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

bioRxiv, March 2023

36945633

Ziyuan Chen, Melissa Seman, Ali Farhat, Yekaterina Fyodorova, Saikat Biswas, Alexander Levashkevich, P Lydia Freddolino, Julie S Biteen, Kaushik Ragunathan

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 identified in 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. During differentiation, epigenetic silencing is essential for preserving cellular identity. Establishing and maintaining epigenetic silencing depends on histone H3 lysine 9 methylation, which HP1 proteins recognize and bind with low micromolar affinity and millisecond-scale kinetics. HP1 proteins also recruit diverse histone modifiers to maintain gene silencing. HP1 protein biochemistry has revealed what can happen, but the state-of-the-art in this field includes little about what does happen in the complex and crowded environment of the nucleus. Using single-molecule tracking of HP1 proteins and their binding partners, we identified the rules that govern their complex formation in the native chromatin context, and we found that chromatin- previously thought to be an inert platform-enhances complex formation between HP1 and its binding partners.