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

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

研究成果: Article

17 引用 (Scopus)

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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 (H4K20me1). The specificity of the H4K20me1-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 H4K20me1 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 H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

元の言語English
ページ(範囲)3885-3902
ページ数18
ジャーナルJournal of Molecular Biology
428
発行部数20
DOI
出版物ステータスPublished - 2016 10 9

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ASJC Scopus subject areas

  • Molecular Biology

これを引用

Sato, Y., Kujirai, T., Arai, R., Asakawa, H., Ohtsuki, C., Horikoshi, N., Yamagata, K., Ueda, J., Nagase, T., Haraguchi, T., Hiraoka, Y., Kimura, A., Kurumizaka, H., & Kimura, H. (2016). A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation. Journal of Molecular Biology, 428(20), 3885-3902. https://doi.org/10.1016/j.jmb.2016.08.010