Rizzo Jason M, Sinha Satrajit
Department of Biochemistry, State University of New York at Buffalo, Buffalo, NY, 14203, USA.
Methods Mol Biol. 2014;1195:49-59. doi: 10.1007/7651_2014_77.
Eukaryotic DNA is wrapped around histone octamers, known as nucleosomes, in an orderly fashion that provides the primary structure of chromatin organization. The compaction of DNA into nucleosomal repeats not only allows the tight packaging of the large eukaryotic genomes into the nucleus, it also dictates the accessibility of genetic information. Thus, in order to understand how nucleosomes can affect the dynamics of DNA-protein interactions, such as those associated with transcriptional regulatory mechanisms, it is important to define nucleosomal positioning and occupancy along genomic DNA. Here we describe a method that relies on the enzymatic activity of micrococcal nuclease (MNase) to determine nucleosomal footprints and boundaries. By pairing this technique with next generation sequencing techniques (i.e., MNase-seq), it is possible to generate a genome-wide detailed map of chromatin architecture.
真核生物的DNA以有序的方式缠绕在称为核小体的组蛋白八聚体周围，这提供了染色质组织的主要结构。DNA压缩成核小体重复序列不仅允许将庞大的真核生物基因组紧密包装进细胞核，还决定了遗传信息的可及性。因此，为了理解核小体如何影响DNA-蛋白质相互作用的动态过程，比如那些与转录调控机制相关的相互作用，定义核小体在基因组DNA上的定位和占有率很重要。在这里，我们描述了一种依靠微球菌核酸酶（MNase）的酶活性来确定核小体足迹和边界的方法。通过将该技术与下一代测序技术（即MNase-seq）相结合，就有可能生成全基因组范围的染色质结构详细图谱。
