Duband-Goulet Isabelle, Ouararhni Khalid, Hamiche Ali
Institut Andre Lwoff, CNRS UPR 9079, Equipe : fonction et dynamique de la chromatine, Bat B, 2ème Etage, 07 rue Guy Moquet, 94800 Villejuif, France.
Methods. 2004 May;33(1):12-7. doi: 10.1016/j.ymeth.2003.10.015.
Packaging of the DNA in nucleosomes restricts its accessibility to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a short linear DNA fragment allows gel fractionation of nucleosomes according to their translational positions, whose locations are dependent on the underlying DNA sequence. Nucleosome mobilization by chromatin remodeling factors is easily detected by observing band disappearance in gel, which in turn provides evidence for histone octamer displacement. Here, we provide methods for chromatin assembly that we have been using in our analysis for nucleosome mobilization by chromatin remodeling factors. These methods are straightforward and easy to follow. Thus, they may provide a good starting assay system for analysis of nucleosome movements by other chromatin remodeling machines.
DNA在核小体中的包装限制了其对调控因子和酶复合物的可及性，使得核小体结构的局部重塑成为建立蛋白质-DNA相互作用的先决条件。使用在短线性DNA片段上重构一个核小体的实验系统，可以根据核小体的平移位置对其进行凝胶分级分离，这些位置取决于潜在的DNA序列。通过观察凝胶中条带的消失很容易检测到染色质重塑因子引起的核小体移动，这反过来又为组蛋白八聚体的位移提供了证据。在这里，我们提供了我们在分析染色质重塑因子引起的核小体移动时一直在使用的染色质组装方法。这些方法简单易懂。因此，它们可能为分析其他染色质重塑机器引起的核小体移动提供一个良好的起始检测系统。
