A flexible and unstructured hinge region connects the Swi6 CD and CSD domains. In addition, HP1 proteins are posttranslationally modified, and these modifications affect both nucleosome binding and HP1 proteinвЂ“protein interactions ( 15, 16). The HP1 CSD domain promotes protein-protein and protein-nucleosome interactions and oligomerizes to form higher-order, phase-separated HP1-containing chromatin complexes that exhibit liquid-like properties ( 12вЂ“ 14). The HP1 CD domain binds to H3K9me peptides with low micromolar affinity (1 to 10 ОјM) ( 10, 11). HP1 proteins have a distinct architecture that consists of two conserved structural domains: (i) an N-terminal chromodomain (CD) that recognizes H3K9me nucleosomes and (ii) a C-terminal chromoshadow domain (CSD) that promotes dimerization ( Fig. These diverse cellular functions associated with heterochromatin are critically dependent on an evolutionarily conserved HP1 family of proteins that recognize and bind to H3K9me nucleosomes ( 8). Heterochromatin establishment is critical for chromosome segregation, sister chromatid cohesion, transposon silencing, and maintaining lineage-specific patterns of gene expression ( 7). H3K9 methylation (H3K9me) is a conserved epigenetic modification that is associated with transcriptional silencing and heterochromatin formation ( 6). Our studies indicate that HP1 oligomerization is essential to form dynamic, higher-order complexes that outcompete nucleic acid binding to enable specific H3K9me recognition. 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. We estimate that Swi6 recognizes H3K9me3 with ~94-fold specificity relative to unmodified nucleosomes in living cells. Using Swi6 mutants that perturb H3K9me recognition, oligomerization, or nucleic acid binding, we determine how each biochemical property affects protein dynamics. By tracking single Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Here, we visualize single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. 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.

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