Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity

Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role.