In vitro and in vivo assays for studying histone ubiquitination and deubiquitination.
In vitro and in vivo assays for studying histone ubiquitination and deubiquitination.
复制标题
用于研究组蛋白泛素化和去泛素化的体外和体内测定。
DOI：
10.1007/978-1-59745-190-1_20
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Wang,Hengbin
中科院分区：
文献类型：
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作者：
Zhai,Ling;Joo,Heui-Yun;Wang,Hengbin
翻译后组蛋白修饰在调节染色质结构和功能中发挥重要作用（Martin and Zhang, Nat Rev Mol Cell Biol:838 - 849, 2005; Jenuwein and Allis, Science: 293:1074-1080, 2001）。这种修饰的一个例子是组蛋白泛素化，主要发生在H2A和H2B上。最近的研究强调了H2A泛素化在Polycomb蛋白介导的基因沉默中的重要调控作用（Wang et al., Nature 431:873-878, 2004; Joo et al., Nature 449:1068-1072, 2007）和H2B泛素化在转录、H3甲基化和DNA甲基化中的重要调控作用（Zhang, Genes Dev 17:2733-2740, 2003; Sun and Allis, Nature 418:104 - 108,2002； Sridhar等，Nature 447:735-738, 2007）。在这里，我们描述了体外组蛋白泛素化和去泛素化测定的方法。我们还描述了研究一种假定的组蛋白泛素连接酶和去泛素酶的体内功能的方法。这些实验程序在很大程度上是基于我们对哺乳动物细胞的研究。这些方法应该为研究这种庞大的组蛋白修饰提供有用的工具。
Posttranslational histone modifications play important roles in regulating chromatin structure and function (Martin and Zhang, Nat Rev Mol Cell Biol 6:838–849, 2005; Jenuwein and Allis, Science 293:1074–1080, 2001). One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Recent studies have highlighted important regulatory roles of H2A ubiquitination in Polycomb group proteins-mediated gene silencing (Wang et al., Nature 431:873–878, 2004; Joo et al., Nature 449:1068–1072, 2007) and H2B ubiquitination in transcription, H3 methylation, and DNA methylation (Zhang, Genes Dev 17:2733–2740, 2003; Sun and Allis, Nature 418:104–108, 2002; Sridhar et al., Nature 447:735–738, 2007). Here we describe methods for in vitro histone ubiquitination and deubiquitination assays. We also describe approaches to investigate the in vivo function of a putative histone ubiquitin ligase and deubiquitinase. These experimental procedures are largely based on our studies in mammalian cells. These methods should provide useful tools for studying this bulky histone modification.