Project description: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-CHM13) genome assembly containing the first complete human centromere sequences provides a new resource for examining CENP-A position. Mapping CENP-A position in clones of the same cell line to T2T-CHM13 identified highly similar CENP-A position following 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 site of kinetochore recruitment varies among human cells. Across the cell cycle, CENP-A molecules deposited in G1 phase are maintained at 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.