Sims Hillel I, Pham Chuong D, Schnitzler Gavin R
Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA.
Methods Mol Biol. 2012;833:311-36. doi: 10.1007/978-1-61779-477-3_19.
Positioning of nucleosomes regulates the access of DNA binding factors to their consensus sequences. Nucleosome positions are determined, at least in part by the effects of DNA sequence during nucleosome assembly. Nucleosomes can also be repositioned (moved in cis) by ATP-dependent nucleosome remodeling complexes. Most studies of repositioning have used short DNA fragments containing a single nucleosome. It is difficult to use this type of template to analyze the role of DNA sequence in repositioning, however, because the many remodeling complexes are strongly influenced by nearby DNA ends. Mononucleosomal templates also cannot provide information about how repositioning occurs in the context of chromatin, where the presence of flanking nucleosomes could limit repositioning options. This protocol describes a newly developed method that allows the mapping of nucleosome positions (with and without remodeling) on any chosen region of a plasmid polynucleosomal template in vitro. The approach uses MNase digestion to release nucleosome-protected DNA fragments, followed by restriction enzyme digestion to locally unique sites, and Southern blotting, to provide a comprehensive map of nucleosome positions within a probe region. It was developed as part of studies which showed that human remodeling enzymes tended to move nucleosomes away from high affinity nucleosome positioning sequences, and also that there were differences in repositioning specificity between different remodeling complexes.
核小体的定位调节DNA结合因子对其共有序列的可及性。核小体的位置至少部分由核小体组装过程中DNA序列的作用决定。核小体也可以通过依赖ATP的核小体重塑复合物重新定位（顺式移动）。大多数重新定位的研究使用含有单个核小体的短DNA片段。然而，使用这种类型的模板来分析DNA序列在重新定位中的作用很困难，因为许多重塑复合物受到附近DNA末端的强烈影响。单核小体模板也无法提供关于在染色质背景下重新定位如何发生的信息，在染色质中侧翼核小体的存在可能会限制重新定位的选择。本方案描述了一种新开发的方法，该方法允许在体外绘制质粒多聚核小体模板的任何选定区域上的核小体位置（有无重塑）。该方法使用微球菌核酸酶消化来释放核小体保护的DNA片段，然后用限制性内切酶消化到局部独特位点，并通过Southern印迹法，以提供探针区域内核小体位置的综合图谱。它是作为研究的一部分而开发的，这些研究表明人类重塑酶倾向于将核小体从高亲和力的核小体定位序列移开，并且不同重塑复合物在重新定位特异性方面也存在差异。
