Mizuguchi Gaku, Wu Wei-Hua, Alami Samar, Luk Ed
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
Methods Enzymol. 2012;512:275-91. doi: 10.1016/B978-0-12-391940-3.00012-3.
The evolutionarily conserved histone variant H2A.Z has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. Saccharomyces cerevisiae Swr1, a Swi2/Snf2-related ATPase, is the catalytic core of a multisubunit chromatin remodeling enzyme, called the SWR1 complex, that efficiently replaces conventional histone H2A in nucleosomes with histone H2A.Z. Swr1 is required for the deposition of histone H2A.Z at stereotypical promoter locations in vivo, and Swr1 and H2A.Z commonly regulate a subset of yeast genes. Here, we describe an integrated nucleosome assembly-histone replacement system whereby histone exchange by chromatin remodeling activities may be analyzed in vitro. The system demonstrates ATP- and SWR1-complex-dependent replacement of histone H2A for histone H2A.Z on a preassembled nucleosome array. This system may also be adapted to analyze dynamic interactions between chromatin remodeling and modifying enzymes, histone chaperones, and nucleosome substrates containing canonical, variant, or covalently modified histones.
进化上保守的组蛋白变体H2A.Z在基因表达调控以及建立沉默异染色质扩散的缓冲机制中发挥着重要作用。酿酒酵母中的Swr1是一种与Swi2/Snf2相关的ATP酶，它是一种多亚基染色质重塑酶（称为SWR1复合物）的催化核心，该复合物能有效地将核小体中的常规组蛋白H2A替换为组蛋白H2A.Z。Swr1是体内组蛋白H2A.Z在典型启动子位置沉积所必需的，并且Swr1和H2A.Z共同调控酵母基因的一个子集。在这里，我们描述了一种整合的核小体组装-组蛋白替换系统，通过该系统可以在体外分析染色质重塑活性引起的组蛋白交换。该系统证明了在预组装的核小体阵列上，ATP和SWR1复合物依赖的组蛋白H2A被组蛋白H2A.Z替换。该系统也可用于分析染色质重塑和修饰酶、组蛋白伴侣以及含有经典、变体或共价修饰组蛋白的核小体底物之间的动态相互作用。
