Understanding how genetically encoded tags and crowding agents affect phase separation by heterochromatin protein HP1α

The heterochromatin protein HP1α (heterochromatin protein 1 alpha) phase separates in vitro and displays properties compatible with phase separation in cells. Phase separation of HP1α in cells is typically studied using genetically encoded fluorescent tags such as green fluorescent protein (GFP). Whether such tags affect the intrinsic phase separation properties of HP1α is understudied. We assessed how tag size and linker length affect phase separation by HP1α in vitro. GFP tags inhibited phase separation by HP1α. In contrast, an UnaG tag with a 16 amino acid glycine-glycine-serine (GGS) linker minimally perturbed HP1α phase separation in vitro and could be used to visualize HP1α dynamics in cells. We further investigated the effects of a commonly used crowding agent, polyethylene glycol (PEG). PEG induced phase separation of proteins with no propensity to phase separate under physiological buffer conditions and dampened the effects of HP1α mutations. Therefore, phase separation of biological macromolecules with PEG-containing crowding agents should be interpreted with caution.