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首页> 外文期刊>Journal of Molecular Biology >A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation
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A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation

机译：H4K20单甲基化的活细胞成像的遗传编码探针。

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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 (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/).
机译：真核基因表达在染色质的背景下受到调节。翻译后组蛋白修饰的动态变化被认为在基本细胞功能中起关键作用，例如调节细胞周期，发育和分化。为了阐明组蛋白修饰与细胞功能之间的关系，重要的是监测单个活细胞中修饰的动力学。已经提出了一种称为薄荷抗体（修饰特异性细胞内抗体）的遗传编码探针，该探针是标记有荧光蛋白的单链可变片段，是一种有用的可视化工具。然而，抗体片段在细胞内表达的功效受到限制，部分原因是与分泌蛋白如抗体折叠的内质网相比，细胞质中的环境条件不同。在这项研究中，我们开发了一种新的针对组蛋白H4 Lys20单甲基化（H4K2Ome1）的薄荷体。 H4K2Ome1-mintbody在活细胞中的特异性使用酵母突变体和哺乳动物细胞进行了验证，其中该目标修饰被减弱了。 H4K20me1-mintbody的表达使我们能够监测H4K2Ome1水平在细胞周期中的振荡。此外，在小鼠和线虫细胞中使用H4K20me1-mintbody可以看到剂量补偿的X染色体。使用X射线晶体学和突变分析，我们确定了有助于稳定和/或适当折叠薄荷体的关键氨基酸。综上所述，这些数据为旨在开发功能性细胞内抗体的未来研究提供了重要的启示。特别是，H4K2Ome1-薄荷糖体提供了一个强大的工具，可以追踪活细胞和生物体中这种特定的组蛋白修饰。 （C）2016作者。由Elsevier Ltd.发行。这是CC BY许可下的开放访问文章（http://creativecommons.org/licenses/by/4.0/）。

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