HP1 Oligomerization Compensates for Low-Affinity H3K9me Recognition and Provides a Tunable Mechanism for Heterochromatin-Specific Localization

HP1 proteins bind with low affinity but high specificity to histone H3 lysine 9 methylation (H3K9me), forming transcriptionally inactive genomic compartments referred to as heterochromatin. How HP1 proteins traverse a complex and crowded chromatin landscape on the millisecond timescale and yet recognize H3K9me with high specificity remains paradoxical. Here, we visualize the single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By analyzing the motions of individual Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Using mutants that perturb Swi6 H3K9me recognition, oligomerization, or nucleic acid binding, we parse how each biochemical property affects protein dynamics in living cells. Based on in vivo kinetic measurements, we estimate that Swi6 recognizes H3K9me in cells with ~94-fold specificity. While nucleic acid binding suppresses Swi6 oligomerization, as few as four tandem chromodomains are sufficient to overcome these inhibitory effects to facilitate Swi6 localization at sites of heterochromatin formation. Our studies propose that HP1 oligomerization is essential to form dynamic, higher-order complexes that can outcompete the inhibitory effects of nucleic acid binding to enable highly specific H3K9me recognition in living cells.