Galloy Maxime, Lachance Catherine, Cheng Xue, Distéfano-Gagné Félix, Côté Jacques, Fradet-Turcotte Amelie
St-Patrick Research Group in Basic Oncology, Oncology Division, Centre Hospitalier Universitaire (CHU) de Québec-Université Laval Research Center, Québec, QC, Canada.
Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Université Laval, Québec, QC, Canada.
Front Cell Dev Biol. 2021 Sep 16;9:729338. doi: 10.3389/fcell.2021.729338. eCollection 2021.
The modification of histones-the structural components of chromatin-is a central topic in research efforts to understand the mechanisms regulating genome expression and stability. These modifications frequently occur through associations with multisubunit complexes, which contain active enzymes and additional components that orient their specificity and read the histone modifications that comprise epigenetic signatures. To understand the functions of these modifications it is critical to study the enzymes and substrates involved in their native contexts. Here, we describe experimental approaches to purify native chromatin modifiers complexes from mammalian cells and to produce recombinant nucleosomes that are used as substrates to determine the activity of the complex. In addition, we present a novel approach, similar to the yeast anchor-away system, to study the functions of essential chromatin modifiers by quickly inducing their depletion from the nucleus. The step-by-step protocols included will help standardize these approaches in the research community, enabling convincing conclusions about the specificities and functions of these crucial regulators of the eukaryotic genome.
组蛋白（染色质的结构成分）修饰是研究调控基因组表达和稳定性机制的核心课题。这些修饰通常通过与多亚基复合物结合发生，该复合物包含活性酶以及其他成分，这些成分决定其特异性并读取构成表观遗传特征的组蛋白修饰。为了解这些修饰的功能，在其天然环境中研究相关酶和底物至关重要。在此，我们描述了从哺乳动物细胞中纯化天然染色质修饰复合物以及制备用作底物以确定复合物活性的重组核小体的实验方法。此外，我们提出了一种类似于酵母锚定去除系统的新方法，通过快速诱导关键染色质修饰因子从细胞核中耗尽来研究其功能。所包含的分步方案将有助于在研究界规范这些方法，从而就这些真核基因组关键调节因子的特异性和功能得出令人信服的结论。
