Understanding of the mechanism underlying gene expression rhythms using a gene circuit model with engineered Cas9 transcription factors

• Project/Area Number: 16KT0175
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 特設分野
• Research Field: Constructive Systems Biology
• Research Institution: Kyoto Prefectural University of Medicine
• Principal Investigator: Tsuchiya Yoshiki 京都府立医科大学, 医学(系)研究科(研究院), 講師 (30456777)
• Research Collaborator: YAGITA Kazuhiro
• Project Period (FY): 2016-07-19 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 概日リズム / 人工転写因子 / 人工遺伝子回路 / 転写フィードバックループ

Outline of Final Research Achievements

In this study, I aimed to reveal the principle underlying cell-autonomous oscillation of gene expression. I designed a gene circuit that is based on transcriptional-translational feedback loop in the circadian clock and regulated by engineered Cas9 transcription factors. Although establishment of the gene circuit has not yet been succeeded, engineered Cas9 transcription factors has been shown to be able to regulate endogenous gene expression. In addition, to know the necessity of the REV-ERBα/β-mediated secondary loop in the mammalian circadian transcriptional feedback loop, I have established the Rev-erbα/Rev-erbβ double knockout (DKO) mouse embryonic stem cells. Circadian oscillation of PER2 expression in differentiated Rev-erb-DKO ES cells was comparable to that in wild-type cells. These results indicate that the REV-ERBα/β-mediated secondary loop is dispensable in regulation of the mammalian circadian clock.

Academic Significance and Societal Importance of the Research Achievements

本研究により、哺乳類型概日時計の転写フィードバックループによる遺伝子発現振動について、2次ループに依存せずに単一の転写ループで強固なリズムを維持できることの新たな証拠が得られた。今後、人工転写回路によって単一の転写ループのみでの安定的な遺伝子発現リズムの再構築が実現すれば、遺伝子発現振動および振動周期長や頑健性の原理解明に繋がることが期待できる。

Research Products

• [Journal Article] Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth. (2019)
  • Author(s): Oshima T, Niwa Y, Kuwata K, Srivastava A, Hyoda T, Tsuchiya Y, Kumagai M, Tsuyuguchi M, Tamaru T, Sugiyama A, Ono N, Zolboot N, Aikawa Y, Oishi S, Nonami A, Arai F, Hagihara S, Yamaguchi J, Tama F, Kunisaki Y, Yagita K, Ikeda M, Kinoshita T, Kay SA, Itami K, Hirota T.
  • Journal Title: Science Advance Volume: 23 Issue: 1 Pages: 9060-9060
  • DOI: 10.1126/sciadv.aau9060
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 時計遺伝子ノックアウトES細胞を用いた哺乳類概日時計制御機構の解析 (2019)
  • Author(s): 土谷 佳樹
  • Organizer: 日本ゲノム編集学会 第4回大会
• [Presentation] The Role of REV-ERBs as Key Factors of Circadian Output. (2018)
  • Author(s): Yoshiki Tsuchiya
  • Organizer: International Symposium on Biological Rhythms ~20 years since Discovery of Mammalian Clock Genes~
  • Int'l Joint Research