DNA metabolic processes in the lens placode (2021)
  • Author(s): Yamada R, Oguri A, Fujiki K, Shirahige K, Hirate Y, Kanai-Azuma M, Takezoe H, Akimoto Y, Takahashi N, Kanai Y.
  • Journal Title: Dis Model Mech Volume: 14 Issue: 12
  • DOI: 10.1242/dmm.049251
  • Peer Reviewed / Open Access
• [Journal Article] Codependency and mutual exclusivity for gene community detection from sparse single-cell transcriptome data (2021)
  • Author(s): Nakajima N, Hayashi T, Fujiki K, Shirahige K, Akiyama T, Akutsu T, Nakato R.
  • Journal Title: Nucleic Acids Res Volume: 49 Issue: 18 Pages: e104-e104
  • DOI: 10.1093/nar/gkab601
  • Peer Reviewed / Open Access
• [Journal Article] Small extracellular vesicles derived from interferon-γ pre-conditioned mesenchymal stromal cells effectively treat liver fibrosis (2021)
  • Author(s): Takeuchi S, Tsuchiya A, Iwasawa T、Nojiri S、Watanabe T、Ogawa M、Yoshida T、Fujiki K、Koui Y、Kido T、Yoshioka Y、Fujita M、Kikuta J、Itoh T、Takamura M、Shirahige K、Ishii M、Ochiya T、Miyajima A、Terai S
  • Journal Title: npj Regenerative Medicine Volume: 6 Issue: 1
  • DOI: 10.1038/s41536-021-00132-4
  • Peer Reviewed / Open Access
• [Journal Article] Single-cell transcriptional analysis reveals developmental stage-dependent changes in retinal progenitors in the murine early optic vesicle (2021)
  • Author(s): Yamada R, Oguri A, Fujiki K, Shirahige K, Takezoe H, Takahashi N, Kanai Y.
  • Journal Title: Biochem Biophys Res Commun Volume: 543 Pages: 80-86
  • DOI: 10.1016/j.bbrc.2021.01.043
  • Peer Reviewed / Open Access
• [Journal Article] Age-Dependent Ribosomal DNA Variations in Mice (2020)
  • Author(s): Watada Eriko、Li Sihan、Hori Yutaro、Fujiki Katsunori、Shirahige Katsuhiko、Inada Toshifumi、Kobayashi Takehiko
  • Journal Title: Molecular and Cellular Biology Volume: 40 Issue: 22
  • DOI: 10.1128/mcb.00368-20
• [Journal Article] Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity (2020)
  • Author(s): Tanaka, S. Ise, W. Inoue, T. Ito, A. Ono, C. Shima, Y. Sakakibara, S. Nakayama, M. Fujii, K. Miura, I. Sharif, J. Koseki, H. Koni, P. A. Raman, I. Li, Q. Z. Kubo, M. Fujiki, K. Nakato, R. Shirahige, K. Araki, H. Miura, F. Ito, T. Kawakami, E. Baba, Y. Kurosaki, T.
  • Journal Title: Nature Immunology Volume: 21 Issue: 8 Pages: 950-961
  • DOI: 10.1038/s41590-020-0700-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] シングルセルレベルにおける黒髪と白髪の発現変動解析 (2021)
  • Author(s): 渡邉 紘介、藤木 克則、須谷 尚史、白髭 克彦
  • Organizer: 第44回日本分子生物学会年会
  • Int'l Joint Research
• [Presentation] 多点間クロマチン相互作用検出技術によるゲノム高次構造の解析 (2020)
  • Author(s): 藤木克則、白髭克彦
  • Organizer: 第48回日本臨床免疫学会総会
  • Invited