Molecular basis for CPC-HP1 interaction: Implications for chromatin association and phase separation

Abstract

The ultimate goal of mitosis is the accurate transmission of the genome into daughter cells. The Chromosomal Passenger Complex (CPC, comprised by Borealin, Survivin, INCENP and Aurora B) is essential for the faithful segregation of chromosomes. Among others, its main biological tasks include chromosome condensation, correction of kinetochoremicrotubule malattachments, activation of the spindle assembly checkpoint and cleavage furrow formation during cytokinesis. In early prophase, CPC localises to the inner centromere through its indirect binding to Sgo1 via Borealin, H3T3ph via Survivin and also via Heterochromatin Protein 1 (HP1). While the interactions of CPC with H3 and Sgo1 have been relatively better characterised, the molecular mechanism that regulates CPC binding to HP1 remains unexplained. This study aims to provide the molecular basis for the interaction between CPC and HP1 and to dissect how HP1 assists CPC in the fulfilment of its cellular functions.

To identify the intermolecular interactions between CPC and HP1, the recombinant proteins were expressed and purified from E. coli and CPCHP1α complex could be reconstituted in vitro. SEC studies showed that the binding of the INCENP PxVxL/I motif to the HP1α chromoshadow domain is essential for CPC-HP1α interaction. However, crosslinking/ mass spectrometry analysis revealed a more elaborate network of intermolecular interactions between CPC and HP1α, with numerous crosslinks between Borealin/INCENP and HP1α. Measurement of the binding affinity using truncated versions of the proteins showed that the new contacts identified in this study are required for robust complex formation. Interestingly, HP1α increases CPC's intrinsic binding affinity for reconstituted nucleosomes, suggesting that the association of CPC-HP1α might have an effect on chromatin architecture. Finally, the contribution of these multivalent intermolecular interactions in promoting liquid-liquid phase separation was assessed. Altogether, the results presented support the idea that HP1α is a crucial CPC binding partner, and that this interaction may have considerable implications in CPC ability to associate with chromatin and drive phase separation.