Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C

Abstract

INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodelling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional class averaging to demonstrate that these remodelling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic 'tail' domain and a compact 'head' that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome-binding affinity but is largely dispensable for remodelling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodelling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodelling.

Figure 1. EM analysis of yeast INO80-C and SWR-C

(a) Upper panel. EM image of negatively stained INO80-C, showing particles in extended (black circles) and compact (white circles) conformations. Scale bar is 100 nm. Lower panels. Selected class averages of INO80-C showing the complex in extended (upper panels) and compact conformations (lower panels). The numbers are percentages of each conformation in the entire population (10,128 particles) (see Supplementary Figure 2 for all averages). Side length of individual panels is 57.3 nm. (b) Upper panel. EM image of negatively stained SWR-C. Scale bar is 100 nm. Lower panels. Selected class averages of SWR-C showing the complex in extended (upper panels) and compact conformations (lower panels). The numbers are percentages of each conformation in the entire population (20,412 particles) (see Supplementary Fig. S2 for all averages). Side length of individual panels is 57.3 nm.

Figure 2. INO80-C and SWR-C contain single hexameric rings of Rvb1/Rvb2

(a) Selected class averages of the head domain of INO80-C. Top view (top panel) shows a hexameric ring structure of Rvb1/Rvb2 (no symmetry was applied). Side view (bottom panel) depicts two layers of density. (b) Selected class averages of the head domain of SWR-C. Top view (top panel) shows a hexameric ring structure of Rvb1/Rvb2 (no symmetry was applied). Side view (bottom panel) depicts two layers of density. Side length of individual panels in (a) and (b) is 57.3 nm. (c, d) Rvb1 and Rvb2 subunit stoichiometries determined from mass spectrometry analyses for INO80-C (c) and SWR-C (d). Each error bar represents the standard deviation from three independent preparations.

Figure 3. Functional analysis of the INO80-C^arp8 subcomplex

(a) SDS-PAGE gel of INO80-C, INO80-C^arp8 and INO80-C^ies6 subcomplexes. (b) Selected class averages of negatively stained INO80-C^arp8 subcomplex. Top view (left panel) and side view (middle and right panels). Side length of individual panels is 57.3 nm. (c) Nucleosome-stimulated ATPase activity of the INO80-C^arp8 subcomplex. (d) DNA-stimulated ATPase activity of the INO80-C^arp8 subcomplex. Each error bar represents the standard deviation from three independent experiments.

Figure 4. The INO80-C^arp8 subcomplex is active for nucleosome remodeling

(a) Mononucleosome-sliding activity of the INO80-C^arp8 subcomplex. End-positioned mononucleosomes were incubated with 2-fold serial dilutions of INO80-C or INO80-C^arp8 (2-fold higher concentration than WT), and reactions were analyzed by Native-PAGE. (b) Remodeling activity of the INO80-C^arp8 subcomplex on an 11-mer nucleosomal array substrate. Remodeling rates (min⁻¹) are measured at 5 nM or 50 nM array concentration. (c) Dimer-exchange activity of the INO80-C^arp8 subcomplex. 5 nM INO80-C or INO80-C^arp8 were incubated with 100 nM H2A.Z mononucleosomes and 50 nM FLAG-tagged H2A/H2B dimers for 30 min, 60 min or 120 min in the presence of 2 mM ATP. Reactions were analyzed by Native-PAGE, and H2A incorporation was monitored by Western blot analysis.

Figure 5. Structural and functional analysis of INO80-C^ies6 and SWR-C^swc2 subcomplexes

(a) Selected class averages of negatively stained INO80-C^ies6 and SWR-C^swc2 subcomplexes showing extended (upper panels) and compact conformations (lower panels). The numbers are percentages of each conformation in the entire populations (10,202 particles for INO80-C^ies6, 10,482 particles for SWR-C^swc2) (see Supplementary Figures 7b and 8b for all averages). Side length of individual panels is 62.6 nm. (b) Nucleosome-stimulated ATPase activity of the INO80-C^ies6 subcomplex. (c) DNA-stimulated ATPase activity of the INO80-C^ies6 subcomplex. Each error bar represents the standard deviation from three independent experiments.

Figure 6. The INO80-C^ies6 subcomplex is inactive in remodeling assays

(a) Nucleosome-binding activity of INO80-C, and the INO80-C^arp8 and INO80-C^ies6 subcomplexes was analyzed by a gel-shift assay. 2 nM 154bp-mononucleosomes were incubated with each complex and subjected to 4% Native-PAGE. (b) Mononucleosome-sliding activity of the INO80-C^arp8 subcomplex. The assays were performed as described in Fig. 3e. (c) Dimer-exchange activity of the INO80-C^ies6 subcomplex. 5 nM INO80-C WT and INO80-C^ies6 were incubated with 100 nM H2A.Z containing mononucleosomes and 50 nM FLAG-tagged H2A/H2B dimers for 60 min in the presence or absence of 2 mM ATP.

Figure 7. INO80-C and SWR-C lack helicase activity

Upper panel shows a schematic diagram of substrates used for the DNA helicase assay. Radiolabeled 32-mer and 24-mer oligonucleotides were annealed to linear, single-stranded ΦX174 DNA. The star denotes the location of the ³²P label. Native-PAGE (lower panel) showed that both INO80-C and SWR-C were not able to displace any oligonuleotides, while human BLM helicase displaced only the 24-mer oligonucleotide showing a 3’ to 5’ helicase activity.

Figure 8. Model depicting the proposed conformational change of INO80-C that is coupled to nucleosome binding

The model proposes that the Ies6/Arp5 module helps to maintain an active, extended form that is competent for functional interactions with a nucleosome. This module is postulated to function as a “hinge” that responds to nucleosome binding, catalyzing productive interactions with the catalytic ATPase domain.