Spatio-temporal chromosomal arrangements by late-replicating heterochromatin

Centromere clustering orchestrates the three-dimensional genome organization and, in turn, impacts genome stability ^1-4 . In this study, we report the principles governing the chromosome-scale architectural changes associated with centromere clustering and declustering in Cryptococcus neoformans , an opportunistic human fungal pathogen. We demonstrate that centromeres and telomeres in C. neoformans are scattered around the nuclear periphery during interphase ^G1 , resembling metazoans rather than model budding yeasts. Hi-C data of the synchronised cells reveal centromere and telomere-associated heterochromatic regions are organized as separate compartments at the nuclear periphery, distinct from the euchromatic regions, that eventually shape the global organization of interphase ^G1 chromosomes. Polymer modeling of the Hi-C data at interphase and anaphase stages strongly supports a global reorganization of chromosomes from the non-Rabl to the Rabl-like chromosomal arrangement. Most strikingly, while centromeres are clustered and replicate early in most yeasts, our sort-seq studies provide evidence that C. neoformans centromeres replicate late during the S-phase. In summary, we uncover yet unknown principles governing the chromosome-scale architecture that link centromere DNA replication timing, spatial centromere positioning, functional compartmentalization of chromosomes, and dynamic changes in the global three-dimensional genome architecture in a species that shared a common ancestor with metazoans more than a billion years ago.