DNase I Chromatin Accessibility Analysis.

Chromatin consists of compacted DNA in complex with proteins and contributes to the organization of DNA and its stability. Furthermore, chromatin plays key roles in regulating cellular processes such as DNA replication, transcription, DNA repair, and mitosis. Chromatin assumes more compact (inaccessible) or decondensed (accessible) conformations depending on the function that is being supported in the genome, either locally or globally. The activity of nucleases has been used previously to assess the accessibility of specific genomic regions in vitro, such as origins of replication at varying points in the cell cycle. Here, we provide an assay to determine the accessibility of specific human genomic regions (example used herein: Lamin B2 origin of DNA replication) by measuring the effect of DNase I nuclease on qPCR signal from the studied site. This assay provides a powerful method to interrogate the molecular mechanisms that regulate chromatin accessibility, and how these processes affect various cellular functions involving the human genome that require manipulation of chromatin conformation.

染色质由与蛋白质复合的压缩DNA组成，有助于DNA的组织及其稳定性。此外，染色质在调节细胞过程中起着关键作用，例如DNA复制、转录、DNA修复和有丝分裂。染色质根据基因组中局部或整体所支持的功能呈现更紧密（难以接近）或去浓缩（可接近）的构象。核酸酶的活性先前已被用于在体外评估特定基因组区域的可接近性，例如细胞周期不同阶段的复制起点。在此，我们提供一种测定方法，通过测量DNA酶I核酸酶对所研究位点的定量聚合酶链反应（qPCR）信号的影响，来确定特定人类基因组区域（本文使用的例子：DNA复制的核纤层蛋白B2起点）的可接近性。这种测定方法提供了一种强有力的手段，用于探究调节染色质可接近性的分子机制，以及这些过程如何影响涉及人类基因组且需要操纵染色质构象的各种细胞功能。