<i>In vitro</i> reconstitution of heterochromatin compartments reveals the spontaneous formation of tunable liquid–liquid interfaces

Compartmentalization of the genome into heterochromatin and euchromatin is a highly conserved and essential process across eukaryotes. Constitutive heterochromatin (C-Het) packages the repetitive regions of the genome within a biomolecular condensate formed through the enrichment of histone modification H3K9me3 and recruitment of its cognate reader protein heterochromatin protein-1 (HP1a). Linking the function of C-Het to its structure requires methods to assess the individual and combinatorial contributions of H3K9me3 and HP1a on the biophysical properties of C-Het. To this end, this study implements a minimal reconstitution system composed of in vitro assembled nucleosome arrays with and without H3K9me3 modifications (Me, methylated, and U, unmodified, respectively) and purified Drosophila HP1a. This minimal system reveals that H3K9me3 limits condensate coalescence and promotes intra-array interactions. Importantly, HP1a dramatically increases both Me- and U-chromatin condensate liquidity and is required for compartmentalization of U- and Me-chromatin. Heterochromatin compartmentalization is shown to be spontaneous and reversible, giving rise to liquid-liquid interfaces that can be tuned through mutations that alter discrete HP1a valencies. These direct measurements demonstrate that nuclear compartmentalization is an energetically favorable process, where HP1a mediates the differential solvation of the underlying chromatin, resulting in the formation of discrete and tunable compartmental interfaces.