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.