The role of electrostatic interactions in the phase separation of HP1α and its protein binding partners.

Heterochromatin protein 1α (HP1α) is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1α functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1α can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1α homodimer, the functional form of the protein. It has been shown that some HP1α binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1α and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1α phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1α in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1α, while electrostatic interactions dominate the regulation of phosphorylated HP1α. These results suggest that HP1α's binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts.