Molecular basis for CPC-HP1 interaction: Implications for chromatin association and phase separation
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Date
31/07/2021Item status
Restricted AccessEmbargo end date
31/07/2022Author
Jiménez Sánchez, Ignacio
Metadata
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.