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

Abstract

Heterochromatin protein 1 (HP1) 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 HP1 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. HP1 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 HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 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 HP1 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 HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1s binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts. Statement of significanceCellular nuclei are organized into euchromatin and heterochromatin domains, encompassing actively transcribed genes or transcriptionally silent regions, respectively. The regulation of these domains is an active area of study with vast consequences for human health and disease. Here, we focus on the HP1 protein, a key component of heterochromatin compartments that is believed to function through a phase separation mechanism. HP1 interacts with a multitude of binding partners that recognize a specific interaction surface on the protein. While these specific interactions are important, our results suggest that the overall electrostatic network that forms between the proteins has a profound effect on the properties of HP1. These results have important implications for understanding how HP1 function is regulated in heterochromatin environments.