Purification of histone ubiquitin ligases from HeLa cells.
Purification of histone ubiquitin ligases from HeLa cells.
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DOI：
10.1016/j.ymeth.2011.03.003
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发表时间：
2011-07
期刊：
影响因子：
4.8
通讯作者：
Wang, Hengbin
中科院分区：
文献类型：
--
作者：
Jones, Amanda;Joo, Heui-Yun;Robbins, Woody;Wang, Hengbin
翻译后组蛋白修饰在调节包括转录在内的基于染色质的核过程中起着重要作用。在这些修饰中，组蛋白泛素化是最不为人所知的。组蛋白泛素化主要针对组蛋白H2A和H2B。虽然从出芽酵母到哺乳动物，H2B的泛素化在进化上是保守的，但在出芽酵母、蠕虫或植物中尚未检测到H2A的泛素化。直到最近，对组蛋白泛素化的研究远远落后于对其他组蛋白修饰的研究，主要是因为泛素化组蛋白的特异性抗体很难产生。尽管存在这一障碍，对组蛋白泛素化的酶机制的识别，以及成功地使用遗传和免疫印迹方法相结合来检测泛素化组蛋白，有助于揭示这种修饰在转录起始和延伸、细胞周期进展和DNA损伤反应中的重要调节作用。在最近开发的泛素化组蛋白特异性抗体的帮助下，组蛋白泛素化与癌症发展之间建立了一个有趣的联系。虽然参与H2B泛素化的酶首先在出芽酵母中被鉴定，随后在基于基因同源性的高等生物中被鉴定，但参与H2A泛素化的酶机制的鉴定在很大程度上依赖于生化纯化方法。针对组蛋白的泛素连接酶的无偏搜索也导致了H3和H4泛素连接酶的鉴定。在这里，我们详细介绍了一种从HeLa细胞中鉴定组蛋白泛素连接酶的生化方法。类似的方法已经成功地用于鉴定组蛋白甲基转移酶、组蛋白去甲基化酶、染色质重塑因子和一般转录因子。只要可以建立体外酶分析，我们描述的方法可以很容易地适应于识别其他组蛋白和非组蛋白修饰酶。
Posttranslational histone modifications play an important role in regulating chromatin based nuclear processes including transcription. Of these modifications, histone ubiquitination is among the least understood. Histone ubiquitination predominately targets histones H2A and H2B. While ubiquitination of H2B is evolutionarily conserved from budding yeast to mammals, ubiquitination of H2A has not been detected in budding yeast, worms, or plants. Until recently, studies of histone ubiquitination lagged far behind the study of other histone modifications, largely because antibodies specific for ubiquitinated histones are difficult to generate. Despite this obstacle, the identification of the enzymatic machineries involved in histone ubiquitination, together with the successful use of a combination of genetic and immunoblot approaches to detect ubiquitinated histones, have helped to reveal important regulatory roles for this modification in transcriptional initiation and elongation, cell cycle progression, and DNA damage response. With the aid of the recently developed ubiquitinated histone-specific antibodies, an intriguing link between histone ubiquitination and cancer development has been established. While the enzymes involved in H2B ubiquitination were identified first in budding yeast and subsequently in higher organisms based on gene homology, the identification of the enzymatic machineries involved in H2A ubiquitination largely depended on a biochemical purification approach. The unbiased search for ubiquitin ligases targeting histones also led to the identification of a H3 and H4 ubiquitin ligase. Here we detail a protocol for the biochemical approach to identify histone ubiquitin ligase(s) from HeLa cells. Similar approaches have been successfully used to identify histone methyltransferases, histone demethylases, chromatin remodeling factors, and general transcription factors. So long as an in vitro enzymatic assay can be established, the approach we describe can be easily adapted to identify other histone and non-histone modifying enzymes.
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DOI：
10.1007/978-1-59745-540-4_10
发表时间：
2009-01-01
期刊：
YEAST FUNCTIONAL GENOMICS AND PROTEOMICS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Jackson, Jessica;Shilatifard, Ali
通讯作者：
Shilatifard, Ali
影响因子：
4.8
作者：
Devroe, E;Erdjument-Bromage, H;Silver, PA
通讯作者：
Silver, PA
影响因子：
2.7
作者：
Koken, MHM;Hoogerbrugge, JW;Hoeijmakers, JHJ
通讯作者：
Hoeijmakers, JHJ
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
11.8
作者：
Akkers, Robert C.;van Heeringen, Simon J.;Jacobi, Ulrike G.;Janssen-Megens, Eva M.;Francoijs, Kees-Jan;Stunnenberg, Hendrik G.;Veenstra, Gert Jan C.
通讯作者：
Veenstra, Gert Jan C.