Understanding how genetically encoded tags affect phase separation by Heterochromatin Protein HP1α

Liquid-liquid phase separation (LLPS) is driven by weak multi-valent interactions. Such interactions can result in the formation of puncta in cells and droplets in vitro. The heterochromatin protein HP1 forms droplets with chromatin in vitro and is found in puncta in cells. A common approach to visualize the dynamics of HP1 in cells is to genetically encode fluorescent tags on the protein. HP1 modified with tags such as GFP has been shown to localize to dynamic puncta in vivo. However, whether tagged HP1 retains its intrinsic phase separation properties has not been systematically studied. Here, using different C-terminal tags (AID-sfGFP, mEGFP, and UnaG), we assessed how tag size and linker length affected the phase separation ability of HP1 with DNA in vitro. We found that the AID-sfGFP tag (52 kDa) promoted HP1 phase-separation, possibly driven by the highly disordered AID degron. The mEGFP tag (27 kDa) inhibited phase-separation by HP1, whereas an UnaG tag (13 kDa) with a 16 amino acid linker showed minimal perturbation. The UnaG tag can thus be used in cellular studies of HP1 to better correlate in vitro and in vivo studies. To test if cellular crowding overcomes the negative effects of large tags in vivo, we used polyethylene glycol (PEG) to mimic crowding in vitro. We found that addition of 10% PEG8000 or PEG4000 enables phase separation by GFP-tagged HP1 at comparable concentrations as untagged HP1. However, these crowding agents also substantially dampened the differences in phase-separation between wild-type and mutant HP1 proteins. PEG further drove phase-separation of Maltose Binding Protein (MBP), a tag often used to solubilize other proteins. These results suggest that phase-separation of biological macromolecules with PEG should be interpreted with caution as PEG-based crowding agents may change the types of interactions made within the phases.