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

SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.

A septin-Hof1 scaffold at the yeast bud neck binds and organizes actin cables.

Cellular actin arrays are often highly organized, with characteristic patterns critical to their in vivo functions, yet the mechanisms for establishing these higher order geometries remain poorly understood. In Saccharomyces cerevisiae, formin-polymerized actin cables are spatially organized and aligned along the mother-bud axis to facilitate polarized vesicle traffic. Here, we show that the bud neck-associated F-BAR protein Hof1, independent of its functions in regulating the formin Bnr1, binds to actin filaments and organizes actin cables in vivo. Hof1 bundles actin filaments and links them to septins in vitro. F-actin binding is mediated by the "linker" domain of Hof1, and its deletion leads to cable organization defects in vivo. Using superresolution imaging, we show that Hof1 and septins are patterned at the bud neck into evenly spaced axial pillars (∼200 nm apart), from which actin cables emerge and grow into the mother cell. These results suggest that Hof1, while bound to septins at the bud neck, not only regulates Bnr1 activity, but also binds to actin cables and aligns them along the mother-bud axis. More broadly, these findings provide a strong example of how an actin regulatory protein can be spatially patterned at the cell cortex to govern actin network geometry.

Actin-related proteins regulate the RSC chromatin remodeler by weakening intramolecular interactions of the Sth1 ATPase.

The catalytic subunits of SWI/SNF-family and INO80-family chromatin remodelers bind actin and actin-related proteins (Arps) through an N-terminal helicase/SANT-associated (HSA) domain. Between the HSA and ATPase domains lies a conserved post-HSA (pHSA) domain. The HSA domain of Sth1, the catalytic subunit of the yeast SWI/SNF-family remodeler RSC, recruits the Rtt102-Arp7/9 heterotrimer. Rtt102-Arp7/9 regulates RSC function, but the mechanism is unclear. We show that the pHSA domain interacts directly with another conserved region of the catalytic subunit, protrusion-1. Rtt102-Arp7/9 binding to the HSA domain weakens this interaction and promotes the formation of stable, monodisperse complexes with DNA and nucleosomes. A crystal structure of Rtt102-Arp7/9 shows that ATP binds to Arp7 but not Arp9. However, Arp7 does not hydrolyze ATP. Together, the results suggest that Rtt102 and ATP stabilize a conformation of Arp7/9 that potentiates binding to the HSA domain, which releases intramolecular interactions within Sth1 and controls DNA and nucleosome binding.

Subunit Rtt102 controls the conformation of the Arp7/9 heterodimer and its interactions with nucleotide and the catalytic subunit of SWI/SNF remodelers.

Chromatin-remodeling complexes are assembled around a catalytic subunit that contains a central ATPase domain and flanking sequences that recruit auxiliary subunits. The catalytic subunits of SWI/SNF remodelers recruit Arp7/9 through a helicase/SANT-associated (HSA) domain N-terminal to the ATPase domain. Arp7/9-containing remodelers also carry the auxiliary subunit Rtt102, but the role of this subunit is poorly understood. Here, we show that Rtt102 binds with nanomolar affinity to the Arp7/9 heterodimer and modulates its conformation and interactions with the ATPase subunit and nucleotide. When bound to Rtt102, Arp7/9 interacts with a shorter segment of the HSA domain. Structural analysis by small-angle x-ray scattering further shows that when bound to Rtt102, the complex of Arp7/9 with the catalytic subunit assumes a more stable compact conformation. We also found that Arp7, Arp9, and Arp7/9 interact very weakly with ATP, but Rtt102 promotes high-affinity ATP binding to a single site in the heterodimer. Collectively, the results establish a function for subunit Rtt102 as a stabilizing factor for the Arp7/9 heterodimer, enhancing its interaction with nucleotide and controlling the conformation of SWI/SNF remodelers in an Arp7/9-dependent manner.