Tuning the phase separation properties of HP1α through ligand interactions.

Abstract

Heterochromatin protein 1α (HP1α) is a crucial component for gene repression and the structural integrity of heterochromatin. It is proposed that HP1α functions through a liquid-liquid phase separation (LLPS) mechanism where HP1α compacts nucleosome arrays into a liquid compartment and prevents transcription factors from accessing chromatin. HP1α can undergo LLPS via phosphorylation of the N-terminus extension (NTE) or through interactions with DNA/nucleosomes. The multidomain architecture of HP1α allows for simultaneous multivalent interactions with other HP1α proteins and multiple binding partners. These multivalent interactions are believed to be important for forming and maintaining heterochromatin structure but their exact role in HP1α LLPS is not well understood. In our study, we used experimental and computational approaches to investigate how a series of peptides from HP1α binding partners can fine tune the ability of the protein to undergo phase separation. Our results showed that in phosphorylation-driven LLPS, positively charged peptides enhance phase separation, while negatively or near neutral charged peptides disrupt the process. DNA-driven LLPS can also be inhibited by negatively or near neutral charged ligands. However, in this case, positively charged peptides compete with HP1α for DNA interactions, which also disrupts LLPS. Our results suggest that phosphorylation-driven LLPS has a larger tunability range compared to phase separation with DNA, and that the electrostatic properties of HP1α ligands play an important role in modulating this process. Our study provides insights into the potential mechanisms of heterochromatin regulation where binding partners may modulate the biophysical properties of HP1α to control access to chromatin and the genetic information in the cell.