A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation
A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation
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DOI：
10.1016/j.jmb.2016.08.010
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发表时间：
2016-10-09
影响因子：
5.6
通讯作者：
Kimura, Hiroshi
中科院分区：
文献类型：
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作者：
Sato, Yuko;Kujirai, Tomoya;Kimura, Hiroshi
真核基因的表达在染色质的背景下受到调控。翻译后组蛋白修饰的动态变化被认为在调节细胞周期、发育和分化等基本细胞功能中发挥关键作用。为了阐明组蛋白修饰与细胞功能之间的关系，监测单个活细胞中组蛋白修饰的动力学是很重要的。一种被称为修饰特异性细胞内抗体(MintBody)的基因编码探针，是一种标记有荧光蛋白的单链可变片段，已被认为是一种有用的可视化工具。然而，抗体片段在细胞内表达的效果一直是有限的，部分原因是与内质网相比，细胞质中的环境条件不同，内质网中分泌的蛋白质如抗体被折叠。在这项研究中，我们开发了一种新的针对组蛋白H4Lys20单甲基化(H4K2Ome1)的薄荷抗体。用酵母突变体和这种靶向修饰减弱的哺乳动物细胞验证了H4K2Ome1亚基在活细胞中的特异性。H4K20me1抗体的表达使我们能够监测H4K2Ome1水平在细胞周期中的振荡。此外，还利用H4K20me1-微体在小鼠和线虫细胞中显示了剂量补偿的X染色体。使用X射线结晶学和突变分析，我们确定了有助于稳定和/或正确折叠薄荷体的关键氨基酸。综上所述，这些数据为未来旨在开发功能性细胞内抗体的研究提供了重要的启示。具体地说，H4K2Ome1-薄荷体提供了一个强大的工具来追踪活细胞和生物体中这种特殊的组蛋白修饰。(C)2016年提交人。由爱思唯尔有限公司出版。这是一篇在CC by License(http://creativecommons.org/licenses/by/4.0/).下的开放获取文章
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/).