Li Q, Wrange O
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
Methods. 1997 May;12(1):96-104. doi: 10.1006/meth.1997.0451.
Several promoters have been shown to have sequence specifically positioned nucleosomes that determine the architecture of the promoter. DNA binding proteins that regulate gene expression are in many cases known to bind to their cognate DNA segments organized within such positioned nucleosomes. It has become increasingly evident that the cooperation of chromatin and transcription factors results in an efficient and fine-tuned regulation of transcription. The first step in a gene induction event must be the access of transcription factors for the regulatory promoter/enhancer target sites. In this perspective it becomes interesting to evaluate the affinity of DNA binding proteins for their cognate binding site in a nucleosome context. Here we describe the preparation of nucleosome probe, a method for in vitro nucleosome reconstitution by salt dilution, purification of the reconstituted mononucleosomes, and characterization of the translational and rotational positions of the nucleosomal DNA. In addition, methods for affinity determination and characterization of protein-nucleosomal DNA interaction, such as methylation protection and methylation interference by dimethyl sulfate, quantitative DNase I footprinting, and electrophoretic mobility shift assay, are described.
已证实几种启动子具有序列特异性定位的核小体，这些核小体决定了启动子的结构。在许多情况下，已知调节基因表达的DNA结合蛋白会与其在此类定位核小体内组织的同源DNA片段结合。越来越明显的是，染色质和转录因子的协同作用导致了高效且精细的转录调控。基因诱导事件的第一步必定是转录因子进入调控启动子/增强子靶位点。从这个角度来看，评估DNA结合蛋白在核小体环境中与其同源结合位点的亲和力变得很有趣。在此，我们描述了核小体探针的制备、通过盐稀释进行体外核小体重组的方法、重组单核小体的纯化以及核小体DNA平移和旋转位置的表征。此外，还描述了蛋白质 - 核小体DNA相互作用的亲和力测定和表征方法，如甲基化保护和硫酸二甲酯甲基化干扰、定量DNase I足迹分析以及电泳迁移率变动分析。
