Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry.
Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry.
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使用原位邻近连接分析和 Click-It 化学检测和定量复制 DNA 时的乙酰化组蛋白。
DOI：
10.1007/978-1-4939-9434-2_3
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Sidorova,Julia
中科院分区：
文献类型：
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作者：
Lazarchuk,Pavlo;Roy,Sunetra;Schlacher,Katharina;Sidorova,Julia
组蛋白乙酰化在DNA转录、修复和复制调控中起重要作用。在这里，我们详细介绍了一种定量检测在体内培养细胞中复制叉处或后面的新生染色质中特定组蛋白修饰的方法。该方法包括用EdU标记DNA，使用Click化学将DNA中的EdU部分生物素化，然后使用原位接近连接试验（PLA）选择性地可视化EdU与修饰特异性抗体识别的修饰组蛋白的共定位。我们专注于检测乙酰化组蛋白H3和H4在培养的人类细胞的新生染色质作为该方法应用的一个具体例子。值得注意的是，该方法完全适用于研究预期存在于新生DNA或复制叉上的组蛋白或非组蛋白，并已成功用于模式生物和人类组织培养。
Histone acetylation plays important roles in the regulation of DNA transcription, repair, and replication. Here we detail a method for quantitative detection of specific histone modifications in the nascent chromatin at or behind replication forks in vivo in cultured cells. The method involves labeling DNA with EdU, using Click chemistry to biotinylate EdU moieties in DNA, and then using in situ proximity ligation assay (PLA) to selectively visualize co-localization of EdU with a modified histone of choice recognized by a modification-specific antibody. We focus on detection of acetylated histones H3 and H4 in the nascent chromatin of cultured human cells as a specific example of the method’s application. Notably, the method is fully applicable to studies of histones or nonhistone proteins expected to be present on nascent DNA or at replication forks, and has been successfully used in model organisms and human tissue culture.