A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assembly

Abstract

A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.

Figure 1. Dissecting Swi6 self-association equilibria

a, Swi6 domains. N: N-terminal region; CD: chromodomain; H: Hinge; CSD: chromoshadow domain. b, Sedimentation Equilibrium (SE) AUC analysis of Swi6^WT self-association. Interference profiles at different rotor speeds shown. Every 20^th point is shown. c, Sedimentation Velocity (SV) AUC analysis of Swi6^WT self-association. . For b, and c, best fits for global analysis using an isodesmic self-association model are shown. d, Model of Swi6 self-association (monomer: S). e, [Swi6^WT ] = 20μM. Sedimentation coefficients: dimer (S₂) ~4S; tetramer (S₄) ~5.2S. f, Top: Swi6 CD modeled on drosophila HP1 CD with H3K9me3 peptide (PDB: 1KNE). Bottom: H3-tail (aa 4-14) and CD loop regions of Swi6 (aa 72-97), dHP1α, hHP1α and hHP1β. Conserved lysine in red. g, Top: Models for CD-CD and CD-loop interactions. H3 tail: green line; methylation: red circle. Middle: Schematic of Swi6:H3 tail interactions (Left) and of hypothetical CD:CD interactions (Right). Grey oval: region of negative charge; brown oval: π-cation interactions. Bottom: mutants used.

Figure 2. Impact of disrupting H3 tail mimic-CD interaction

a, Isodesmic association constant ($K_{obs}^{\mathrm{iso}}$) and b, dimerization association constant ($K_{obs}^{\dim }$) for Swi6 mutants (Values in Supplementary Figure 4). c, Model for the self-association of Swi6. (K^conf = [open]/closed]). K^oligo is isodesmic association constant for oligomerization from the open state. For Swi6^WT, $K_{obs}^{\dim }=\frac{K^{csd}}{K^{conf}}$ and $K_{obs}^{\mathrm{iso}}=K^{conf}\times K^{oligo}$. d, Affinity constants for H3K9me3 tail peptide measured by tryptophan fluorescence (top) and fluorescence anisotropy (bottom) studies (Values in Supplementary Figure 8). e, Location of MSL probe on G95C (yellow circle). f, SV AUC (left panels) and EPR analyses (right panels) of Swi6^probe-WT and Swi6^probe-LoopX. Representative EPR spectra shown as derivative of absorbance (y-axis) vs. magnetic field (x-axis) . c(M): molar mass distribution. Errors for probe immobilized < 10%. g, Impact of 18mer H3 peptides on probe immobilization. [Swi6^probe-WT ]= 20μM. For all panels, errors (n > 3) represent s.e.m.

Figure 3. EM studies of Swi6 and Swi6-H3K_C9me3 nucleosome complex

a, CFP-Swi6 .b, Swi6-CFP. For a, and b, representative 2D class averages are shown. c, Two different views of 3D reconstruction of the Swi6:H3K_C9me3 nucleosome complex (left), nucleosome (middle), and difference map between the two reconstructions (right). Nucleosome crystal structure (PDB 1KX5) was fitted into reconstruction. Isosurface of nucleosome 3D reconstruction at high threshold in dark blue, and low threshold in light blue (nucleosome type used in methods); H3 in red; difference map in yellow. d, Putative locations of Swi6 domains docked into difference map: CD of Swi6 (black; PDB 2RSO, aa 72-142), CSD domain of Swi6 (red; PDB 1E0B), and Hinge (H). e. Proposed locations of the two unoccupied CDs.

Figure 4. Nucleosome recognition and in vivo impact of disrupting loop-CD interaction

a, Nucleosome binding assayed by fluorescence anisotropy. b, Affinity constants for 20mer DNA. For a, and b, K_d values are in Supplementary figure 8. Errors (n ≥ 3) represent s.e.m. c, Model for conformational switch in Swi6 upon binding methylated nucleosomes. d, Top: Schematics of centromere 1 showing ura4+ reporter. Bottom: Silencing assay using ura4+ reporter. e, swi6^LoopX and swi6^AcidicX mutants decrease H3K9 methylation levels at the centromeric dg in dcr1Δ background. Errors: s.e.m from three independent IPs. f, Model: Conformational versatility of HP1-chromatin platform enables recruitment of diverse regulators that promote (yellow, red, blue and green cartoons) or inhibit (grey cartoon) heterochromatin spread.