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.

HP1 proteins bind with low affinity but high specificity to sites of histone H3 lysine 9 methylation (H3K9me) in the genome. HP1 binding to H3K9me compartmentalizes the genome into transcriptionally inactive heterochromatin and actively transcribed euchromatin. A characteristic feature of HP1 proteins is their dynamic and rapid turnover from sites of heterochromatin formation. How low-affinity H3K9me recognition enables HP1 proteins to rapidly and efficiently traverse a complex and crowded chromatin landscape on the millisecond timescale remains a paradox. Here, we visualize the real-time motions of an HP1 homolog, the fission yeast protein Swi6, in its native chromatin environment. By analyzing the motions of Swi6 with high spatial and temporal resolution, we map individual mobility states that are directly linked to discrete biochemical intermediates. We find that nucleic acid binding titrates Swi6 away from sites of heterochromatin formation, whereas increasing the valency of chromodomain-mediated H3K9me recognition promotes specific chromatin localization. We propose that Swi6 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for protein turnover. Our high-resolution biophysical studies provide a comprehensive framework for in vivo biochemistry and reveal how the competing biochemical properties of Swi6 affect H3K9me recognition in living cells.

Histone H3 lysine 9 methylation (H3K9me) epigenetically silences gene expression by forming heterochromatin. Proteins called HP1, which contain specialized reader domains, bind to H3K9me and recruit factors that regulate epigenetic silencing. Though these interactions have been identifiedin vitro, we do not understand how HP1 proteins specifically and selectively bind to heterochromatin-associated factors within the nucleus. Using fission yeast as a model system, we measured the single-molecule dynamics associated with two archetypal HP1 paralogs, Swi6 and Chp2, and inferred how they form complexes with their interacting partners: Epe1, a putative H3K9 demethylase; Clr3, a histone deacetylase; and Mit1, a chromatin remodeler. Through a series of genetic perturbations that affect H3K9 methylation and HP1-mediated recruitment, we were able to track altered diffusive properties associated with each HP1 protein and its binding partner. Our findings show that the HP1-interacting proteins we investigated only co-localize with Swi6 and Chp2 at sites of H3K9me. When H3K9me is absent, Epe1 and Swi6 exhibit diffusive states consistent with off-chromatin interactions. Our results suggest that histone modifications like H3K9 methylation are not simply inert binding platforms but rather, they can shift the balance of HP1 complex assembly toward a predominantly chromatin-bound state. By inferring protein-protein interactions based on the altered mobilities of proteins in living cells, we propose that H3K9 methylation can stimulate the assembly of diverse HP1-associated complexes on chromatin.

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