Liquid droplet formation by HP1á suggests a role for phase separation in heterochromatin

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands1-3. Here we identify a new property of the human HPla protein: the ability to form phase-separated droplets. While unmodified HPla is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HPla ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HPla droplets, but molecules such as the transcription factor TFIIB show no preference. Using a singlemolecule DNA curtain assay, we find that both unmodified and phosphorylated HPla induce rapid compaction of DNA strands into puncta, although with different characteristics4. We show by direct protein delivery into mammalian cells that an HPla mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HPla. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HPl droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.