Yang Zungyoon, Hayes Jeffrey J
Department of Biochemistry and Biophysics, Box 712 University of Rochester Medical Center, Rochester, NY 14642, USA.
Methods. 2004 May;33(1):25-32. doi: 10.1016/j.ymeth.2003.10.017.
The core histone tail domains are critical regulators of chromatin structure and function and modifications such as acetylation of lysine residues within the tails are central to this regulation. Studies have shown that the removal of core histone tail domains by trypsinization in which one-half to two-thirds of each core histone tail domain are removed in gross aspects mimics the acetylation of core histone tails. In addition, removal of the tails has been useful in understanding general tail function. Thus, removal of native core histone tails by trypsinization is a widely used method. In addition, many in vitro studies now employ core histones site-specifically modified with photo activatable cross-linking probes or fluorescent probes. However, in our experience, standard methods employing trypsinized donor chromatin for reconstitution of nucleosomes containing certain chemically modified histones lacking the core histone tail domains are not uniformly applicable. Here, we describe various methods for preparing nucleosomes containing a core histone modified with a cross-linking agent, APB, and lacking the core histone tail domains.
核心组蛋白尾部结构域是染色质结构和功能的关键调节因子，尾部赖氨酸残基的乙酰化等修饰对于这种调节至关重要。研究表明，通过胰蛋白酶消化去除核心组蛋白尾部结构域（其中每个核心组蛋白尾部结构域的二分之一到三分之二在总体上被去除）在很大程度上模拟了核心组蛋白尾部的乙酰化。此外，去除尾部对于理解尾部的一般功能很有用。因此，通过胰蛋白酶消化去除天然核心组蛋白尾部是一种广泛使用的方法。此外，现在许多体外研究采用经光可激活交联探针或荧光探针进行位点特异性修饰的核心组蛋白。然而，根据我们的经验，使用经胰蛋白酶消化的供体染色质来重建含有某些缺乏核心组蛋白尾部结构域的化学修饰组蛋白的核小体的标准方法并非普遍适用。在这里，我们描述了制备含有用交联剂APB修饰且缺乏核心组蛋白尾部结构域的核心组蛋白的核小体的各种方法。
