HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization

Abstract

HP1 proteins traverse a complex and crowded chromatin landscape to bind with low affinity but high specificity to histone H3K9 methylation (H3K9me) and form transcriptionally inactive genomic compartments called heterochromatin. Here, we visualize single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By tracking single Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Using Swi6 mutants that perturb H3K9me recognition, oligomerization, or nucleic acid binding, we determine how each biochemical property affects protein dynamics. We estimate that Swi6 recognizes H3K9me3 with ~94-fold specificity relative to unmodified nucleosomes in living cells. While nucleic acid binding competes with Swi6 oligomerization, as few as four tandem chromodomains can overcome these inhibitory effects to facilitate Swi6 localization at heterochromatin formation sites. Our studies indicate that HP1 oligomerization is essential to form dynamic, higher-order complexes that outcompete nucleic acid binding to enable specific H3K9me recognition.

Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions

“…To eliminate effects from the spatial correlation between single-molecule steps within the same trajectories, we simulated diffusion trajectories with similar confined area size, average track length, and overall density as experimental trajectories by drawing step lengths from the step size distribution of the corresponding experiment steps. This normalization is reported in previous work (24).…”

“…Unlike the split in the mobility state in Chp2 in swi6∆ cells, we observed a change in the weight fraction of molecules in the chromatin-sampling (β) state when imaging PAmCherry-Swi6 in chp2∆ cells (24). Hence, the deletions of the individual HP1 proteins have very different impacts on protein dynamics.…”

“…For example, in the case of Swi6, nucleosome binding in vivo is inhibited, not enhanced, by interactions with nucleic acids, unlike in in vitro binding assays. This enhancement is because the large excess of DNA in the cell can displace Swi6 from its binding site and promote protein turnover at sites of heterochromatin formation (24). Attempts to bridge the gap between in vitro and in vivo studies such as FRET and two-color imaging measurements rely on protein-protein interactions that are infrequent and transient given the dynamic properties of chromatin-binding proteins.…”

“…Single-molecule microscopy of target protein-photoactivatable fluorescent protein fusions is a powerful tool to study protein dynamics in vivo (26, 27). Live-cell imaging can access the interaction of histone modifiers with their chromatin substrates (24, 28, 29). When combined with critical advances in statistical inference methods for analyzing high spatiotemporal resolution imaging data (Bayesian statistics applied to single-particle tracking) (30, 31), we can map the biophysical mobility states of proteins (as measured by their diffusion coefficients) to their biochemical properties in living cells (24).…”

“…Live-cell imaging can access the interaction of histone modifiers with their chromatin substrates (24, 28, 29). When combined with critical advances in statistical inference methods for analyzing high spatiotemporal resolution imaging data (Bayesian statistics applied to single-particle tracking) (30, 31), we can map the biophysical mobility states of proteins (as measured by their diffusion coefficients) to their biochemical properties in living cells (24). Furthermore, analyzing the probabilities of transitioning between or dissociating from each detected mobility state can provide estimates of the biochemical properties for protein-protein and protein-chromatin interactions in cells (32).…”