Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies.
Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies.
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DOI：
10.1007/s10577-015-9486-4
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Kimura H
中科院分区：
文献类型：
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作者：
Hayashi-Takanaka Y;Maehara K;Harada A;Umehara T;Yokoyama S;Obuse C;Ohkawa Y;Nozaki N;Kimura H
翻译后组蛋白修饰在表观遗传基因调控和基因组维持等基因组功能中起着关键作用。组蛋白H4 n末端的尾部包含几个可以被乙酰化和甲基化的氨基酸。已知其中一些修饰在细胞周期中会发生剧烈变化。在这项研究中，我们生成了一组小鼠单克隆抗体，用于抵抗组蛋白H4修饰，包括K5、K8、K12和K16位点的乙酰化，以及K20位点不同水平的甲基化。它们的特异性通过ELISA和免疫印迹法进行评估，使用合成肽和重组蛋白进行特异性修饰或氨基酸取代。免疫荧光证实了靶修饰的特征性分布。H4K5乙酰化（H4K5ac）特异性抗体CMA405仅在邻近的K8未乙酰化时才与K5ac发生反应。这一独特的特征使我们能够检测到新组装的H4，它在K5和K12上是二乙酰化的，并将其与高乙酰化的H4区分开来，其中K5和K8都是乙酰化的。染色质免疫沉淀结合深度测序（ChIP-seq）显示，H4K8和H4K16的乙酰化在转录起始位点附近富集。这些广泛表征和高度特异性的抗体将为未来的表观遗传学和表观基因组研究提供有用的信息。
Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.