Kinetic analysis of gene expression regulation using visualization and epigenomic manipulation of specific genomic regions

• Project/Area Number: 17K17719
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Cell biology; Molecular biology
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: Handa Tetsuya 東京工業大学, 科学技術創成研究院, 特任助教 (40772570)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: エピジェネティクス / 遺伝子発現制御 / ヒストン修飾 / クロマチン / 生細胞イメージング / CRISPR/Cas9 / エピゲノム編集 / RNA Polymerase 2 / エピゲノム操作 / CRISPR/Cas

Outline of Final Research Achievements

We investigated the dynamics of chromatin structural changes and transcriptional activation in live-cell imaging by combining post-translational modification specific probes against histone and RNA Polymerase 2 (RNAPol2) with CRISPR/Cas9-based visualization of specific genomic regions and manipulation of the epigenome. The histone acetylation including H3K27ac preceded and was independent of activation of RNAPol2. The chromatin structure changed and the mobility correlated with histone modifications and RNAPol2 states.

Academic Significance and Societal Importance of the Research Achievements

本研究課題では、従来の細胞を固定するようなスナップショット的な解析では理解が進んでいなかった、環境応答や細胞分化の際にクロマチン構造がどのように変化し、遺伝子発現が制御されているのかという問題に取り組んだ。反応過程の経路図的な理解にとどまらず、ヒストン修飾を介したクロマチンの運動性制御とRNAPol2活性化との相関など、転写制御の新たな側面の発見につながった。また、人為的な遺伝子発現制御は細胞の形質を操作するための技術として重要であり、将来的な高効率な細胞の分化誘導やリプロクグラミングの基盤技術開発につながることが期待でき、再生医療等にも広く波及効果を及ぼすと考えられる。

Research Products

• [Int'l Joint Research] Colorado State University(米国)
• [Int'l Joint Research] University of Copenhagen(デンマーク)
• [Int'l Joint Research] Institut Curie(フランス)
• [Int'l Joint Research] EMBL Heidelberg(ドイツ)
• [Journal Article] Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II (2021)
  • Author(s): Uchino Satoshi、Ito Yuma、Sato Yuko、Handa Tetsuya、Ohkawa Yasuyuki、Tokunaga Makio、Kimura Hitoshi
  • Journal Title: bioRxiv Volume: - Pages: 441582-441582
  • DOI: 10.1101/2021.04.27.441582
  • Open Access
• [Journal Article] H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing (2021)
  • Author(s): Tjalsma Sjoerd J D、Hori Mayako、Sato Yuko、Bousard Aurelie、Ohi Akito、Raposo Ana Claudia、Roensch Julia、Le Saux Agnes、Nogami Jumpei、Maehara Kazumitsu、Kujirai Tomoya、Handa Tetsuya、Bages‐Arnal Sandra、Ohkawa Yasuyuki、Kurumizaka Hitoshi、da Rocha Simao Teixeira、Zylicz Jan J、Kimura Hiroshi、Heard Edith
  • Journal Title: EMBO reports Volume: 22 Issue: 3
  • DOI: 10.15252/embr.202051989
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Euchromatin factors HULC and Set1C affect heterochromatin organization for mating-type switching in fission yeast Schizosaccharomyces pombe (2021)
  • Author(s): Chavez Alfredo Esquivel、Maki Takahisa、Tsubouchi Hideo、Handa Testuya、Kimura Hiroshi、Haber James E.、Thon Genevieve、Iwasaki Hiroshi
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/2021.03.23.436714
  • Open Access / Int'l Joint Research
• [Journal Article] Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene (2020)
  • Author(s): Forero-Quintero Linda S.、Raymond William、Handa Tetsuya、Saxton Matthew、Morisaki Tatsuya、Kimura Hiroshi、Bertrand Edouard、Munsky Brian、Stasevich Timothy J.
  • Journal Title: bioRxiv Volume: - Pages: 024414-024414
  • DOI: 10.1101/2020.04.03.024414
  • Open Access / Int'l Joint Research
• [Journal Article] Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input. (2020)
  • Author(s): Tetsuya Handa, Akihito Harada, Kazumitsu Maehara, Shoko Sato, Masaru Nakao, Naoki Goto, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Hiroshi Kimura.
  • Journal Title: Nat Protoc. Volume: 15 Issue: 10 Pages: 3334-3360
  • DOI: 10.1038/s41596-020-0375-8
  • Peer Reviewed / Open Access
• [Journal Article] Modeling population size independent tissue epigenomes by ChIL-seq with single-thin sections (2020)
  • Author(s): Maehara Kazumitsu、Tomimatsu Kosuke、Harada Akihito、Tanaka Kaori、Sato Shoko、Okada Seiji、Handa Tetsuya、Kurumizaka Hitoshi、Kimura Hiroshi、Ohkawa Yasuyuki
  • Journal Title: bioRxiv Volume: - Pages: 423434-423434
  • DOI: 10.1101/2020.12.18.423434
  • Open Access
• [Journal Article] Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging (2019)
  • Author(s): Kazuo Yamagata, Kouhei Nagai, Hiroshi Miyamoto, ...., Tetsuya Handa (17番目), Hiroshi Kimura, Yoshihiko Hosoi, Tasuku Mitani, Kazuya Matsumoto, Akira Iritani
  • Journal Title: Scientific Reports Volume: 9 巻 Issue: 1 Pages: 4050-4050
  • DOI: 10.1038/s41598-019-40546-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Detailed protocol ─ Chromatin Integration labeling (2019)
  • Author(s): Tetsuya Handa, Akihito Harada, Kazumitsu Maehara, Yasuyuki Ohkawa, Hiroshi Kimura
  • Journal Title: Protocol Exchange Volume: 122 Pages: 122-122
  • DOI: 10.1038/protex.2018.122
• [Journal Article] A chromatin integration labelling method enables epigenomic profiling with lower input (2018)
  • Author(s): Harada Akihito、Maehara Kazumitsu、Handa Tetsuya、Arimura Yasuhiro、Nogami Jumpei、Hayashi-Takanaka Yoko、Shirahige Katsuhiko、Kurumizaka Hitoshi、Kimura Hiroshi、Ohkawa Yasuyuki
  • Journal Title: Nature Cell Biology Volume: 21 Issue: 2 Pages: 287-296
  • DOI: 10.1038/s41556-018-0248-3
  • Peer Reviewed / Open Access
• [Journal Article] Shelterin promotes tethering of late replication origins to telomeres for replication‐timing control (2018)
  • Author(s): Ogawa Shiho、Kido Sayuri、Handa Tetsuya、Ogawa Hidesato、Asakawa Haruhiko、Takahashi Tatsuro S、Nakagawa Takuro、Hiraoka Yasushi、Masukata Hisao
  • Journal Title: The EMBO Journal Volume: 37 Issue: 15 Pages: 15-15
  • DOI: 10.15252/embj.201898997
  • Peer Reviewed / Open Access
• [Journal Article] Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase (2017)
  • Author(s): Yamazaki Taiga、Hatano Yu、Handa Tetsuya、Kato Sakiko、Hoida Kensuke、Yamamura Rui、Fukuyama Takashi、Uematsu Takayuki、Kobayashi Noritada、Kimura Hiroshi、Yamagata Kazuo
  • Journal Title: PLoS ONE Volume: 12 Issue: 5 Pages: 1-18
  • DOI: 10.1371/journal.pone.0177764
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Multi-ChIL法による少数細胞の複数エピゲノムの同時解析 (2019)
  • Author(s): 半田哲也、原田哲仁、前原一満、大川恭行、木村宏
  • Organizer: 第13回日本エピジェネティクス研究会年会
• [Presentation] 特定ゲノム領域の生細胞イメージングとエピゲノム操作による遺伝子発現制御 (2019)
  • Author(s): 半田哲也、木村宏
  • Organizer: 第19回日本蛋白質科学会年会 第71回日本細胞生物学会大会 合同年次大会
  • Invited
• [Presentation] Chromatin integration labeling technology enables low-input epigenomic profiling (2018)
  • Author(s): Tetsuya Handa, Akihito Harada, Kazumitsu Maehara, Yasuhiro Arimura, Jumpei Nogami, Yoko Hayashi-Takanaka, Katsuhiko Shirahige, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Hiroshi Kimura
  • Organizer: 2018 Cold Spring Harbor meeting: Nuclear Organization & Function
  • Int'l Joint Research
• [Presentation] 特定ゲノム領域の生細胞イメージングとエピゲノム操作による遺伝子発現制御 (2018)
  • Author(s): 半田 哲也
  • Organizer: 日本ゲノム編集学会第3回大会
  • Invited
• [Presentation] A chromatin integration labeling method enables epigenomic profiling with lower input (2018)
  • Author(s): Tetsuya Handa, Akihito Harada, Kazumitsu Maehara, Yasuhiro Arimura, Jumpei Nogami, Yoko Hayashi-Takanaka, Katsuhiko Shirahige, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Hiroshi Kimura
  • Organizer: 3R&3C Symposium
  • Int'l Joint Research
• [Presentation] Dynamics of Histone Modifications and RNA Polymerase II in Heat Shock Response (2017)
  • Author(s): Tetsuya Handa, Hiroshi Kimura
  • Organizer: EMBO Conference: Chromatin and Epigenetics
  • Int'l Joint Research
• [Presentation] 熱ショック応答におけるヒストン修飾とRNA Polymerase 2 の動態解析 (2017)
  • Author(s): 半田哲也、木村宏
  • Organizer: 第11回日本エピジェネティクス研究会年会
• [Presentation] 熱ショック応答におけるヒストン修飾とRNA Polymerase 2 の動態解析 (2017)
  • Author(s): 半田哲也、木村宏
  • Organizer: 第２回クロマチン動構造ワークショップ
• [Book] 実験医学別冊 - エピゲノムをもっと見るための クロマチン解析実践プロトコール (分担執筆) (2020)
  • Author(s): 半田哲也
  • Total Pages: 270
  • Publisher: 羊土社
• [Book] 遺伝子医学 通巻31号 (復刊6号) 「ヒストン修飾解析の最前線」 (2020)
  • Author(s): 半田哲也、木村宏
  • Publisher: (株)メディカルドゥ
• [Remarks] 同一の細胞から複数のエピゲノム情報を同時に検出する技術開発に成功
  • URL: https://www.titech.ac.jp/news/2020/047602.html
• [Remarks] A detailed step-by-step protocol of ChIL-seq…
  • URL: https://protocolsmethods.springernature.com/posts/chromatin-integration-labeling-for-mapping-dna-binding-proteins-and-modifications-with-low-input
• [Remarks] 世界初、単一細胞での遺伝子発現制御解析に成功
  • URL: https://www.titech.ac.jp/news/2018/042981.html