Department of Chemistry, the Pennsylvania State University, University Park, PA 16802, USA.
Department of Chemistry, the Pennsylvania State University, University Park, PA 16802, USA.
Methods. 2023 Jul;215:17-27. doi: 10.1016/j.ymeth.2023.05.003. Epub 2023 May 24.
The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.
核小体是染色质的基本结构单元。发生在核小体水平上的变化是各种酶和因子参与染色质转化的分子基础。这些变化直接或间接受到染色质修饰的调节，如 DNA 甲基化和组蛋白翻译后修饰，包括乙酰化、甲基化和泛素化。核小体的变化通常是随机的、不同步的和异质的，因此很难用传统的集合平均方法进行监测。多种单分子荧光方法已被用于研究核小体在与各种酶相互作用（如 RNA 聚合酶 II、组蛋白伴侣、转录因子和染色质重塑因子）时的结构和结构变化。我们利用多种单分子荧光方法研究伴随这些过程的核小体变化，阐明这些过程的动力学，并最终了解各种染色质修饰如何直接调节这些过程。这些方法包括双色和三色单分子荧光共振能量转移（FRET）、单分子荧光相关光谱和荧光（共）定位。本文报告了我们目前使用的双色和三色单分子 FRET 方法的详细信息。本报告将有助于研究人员设计他们的单分子 FRET 方法，以研究核小体水平的染色质调控。
