HP1β carries an acidic linker domain and requires H3K9me3 for phase separation.

Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1β which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1β mutants defective in H3K9me3 binding were less efficient in phase separationin vitro and failed to accumulate at heterochromatin in vivo. We propose that multivalent interactions of HP1β with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments in vivo.