Establishment of simultaneous and quantitative assay for DNAbinding and transcriptional activity of glucocorticoid receptor.

• Project/Area Number: 23770169
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Hokkaido University
• Principal Investigator: MIKUNI Shintaro 北海道大学, 大学院・医学研究科, 特任助教 (40435954)
• Project Period (FY): 2011 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2011: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
• Keywords: タンパク質 / 核酸の構造・動態・機能 / 転写因子 / バイオイメージング / 分子間相互作用 / 生物物理 / 核酸 / 蛋白質 / 転写活性 / 相関解析 / 相互作用 / 一分子計測 / FCS / FCCS / 核内受容体 / DNA

Research Abstract

Regulation of transcriptional activity could be initiated by associating the transcriptional factors with its specific sequence of genomic DNA. However, the direct relationship between “the affinity for DNA” and “transcriptional activity” of transcriptional factors is not clarified. To clarify this relationship, we tried to establish the simultaneous and quantitative assay for DNA binding and transcriptional activity of glucocorticoid receptor (GR), which is one of transcriptional factors, using fluorescence cross-correlation spectroscopy (FCCS). As a result, the dissociation constant between the purified GR fused with EGFP and the Alexa647-labeled GR specific sequences (glucocorticoid response element, GRE) could be determined as 0.63 μM, and sequence-specificity of GR could be confirmed by FCCS. Moreover, the dissociation constant between the purified RNA-specific binding protein fused withmCherry (red fluorescent protein) and Alexa488-labeled RNA was also determined as 143 nM by FCCS.

Research Products

• [Journal Article] Nonmuscle myosin II folds into a 10S form via two portions of tail for dynamic subcellular loacalization. (2013)
  • Author(s): Kiboku T.
  • Journal Title: Genes to Cells. Volume: 18(2) Issue: 2 Pages: 90-109
  • DOI: 10.1111/gtc.12021
  • Peer Reviewed
• [Journal Article] Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells. (2013)
  • Author(s): Niikura K.
  • Journal Title: Mol Biosyst. Volume: 9(3) Issue: 3 Pages: 501-507
  • DOI: 10.1039/c2mb25420d
  • Peer Reviewed
• [Journal Article] Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate (2011)
  • Author(s): Fan Sun, Shintaro Mikuni, Masataka Kinjo
  • Journal Title: Biochemical and Biophysical Research Communications Volume: Volume 404, Issue 2 Issue: 2 Pages: 706-710
  • DOI: 10.1016/j.bbrc.2010.12.047
  • NAID: 120002834708
  • Peer Reviewed
• [Journal Article] Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement.
  • Author(s): Yamada M, Kumamoto K, Mikuni S, Arai Y, Kinjo M, Nagai T, Tsukasaki Y, Watanabe TM, Fukui M, Jin M, Toba S, Hirotsune S.
  • Journal Title: Scientific reports
  • Peer Reviewed
• [Presentation] 蛍光相関分光法を用いたDNA-転写因子相互作用と転写活性化同時定量法の開発 (2013)
  • Author(s): 三國新太郎
  • Organizer: 先端的光イメージング拠点形成プロジェクト・成果報告シンポジウム
  • Place of Presentation: 北海道大学(札幌市)
  • Year and Date: 2013-03-18
• [Presentation] Determination of dissociation constant between glucocorticoid receptor and DNA using FCCS (2012)
  • Author(s): Mikuni S and Kinjo M
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学(名古屋市)
• [Presentation] Training course for FCS and FCCS Fluorescence Microscopy: from Basics toAdvanced (2012)
  • Author(s): Kinjo M and Mikuni S
  • Place of Presentation: 沖縄科学技術大学院大学(恩納村)
• [Presentation] Determination of dissociation constant between glucocorticoid receptor and DNA using FCCS (2012)
  • Author(s): Shintaro Mikuni
  • Organizer: The 50th Annual Meeting of the Biophysical Society of Japan
  • Place of Presentation: 名古屋大学（名古屋市）
• [Presentation] Training course for FCS and FCCS (2012)
  • Author(s): Shintaro Mikuni
  • Organizer: Fluorescence Microscopy: from Basics to Advanced
  • Place of Presentation: 沖縄科学技術大学院大学（恩納村）
• [Presentation] Analysis for the relationship of intranuclear diffusional landscape and transcriptional activity of glucocorticoid receptor (2011)
  • Author(s): Shintaro Mikuni
  • Organizer: International Symposium on Photonic Bioimaging
  • Place of Presentation: 京王プラザホテル（札幌市）
• [Presentation] 蛍光相関分光法を用いたDNA-転写因子相互作用と転写活性化同時定量法の開発
  • Author(s): 三國 新太郎
  • Organizer: 先端的光イメージング拠点形成プロジェクト・成果報告シンポジウム
  • Place of Presentation: 北海道大学（札幌市）
• [Remarks]
  • URL: http://www.lfsci.hokudai.ac.jp/labs/infmcd/index.html
• [Remarks] 細胞機能科学研究室
  • URL: http://www.lfsci.hokudai.ac.jp/labs/infmcd/index.html