Neocentromeres fail to maintain DNA methylation boundaries, driving CENP-A drift, instability, and chromosome missegregation

How CENP-A is maintained at one location in rapidly evolving centromeric DNA is unknown. Using single-cell derived clones of human cell lines, we demonstrate heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere.

CENP-A heterogeneity is accompanied by heterogenous DNA methylation patterns, with DNA methylation shifting according to CENP-A position. We demonstrate centromere epigenetic plasticity over extended proliferation, with native centromeres maintaining stable DNA methylation boundaries, but neocentromeres exhibiting DNA methylation instability, boundary loss and increased missegregation. Finally, we show that neocentromeres are more sensitive to DNA methylation inhibition than native centromeres, and that this inhibition is accompanied by expanded CENP-A-enriched domains and increased missegregation.