Structural biochemistry of the 180 kDa Arp8-module of INO80 defines its function during chromatin remodelling

Nuclear actin (N-actin) and actin-related proteins (Arps) are critical components of several chromatin modulating complexes, including the chromatin remodeler INO80, but their function is largely elusive. Here, we report the crystal structure of the 180 kDa Arp8-module of S. cerevisiae INO80 and establish its role as an extranucleosomal DNA binding element. The actin-fold of Arp8 engages N-actin in a novel “side-to-front” interaction and specifies thereby recruitment of the Arp4-N-actin heterodimer to the segmented, “two plug” scaffold of the helical HSA domain of Ino80. The HSA domain spans a distance over 120 Å and provides a binding platform for extranucleosomal DNA, which is required for nucleosome sliding and genome-wide nucleosome positioning. Together with the recent cryoEM structure of INO80Core-nucleosome complex, our results provide insights into the mechanism by which INO80 senses 40 bp extranucleosomal entry DNA to conduct highly processive chromatin remodelling. There are 10 samples analyzed in total. Within those 10 samples we used Drosophila histones. Samples consist of the INO80 WT protein and three mutants. Those 10 samples also contain replicates. The salt gradient dialysis samples are used as control samples.

核肌动蛋白（Nuclear actin）与肌动蛋白相关蛋白（Arps）是多种染色质调控复合物的关键组成成分，其中包括染色质重塑因子INO80，但其具体功能迄今仍未完全阐明。本研究解析了酿酒酵母（S. cerevisiae）INO80复合物180 kDa的Arp8模块的晶体结构，并明确其作为核小体外DNA结合元件的功能。Arp8的肌动蛋白折叠结构域（actin-fold）通过一种全新的“侧面朝正面”相互作用模式与核肌动蛋白结合，由此介导Arp4-核肌动蛋白异二聚体招募至Ino80螺旋HSA结构域的分段式“双塞”支架中。HSA结构域的跨度超过120埃（Å），可为核小体外DNA提供结合平台，该平台是核小体滑动与全基因组核小体定位过程所必需的。结合近期解析的INO80核心复合物-核小体冷冻电镜（cryoEM）结构，本研究结果为INO80识别40 bp核小体外进入DNA、进而实现高持续性染色质重塑的机制提供了全新的研究见解。本研究共分析10份实验样本，实验中采用了果蝇（Drosophila）组蛋白。该批样本涵盖INO80野生型（WT）蛋白与三种突变体，且包含生物学重复；其中盐梯度透析样本作为对照样本。