Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement

Abstract

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility 1,2 . The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair 2-4 . Here we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored through the interactions between the N-terminus of Sth1 and the core. Our cryo-EM analysis also shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of Rsc8 SWRIM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes. MainEukaryotes have four major families of chromatin remodelers: SWI/SNF, ISWI, CHD, and INO80 2 . Each of these remodelers plays distinct roles based on how they select and affect target nucleosomes. Together, these remodelers give rise to the distinct chromatin landscapes observed in eukaryotic cells and determine how genetic information is organized, replicated, transcribed, and repaired 5 . In S. cerevisiae there are two members of the SWI/SNF family of chromatin remodelers: RSC and SWI/SNF 3,4 . RSC is essential for viability and is ten times more abundant than SWI/SNF 4 . While both complexes play a role in remodeling nucleosomes during transcription initiation, RSC is also involved in transcription-independent processes such as mitotic division, double stranded break repair, and telomere maintenance 6-12 .RSC is a ~1-MDa complex composed of 17 proteins, with two copies of Rsc8 and one copy of either Rsc1 or Rsc2 (Fig. S1) 4,13 . In order to determine the structure of RSC, we purified the complex from S. cerevisiae and performed cryo-EM analysis (Fig. S2). We find that RSC is composed of five main lobes, three that form a rigid core (head, body and arm) and two that are flexibly attached (leg and tail) ( Fig. 1a, b; Fig. S3, S4). We determined the structure of the core to ~3.1 Å and mapped 14 proteins within this region: Rsc1/2, Rsc3, Rsc4, Rsc6, Rsc8 (2 copies), Rsc9, Rsc30, Rsc58, Ldb7, Npl6, Htl1, Sfh1, and Sth1 (Fig. 1c, d, e). Our negative stain analysis of the yeast SWI/SNF complex shows that, like RSC, it has head, body, and arm regions that define a rigid core, and a flexible leg that occupies similar overall positions (Fig. S5). However, SWI/SNF lacks a tail lobe, indicating that while RSC and SWI/SNF share a conserved core architecture, ...

Structural insights into assembly and function of the RSC chromatin remodeling complex

“…5a, yellow circle). Several studies have recently reported that SWI/SNF remodelers engage the acidic patch on the opposite face of the nucleosome using their Sfh1 (RSC) or Snf5 (SWI/SNF) subunit (Fig 7e) [20][21][22]24,24 . This interaction is vital to the function of the enzyme, as mutations in a conserved basic domain in SMARCB1 (Snf5 homolog) disrupt binding to the nucleosome, reduce remodeling rates, and have defects in cell-type differentiation 40 .…”

“…Fig. 5) [20][21][22][23][24] . The C-terminal end of the HSA helix contacts the ATPase and engages with the post-HSA and P1 domains, as well as the Brace Helices ( Fig.…”

“…Removal of the ARPs from the full RSC complex results in basal remodelling roughly equivalent to that of Sth1 alone, suggesting that they perform a core function in activating remodeling by RSC 18 . A mechanistic understanding of these regulatory interactions is still missing as the recent cryo-EM structures of the yeast RSC [20][21][22] and SWI/SNF 23 complexes, and the BAF complex, the human ortholog of SWI/SNF 24 did not reach high enough resolution in the ARP module or the HSA/post-HSA/P1 regulatory hub. To bridge this gap, we focused on the well-characterized Sth1/Arp7/Arp9/Rtt102 RSC sub-complex ( Fig.1a) that has been used to gain much of our current understanding of the regulation of remodeling in RSC 17,18,[25][26][27] .…”

Interaction of RSC chromatin remodelling complex with nucleosomes is modulated by H3 K14 acetylation and H2B SUMOylationin vivo

“…Recently, the high-resolution structure of the RSC complex bound to a nucleosome has been solved by cryo-electron microscopy, revealing three flexibly connected parts [17][18][19]. The motor domain of the Sth1 subunit binds at superhelical location (SHL) +2, from where it translocates the DNA one base pair at a time into the direction of the dyad, possibly creating a loop that propagates around the nucleosome [20].…”

“…The ARP module, which couples DNA translocation and ATPase activity [21], connects the motor domain to the substrate recognition module (SRM). The latter contains DNA-binding Zn-cluster domains, five bromodomains, a histone-tail binding BAH domain and the nucleosome-binding C-terminal tail of Sfh1 [17][18][19]. Due to the flexible tethering of these domains, their structure, substrate preference, and interaction with the nucleosome remained largely unresolved.…”