Haplotype-Resolved Genomics Reveals Conserved Chromatin Architecture and Epigenetic Constraints of Human Neocentromeres

Hoyt, S. J.; Hartley, G. A.; Antopia, M. C.; Tullius, T. W.; Taylor, D. J.; Tillquist, N. M.; Neph, S.; Pauloski, N.; Munson, K. M.; Hoekzema, K.; Eichler, E. A.; Stergachis, A. B.; Logsdon, G. A.; O'Neill, R. J.

doi:10.64898/2025.12.23.696241

bioRxiv · 10.64898/2025.12.23.696241

Haplotype-Resolved Genomics Reveals Conserved Chromatin Architecture and Epigenetic Constraints of Human Neocentromeres

Hoyt, S. J.·Hartley, G. A.·Antopia, M. C.·Tullius, T. W.·Taylor, D. J.·Tillquist, N. M.·Neph, S.·Pauloski, N.·Munson, K. M.·Hoekzema, K.·Eichler, E. A.·Stergachis, A. B.·Logsdon, G. A.·O'Neill, R. J.

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Abstract

Human neocentromeres are functional centromeres demarcated by CENP-A nucleosomes that form ectopically at alpha satellite-free loci. How neocentromeres reshape local chromatin and which features of native centromeric chromatin are preserved are unknown. We generated gapless, haplotype-resolved assemblies of native and neocentromeres from three patient-derived cell lines. Integrating CpG methylation, CENP-A profiling, and single-molecule chromatin fiber sequencing, we reveal chromatin features that define the essential centromeric architecture reconstituted during neocentromere establishment. We find that a deletion within the satellite array encompassing the hypo-CpG methylation centromere dip regions (CDRs) led to native centromere inactivation, that neocentromeres harbor CDRs and a dichromatin architecture, recapitulating features of alpha-satellite centromeres, and that LINEs demarcate neocentromere boundaries, implicating transposable elements in restricting CENP-A domain spreading. Moreover, neocentromeric chromatin is incompatible with promoter-like chromatin states, redefining the regulatory landscape within genic regions. Finally, using haplotype-specific chromatin footprinting, we resolve CENP-A nucleosome chromatin architecture of active centromeres.

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Hoyt, S. J., Hartley, G. A., Antopia, M. C., Tullius, T. W., Taylor, D. J., Tillquist, N. M., Neph, S., Pauloski, N., Munson, K. M., Hoekzema, K., Eichler, E. A., Stergachis, A. B., Logsdon, G. A., O'Neill, R. J.. 2025-12-25. Haplotype-Resolved Genomics Reveals Conserved Chromatin Architecture and Epigenetic Constraints of Human Neocentromeres. https://doi.org/10.64898/2025.12.23.696241

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