Epigenetically dynamic human centromeres are maintained within a stable DNA methylation signature

Centromere location is specified by CENP-A, a centromere-specific histone that epigenetically propagates centromere identity. How CENP-A is epigenetically 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 and H3K9me3 patterns, with DNA methylation shifting according to CENP-A position. We further reveal temporary precise CENP-A maintenance but evolution of CENP-A, DNA methylation and H3K9me3 position over prolonged proliferation, with neocentromeres exhibiting DNA methylation instability, H3K9me3 gain, boundary loss and increased epigenetic heterogeneity. Lastly, prolonged CENP-A and HJURP overexpression leads to centromere and neocentromere expansion, gradual CENP-A depletion, neocentromere destabilization and re-localization. We reveal the naturally evolving epigenetic plasticity of human centromeres and neocentromeres and highlight the importance of repressive chromatin boundaries in maintaining centromere stability across cellular proliferation.