Trotter Kevin W, Archer Trevor K
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.
Methods Mol Biol. 2012;833:89-102. doi: 10.1007/978-1-61779-477-3_6.
The packaging of eukaryotic DNA into nucleosomes, the fundamental unit of chromatin, creates a barrier to nuclear processes, such as transcription, DNA replication, recombination, and repair. This obstructive nature of chromatin can be overcome by the enzymatic activity of chromatin remodeling complexes, which create a more favorable environment for the association of essential factors and regulators to sequences within target genes. Here, we describe a detailed approach for analyzing chromatin architecture and remodeling by restriction endonuclease hypersensitivity assay. This procedure uses restriction endonucleases to characterize changes in chromatin that accompany nucleosome remodeling. The specific experimental example described in this article is the BRG1 complex-dependent chromatin remodeling of the steroid hormone-responsive mouse mammary tumor virus promoter. Through the use of these methodologies one is able to quantify changes at specific nucleosomes in response to regulatory signals.
真核生物的DNA包装成染色质的基本单位核小体，对转录、DNA复制、重组和修复等核过程形成了障碍。染色质重塑复合物的酶活性可以克服染色质的这种阻碍性质，该复合物为必需因子和调节因子与靶基因内的序列结合创造了更有利的环境。在这里，我们描述了一种通过限制性内切酶超敏反应分析染色质结构和重塑的详细方法。该程序使用限制性内切酶来表征伴随核小体重塑的染色质变化。本文描述的具体实验例子是类固醇激素反应性小鼠乳腺肿瘤病毒启动子的BRG1复合物依赖性染色质重塑。通过使用这些方法，人们能够量化特定核小体响应调节信号的变化。
