In vivo cross-linking and immunoprecipitation for studying dynamic protein:: DNA associations in chromatin environment
In vivo cross-linking and immunoprecipitation for studying dynamic protein:: DNA associations in chromatin environment
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DOI：
10.1006/meth.1999.0879
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发表时间：
1999-11-01
期刊：
影响因子：
--
通讯作者：
Allis, CD
中科院分区：
文献类型：
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作者：
Kuo, MH;Allis, CD
染色质结构在许多DNA模板过程中起着重要的作用，如转录、复制和重组，最近在鉴定与这些核过程有关的大的、多亚单位复合体方面取得了相当大的进展，所有这些都发生在核小体模板上。绘制特定的基因组位置相对于选择性修饰或独特的组蛋白变体或非组蛋白组分的位置，为了解这些蛋白质(及其修饰)在正常染色质环境中的功能提供了有价值的见解。在这里，我们描述了一种通用的、高分辨率的方法，它包括两个基本步骤：(1)完整细胞的体内甲醛交联，然后(2)选择性免疫沉淀具有特定抗体的蛋白质-DNA复合体。这种方法允许在天然染色质环境中详细分析蛋白质-DNA相互作用。最近，这一技术已成功地用于绘制靶基因上特定修饰(如乙酰化)的组蛋白的边界，以惊人的精度定义依赖于起源的复制和着丝粒特异性复合体的细胞周期调控组装，以及定位相当罕见的转录因子在同源DNA位点上的体内位置。因此，基本的染色质免疫沉淀技术是非常多才多艺的，现在已经被广泛应用于各种细胞类型，包括发芽酵母、苍蝇和人类细胞。因此，更多围绕染色质结构和天然环境中蛋白质-DNA相互作用的研究似乎可能会从这项技术中受益。在这篇文章中，简要回顾了这一强有力的方法的历史，并讨论了基本方法。还介绍了蛋白质回收的步骤，以及该方法的局限性和扩展。(C)1999年学术出版社。
Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination, Considerable progress has recently been made in the identification of large, multisubunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates. Mapping specific genomic loci relative to the position of selectively modified or unique histone variants or nonhistone components provides valuable insights into how these proteins (and their modifications) function in their normal chromatin context. Here we describe a versatile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde crosslinking of intact cells followed by (2) selective immunoprecipitation of protein-DNA complexes with specific antibodies. This method allows for detailed analyses of protein-DNA interactions in a native chromatin environment. Recently, this technique has been successfully employed to map the boundaries of specifically modified (e.g., acetylated) histones along target genes, to define the cell cycle-regulated assembly of origin-dependent replication and centromere-specific complexes with remarkable precision, and to map the in vivo position of reasonably rare transcription factors on cognate DNA sites. Thus, the basic chromatin immunoprecipitation technique is remarkably versatile and has now been used in a wide range of cell types, including budding yeast, fly, and human cells. As such, it seems likely that many more studies, centered around chromatin structure and protein-DNA interactions in its native setting, will benefit from this technique. In this article, a brief review of the history of this powerful approach and a discussion of the basic method are provided. Procedures for protein recovery as well as limitations and extensions of the method are also presented. (C) 1999 Academic Press.