T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.
Curr Protoc. 2022 Aug;2(8):e518. doi: 10.1002/cpz1.518.
The positions of nucleosomes along genomic DNA play a role in defining patterns of gene expression and chromatin organization. Determination of nucleosome positions in vivo and in vitro, as revealed by the locations of histones on DNA, has provided insight into mechanisms of nucleosome sliding, spacing, assembly, and disassembly. Here, we describe methods for the in vitro determination of histone-DNA contacts at base-pair (bp) resolution. The protocol involves the labeling of histones with ortho-phenanthroline (OP), site-specific cleavage of nucleosomal DNA, and processing and analysis of the resulting DNA fragments. This methodology provides an efficient and high-resolution means for studying kinetics and behavior of enzymes that alter nucleosome structure and/or positioning, and can be used to identify preferred distributions of nucleosomes on natural DNA sequences. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cysteine-specific chemical modification of folded histones with ortho-phenanthroline (OP) Basic Protocol 2: Nucleosome sliding assay adapted for OP mapping of histone-DNA contacts Basic Protocol 3: OP-mediated cleavage, processing, and analysis of DNA fragments using a sequencing gel Support Protocol 1: Preparation of dideoxy sequencing ladders Support Protocol 2: Preparation and running of a denaturing DNA sequencing gel.
核小体在基因组 DNA 上的位置在决定基因表达和染色质组织模式方面发挥作用。通过 DNA 上组蛋白的位置揭示了体内和体外核小体位置的确定，为核小体滑动、间隔、组装和拆卸的机制提供了深入了解。在这里，我们描述了在体外确定碱基对 (bp) 分辨率下组蛋白-DNA 接触的方法。该方案涉及用邻菲咯啉 (OP) 标记组蛋白、核小体 DNA 的特异性切割以及所得 DNA 片段的处理和分析。这种方法学为研究改变核小体结构和/或定位的酶的动力学和行为提供了一种高效和高分辨率的手段，并且可以用于鉴定天然 DNA 序列上核小体的优先分布。© 2022 年 Wiley 期刊 LLC。基本方案 1：用邻菲咯啉 (OP) 对折叠组蛋白进行半胱氨酸特异性化学修饰基本方案 2：适用于 OP 作图的核小体滑动测定法组蛋白-DNA 接触基本方案 3：使用测序凝胶进行 OP 介导的切割、处理和 DNA 片段分析支持方案 1：制备双脱氧测序梯支持方案 2：变性 DNA 测序凝胶的制备和运行。
