Chromatin Immunoprecipitation (ChIP) of Heat Shock Protein 90 (Hsp90).
Chromatin Immunoprecipitation (ChIP) of Heat Shock Protein 90 (Hsp90).
复制标题
热休克蛋白 90 (Hsp90) 的染色质免疫沉淀 (ChIP)
DOI：
10.1007/978-1-4939-7477-1_17
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发表时间：
2018
影响因子：
--
通讯作者：
Sawarkar R
中科院分区：
文献类型：
--
作者：
Yoveva A;Sawarkar R
染色质免疫沉淀测序（ChIP-seq）是一种广泛用于体内蛋白质-DNA相互作用和表观遗传标记的全基因组作图的技术。最近的研究表明，热休克蛋白90（Hsp 90）在染色质中的重要作用。这种分子伴侣帮助其他蛋白质获得它们的成熟和功能构象，并帮助组装许多复合物。在本章中，我们提供了如何从果蝇Schneider（S2）细胞中进行Hsp 90 ChIP-seq的具体细节。简言之，细胞同时裂解并可逆交联以稳定蛋白质-DNA相互作用。染色质由分离的细胞核制备，并通过超声处理进行剪切。免疫沉淀Hsp 90结合位点，纯化相应的DNA片段并测序。所描述的方法显示，热休克蛋白90结合接近约三分之一的所有果蝇编码基因的转录起始位点，并在染色质的伴侣的作用的特点。
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique for genome-wide mapping of protein-DNA interactions and epigenetic marks in vivo. Recent studies have suggested an important role of heat shock protein 90 (Hsp90) at chromatin. This molecular chaperone assists other proteins to acquire their mature and functional conformation and helps in the assembly of many complexes. In this chapter, we provide specific details on how to perform Hsp90 ChIP-seq from Drosophila Schneider (S2) cells. Briefly, the cells are simultaneously lyzed and reversibly cross-linked to stabilize protein–DNA interactions. Chromatin is prepared from isolated nuclei and sheared by sonication. Hsp90-bound loci are immunoprecipitated and the corresponding DNA fragments are purified and sequenced. The described approach revealed that Hsp90 binds close to the transcriptional start site of around one-third of all Drosophila coding genes and characterized the role of the chaperone at chromatin.