1UPMC Hillman Cancer Center, Pittsburgh, PA, USA 2Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA 3

Centromere identity is defined and maintained epigenetically by the presence of the histone variant CENP-A. How centromeric CENP-A position is specified and precisely maintained through DNA replication is not fully understood. The recently released Telomere-to-Telomere (T2T) genome assembly containing the first complete human centromere sequences provides a new resource for examining CENP-A position. Mapping CENP-A posi-tion in clones of the same cell line to the T2T assembly identified highly similar CENP-A position after multiple cell divisions. In contrast, centromeric CENP-A epialleles were evident at several centromeres of different human cell lines, demonstrating the location of CENP-A enrichment and the site of kinetochore re-cruitment vary among human cells. Across the cell cycle, CENP-A molecules deposited in G1 phase are maintained in their precise position through DNA replication. Thus, despite CENP-A dilution during DNA replication, CENP-A is precisely reloaded onto the same sequences within the daughter centromeres, maintaining unique centromere identity among human cells.

Automated summary

Centromere identity is defined and maintained by CENP-A, but how its positioning and maintenance during DNA replication are accomplished remains unclear. The newly released T2T genome assembly provides a valuable tool for studying CENP-A positioning. Mapping CENP-A position to the T2T assembly revealed consistent localization after cell divisions, while variations in CENP-A expression were observed across different human cell lines. Despite CENP-A dilution during DNA replication, it is reliably reloaded onto the same sequences in daughter centromeres, preserving centromere identity in human cells.