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bioRxiv · 10.1101/2022.06.20.496886

Molecular interactions underlying the phase separation of HP1: Role of phosphorylation, ligand and nucleic acid binding

Her, C.·Phan, T. M.·Jovic, N.·Kapoor, U.·Ackermann, B. E.·Rizuan, A.·Kim, Y.·Mittal, J.·Debelouchina, G. T.
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Abstract

Heterochromatin protein 1 (HP1) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1 functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1 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 HP1 and that it involves interactions between the positively charged hinge region of HP1 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 HP1 under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1 binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1 can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1 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 HP1 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 HP1 and that can contribute to heterochromatin regulation and function.

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Her, C., Phan, T. M., Jovic, N., Kapoor, U., Ackermann, B. E., Rizuan, A., Kim, Y., Mittal, J., Debelouchina, G. T.. 2022-06-20. Molecular interactions underlying the phase separation of HP1: Role of phosphorylation, ligand and nucleic acid binding. https://doi.org/10.1101/2022.06.20.496886

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