Molecular interactions underlying the phase separation of HP1α: Role of phosphorylation, ligand and nucleic acid binding

Abstract

Heterochromatin protein 1α (HP1α) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can form phase separated liquid droplets upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. While it is known that LLPS requires homodimerization of HP1α and that it involves interactions between the positively charged hinge region of HP1α and the negatively charged phosphorylated NTE or nucleic acid, the precise molecular details of this process and its regulation are still unclear. Here, we combine computational modeling and experimental approaches to elucidate the phase separation properties of HP1α under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1α binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged peptide ligands such as Sgo1 and H3 and disrupted by the addition of negatively charged or neutral peptides such as LBR and CAF-1. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in the LLPS of HP1α where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine tune the properties of HP1α and that can contribute to heterochromatin regulation and function.