The RSC complex remodels chromatin structure and regulates gene transcription. We used cryo–electron microscopy to determine the structure of yeast RSC bound to the nucleosome. RSC is delineated into the adenosine triphosphatase motor, the actin-related protein module, and the substrate recruitment module (SRM). RSC binds the nucleosome mainly through the motor, with the auxiliary subunit Sfh1 engaging the H2A-H2B acidic patch to enable nucleosome ejection. SRM is organized into three substrate-binding lobes poised to bind their respective nucleosomal epitopes. The relative orientations of the SRM and the motor on the nucleosome explain the directionality of DNA translocation and promoter nucleosome repositioning by RSC. Our findings shed light on RSC assembly and functionality, and they provide a framework to understand the mammalian homologs BAF/PBAF and the Sfh1 ortholog INI1/BAF47, which are frequently mutated in cancers.

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Nucleosomes are basic repeating units of chromatin, and form regularly spaced arrays in cells. Chromatin remodelers alter the positions of nucleosomes, and are vital in regulating chromatin organization and gene expression. Here we report the cryoEM structure of chromatin remodeler ISW1a complex bound to the dinucleosome. Each subunit of the complex recognizes a different nucleosome. The motor subunit binds to the mobile nucleosome and recognizes the acidic patch through two arginine residues, and the DNA-binding module interacts with the entry DNA at the nucleosome edge. This nucleosome-binding mode provides the structural basis for linker DNA sensing of the motor. Notably, the Ioc3 subunit recognizes the disk face of the adjacent nucleosome through the H4 tail, the acidic patch and the nucleosomal DNA, which is important for the spacing activity in vitro, and for nucleosome organization and cell fitness in vivo. Together, these findings support the nucleosome spacing activity of ISW1a, and add a new mode of nucleosome remodeling in the context of a chromatin environment.