論文

査読有り
2016年10月

A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation

Journal of Molecular Biology
  • Yuko Sato
  • ,
  • Tomoya Kujirai
  • ,
  • Ritsuko Arai
  • ,
  • Haruhiko Asakawa
  • ,
  • Chizuru Ohtsuki
  • ,
  • Naoki Horikoshi
  • ,
  • Kazuo Yamagata
  • ,
  • Jun Ueda
  • ,
  • Takahiro Nagase
  • ,
  • Tokuko Haraguchi
  • ,
  • Yasushi Hiraoka
  • ,
  • Akatsuki Kimura
  • ,
  • Hitoshi Kurumizaka
  • ,
  • Hiroshi Kimura

428
20
開始ページ
3885
終了ページ
3902
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1016/j.jmb.2016.08.010
出版者・発行元
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K2Ome1). The specificity of the H4K2Ome1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K2Ome1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K2Ome1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms. (C) 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Web of Science ® 被引用回数 : 18

リンク情報
DOI
https://doi.org/10.1016/j.jmb.2016.08.010
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/27534817
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000385324000001&DestApp=WOS_CPL

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