Development of functional enhancer screening system by integrating genome editing technology with single-cell RNA-seq

• Project/Area Number: 18K19636
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 57:Oral science and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: Hojo Hironori 東京大学, 大学院医学系研究科(医学部), 准教授 (80788422)
• Co-Investigator(Kenkyū-buntansha): 大庭 伸介 長崎大学, 医歯薬学総合研究科(歯学系), 教授 (20466733)
• Project Period (FY): 2018-06-29 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2020: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000); Fiscal Year 2019: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: エンハンサー / ゲノム編集 / 一細胞解析 / 骨芽細胞 / エンハンサースクリーニング / 骨形成 / 転写制御 / 一細胞RNA-seq / 骨発生

Outline of Final Research Achievements

In this study, we aimed at establishment of enhancer screening system by using osteoblast differentiation as a model. We constructed guide RNA library that targets putative osteoblast enhancers. We then expressed the library DNA into osteogenic cells that stably expressed Cas9. After osteoblast induction, cells were isolated and single cell RNA-seq was performed. In the assay, we enabled to detect “genotype” as guide RNA sequencing and “phenotypes” as gene expression profile simultaneously at single cell resolution. Through this analysis we identified a functional enhancer for osteoblast differentiation.

Academic Significance and Societal Importance of the Research Achievements

遺伝子の発現を規定する転写ネットワークは、器官発生や細胞の分化決定だけでなく、疾患の分子病態の理解の根底をなすと考えられています。当該分野において、これまで、「転写ネットワークの全貌解明」を目指す研究が活発に進められてきましたが、「転写ネットワークの中で、どこが重要であるか」を生物学的な実験で効率よく抽出する方法は十分に確立していませんでした。本研究では、転写ネットワークを制御するエンハンサーに着目して、重要なエンハンサーを同定する手法を確立しました。本手法を応用することで、生物発生の成り立ちやヒトの病気のメカニズムの解析に役立つことが期待されます。

Research Products

• [Journal Article] The Progress of Stem Cell Technology for Skeletal Regeneration (2021)
  • Author(s): Tani Shoichiro、Okada Hiroyuki、Chung Ung-il、Ohba Shinsuke、Hojo Hironori
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Issue: 3 Pages: 1404-1404
  • DOI: 10.3390/ijms22031404
  • Peer Reviewed / Open Access
• [Journal Article] Gene regulatory landscape in osteoblast differentiation (2020)
  • Author(s): Hojo Hironori、Ohba Shinsuke
  • Journal Title: Bone Volume: 137 Pages: 115458-115458
  • DOI: 10.1016/j.bone.2020.115458
  • NAID: 120006988169
  • Peer Reviewed
• [Journal Article] Hedgehog Activation Regulates Human Osteoblastogenesis (2020)
  • Author(s): Onodera Shoko、Saito Akiko、Hojo Hironori、Nakamura Takashi、Zujur Denise、Watanabe Katsuhito、Morita Nana、Hasegawa Daigo、Masaki Hideki、Nakauchi Hiromitsu、Nomura Takeshi、Shibahara Takahiko、Yamaguchi Akira、Chung Ung-il、Azuma Toshifumi、Ohba Shinsuke
  • Journal Title: Stem Cell Reports Volume: 15 Issue: 1 Pages: 125-139
  • DOI: 10.1016/j.stemcr.2020.05.008
  • Peer Reviewed / Open Access
• [Journal Article] Understanding paraxial mesoderm development and sclerotome specification for skeletal repair (2020)
  • Author(s): Tani Shoichiro、Chung Ung-il、Ohba Shinsuke、Hojo Hironori
  • Journal Title: Experimental & Molecular Medicine Volume: 52 Issue: 8 Pages: 1166-1177
  • DOI: 10.1038/s12276-020-0482-1
  • Peer Reviewed / Open Access
• [Journal Article] Stepwise strategy for generating osteoblasts from human pluripotent stem cells under fully defined xeno-free conditions with small-molecule inducers (2020)
  • Author(s): Zujur Denise、Kanke Kosuke、Onodera Shoko、Tani Shoichiro、Lai Jenny、Azuma Toshifumi、Xin Xiaonan、Lichtler Alexander C.、Rowe David W.、Saito Taku、Tanaka Sakae、Masaki Hideki、Nakauchi Hiromitsu、Chung Ung-il、Hojo Hironori、Ohba Shinsuke
  • Journal Title: Regenerative Therapy Volume: 14 Pages: 19-31
  • DOI: 10.1016/j.reth.2019.12.010
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Insights into Gene Regulatory Networks in Chondrocytes (2019)
  • Author(s): Hojo Hironori、Ohba Shinsuke
  • Journal Title: International Journal of Molecular Sciences Volume: 20 Issue: 24 Pages: 6324-6324
  • DOI: 10.3390/ijms20246324
  • Peer Reviewed / Open Access
• [Journal Article] Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds (2019)
  • Author(s): Kawata Manabu、Mori Daisuke、Kanke Kosuke、Hojo Hironori、Ohba Shinsuke、Chung Ung-il、Yano Fumiko、Masaki Hideki、Otsu Makoto、Nakauchi Hiromitsu、Tanaka Sakae、Saito Taku
  • Journal Title: Stem Cell Reports Volume: 13 Issue: 3 Pages: 530-544
  • DOI: 10.1016/j.stemcr.2019.07.012
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Generation of orthotopically functional salivary gland from embryonic stem cells (2018)
  • Author(s): Tanaka Junichi、Ogawa Miho、Hojo Hironori、Kawashima Yusuke、Mabuchi Yo、Hata Kenji、Nakamura Shiro、Yasuhara Rika、Takamatsu Koki、Irie Tarou、Fukada Toshiyuki、Sakai Takayoshi、Inoue Tomio、Nishimura Riko、Ohara Osamu、Saito Ichiro、Ohba Shinsuke、Tsuji Takashi、Mishima Kenji
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 4216-4216
  • DOI: 10.1038/s41467-018-06469-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Runx2は骨格発生においてクロマチンランドスケープを規定する (2020)
  • Author(s): 北條宏徳、齋藤琢、小野寺晶子、東俊文、鄭雄一、大庭伸介
  • Organizer: 日本骨代謝学会
• [Presentation] Runx2 defines the osteoblast chromatin landscape in skeletal development (2020)
  • Author(s): Hironori Hojo, Taku Saito, Shoko Onodera, Toshifumi Azuma, Ung-il Chung, Masahide Seki, Yutaka Suzuki, Andrew P. McMahon, Shinsuke Ohba
  • Organizer: American Society for Bone and Mineral Research
  • Int'l Joint Research
• [Presentation] Cell-type-distinct regulatory action of Runx2 on the genome underlies its distinct roles in osteoblasts and chondrocytes. (2019)
  • Author(s): Hojo H, Ohba S, Yamakawa A, Guo Q, He X, Saito T, Onodera S, Azuma T, Chung UI, McMahon AP.
  • Organizer: 2019 Annual Meeting of the American Society for Bone and Mineral Research
  • Int'l Joint Research
• [Presentation] Runx2は骨格発生において細胞種特異的な転写制御領域に作用する (2019)
  • Author(s): 北條宏徳、山川晃、齋藤琢、小野寺晶子、東俊文、鄭雄一、大庭伸介
  • Organizer: 第37回日本骨代謝学会学術集会
• [Presentation] A chondrocyte-specific Indian hedgehog enhancer as an OA-driving enhancer. (2019)
  • Author(s): Yamakawa A, Hojo H, Chung UI, Ohba S
  • Organizer: Orthopedic Research Society 2018 Annual Meeting
  • Int'l Joint Research