Fyodorova, Y. | Search bioRxiv

bioRxiv2026

Substrate recognition, not sequestration, drives the engagement of an H3K9 methyltransferase in living cells

The histone H3K9 methyltransferase Clr4 is essential for heterochromatin formation in Schizosaccharomyces pombe, yet how it searches for and engages chromatin in vivo remains unclear. Using live-cell single-molecule tracking of PAmCherry-Clr4, we quantified how perturbations to Clr4 alter its diffusion, search behavior, and residence times. Chromodomain and SET-domain Clr4 mutants move faster and more isotropically and have reduced residence times at heterochromatin, reflecting impaired substrate recognition. In contrast, deleting Swi6, which has been proposed to sequester Clr4, does not reduce the slow-state fraction or alter Clr4 diffusion, indicating that chromatin engagement is intrinsic to Clr4 rather than HP1-dependent. Anisotropy analysis at short and intermediate displacements indicates that Clr4 does not explore chromatin by simple three-dimensional diffusion but through a guided, distance-dependent search in which it repeatedly samples nearby nucleosomes before disengaging. Across all other perturbations we examined, such as deletion of the CLRC component Rik1, impaired Clr4 ubiquitination, and using cells with an unmethylatable H3K9R substrate, Clr4 dynamics were only modestly affected, and a chromatin-associated population persisted in every background. This robustness indicates that the chromodomain and SET domains are the primary determinants of how Clr4 engages chromatin in vivo, allowing it to continuously sample the genome while maintaining a stable bound population. Our results suggest how the promiscuous sampling of chromatin may also enable Clr4 to establish novel sites of heterochromatin during adaptation.

biophysics↗

bioRxiv2023

H3K9 methylation enhances HP1-associated epigenetic silencing complex assembly and suppresses off-chromatin binding

Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and each of their binding partners based on which we inferred their most likely interaction sites. Our results indicate that H3K9me promotes specific complex formation between HP1 proteins and their interactors in a spatially restricted manner, while attenuating their ability to form off-chromatin complexes. As opposed to being an inert platform or scaffold to direct HP1 binding, our studies propose a novel function for H3K9me as an active participant in enhancing HP1-associated complex formation in living cells.

biophysics↗