Structural basis for the inner centromere regulatory hub assembly critical for error-free chromosome segregation

Abstract

Accurate chromosome segregation during cell division is essential for maintaining genome stability, and errors in this process can result in aneuploidy, polyploidy and disorders such as cancer and infertility. Therefore, understanding the proteins and their interactions at play during this critical event becomes significant. Chromosome segregation is controlled primarily by proteins that are localised at the centromere, the region on the chromosome where kinetochores assemble and attach to microtubules. Within this region, the inner centromere acts as a hub for factors that monitor and control chromosome segregation, including the Chromosomal Passenger Complex (CPC), Heterochromatin Protein 1 (HP1), shugoshins, cohesin, and various kinases and phosphatases that regulate this process. Although these components have been studied extensively, the principles by which they assemble into a functional network remain incompletely understood. This study specifically investigates how the inner centromere interaction network assembles, with the focus on how CPC interacts with H3T3 phosphorylated nucleosome in vitro, and centromeric chromatin in vivo. Using an integrated approach combining biochemical reconstitution, cryo-electron microscopy, cellular localisation and functional assays, and in vitro and in vivo MNase assays, the research characterised the structural and mechanistic basis of CPC association with H3T3ph nucleosome. The cryo-EM structure of CPC-H3T3ph NCP complex revealed how CPC engages with the acidic patch and entry-exit DNA of the NCP via Borealin. EMSA and SPR assays confirmed that the stable and conformationally heterogeneous interactions are important for efficient CPC-NCP binding. Structural findings, supported by biochemical and functional assays, identified a previously unrecognised role of the CPC in protecting nucleosomal DNA against MNase digestion in vitro and in vivo. These results provide mechanistic insight into CPC-mediated chromatin stabilisation and highlight how CPC contributes to maintaining genome integrity. By dissecting the CPC-nucleosome interaction, this study advances the understanding of inner centromere organisation and provides a foundation for further exploration of the assembly and function of the inner centromere interaction network.