 Solution

The first task is to map the DNA associated with the centromeric histone variant CENP-A; the second is to determine how that occupancy is maintained. These require different evidence. A shared DNA sequence is not sufficient evidence for an inherited centromeric state.

Use validated anti-CENP-A chromatin immunoprecipitation sequencing with input DNA and an appropriate negative immunoprecipitation control. Check antibody recognition separately in human, horse and chicken; an endogenous epitope-tagged allele can provide an orthogonal control if its chromosome-segregation function is retained. Native digestion can recover CENP-A-containing nucleosomes, whereas crosslinking can retain indirect associations. CUT&RUN provides a complementary occupancy assay with different recovery biases. Establish centromeric function by colocalization with kinetochore proteins and by normal chromosome segregation, not merely by calling a sequencing peak.

Mapping repetitive DNA is the main analytical obstacle. Use centromere-resolved assemblies where possible, retain information from multimapping reads, and analyse satellite-family enrichment as well as uniquely mappable sequence. Absence of a peak in an incomplete reference is not evidence of absent CENP-A. Long-read sequence assemblies, informative single-nucleotide polymorphisms, and DNA fluorescence in situ hybridization can connect a recovered sequence to a particular chromosome and distinguish homologues.

For precise positioning, compare enriched domains with input-normalized background at several resolutions. Map nucleosome-protected fragments and use fibre immunofluorescence or sequence probes to check domain extent and heterogeneity. A broad population peak may represent alternative positions in different cells or homologues, rather than one broad CENP-A domain on every chromosome.

The expected outcome is a common centromeric function without a universal centromeric DNA sequence. Human CENP-A commonly occupies selected portions of alpha-satellite arrays, but human neocentromeres show that such arrays are not obligatory. Horse chromosome 11 provides a normal satellite-free centromere. Chicken chromosomes include both repeat-rich centromeres and comparatively short non-tandem-repetitive centromeric regions.^[ref][ref]

To test mitotic stability, start from single-cell clones and remap CENP-A on the same haplotype after successive divisions, while checking that the underlying DNA has not changed. Pulse labelling of old versus newly incorporated CENP-A can distinguish retention from replenishment. Follow both chromosome identity and domain position: stable centromeric function does not require an invariant nucleotide boundary. To test meiotic transmission, use informative pedigrees or gamete-and-offspring material and allele-resolved mapping, with matched cell types where possible. An inherited positional state, or bounded movement of that state on unchanged DNA, supports epigenetic centromere inheritance. Artificial centromere deletion and neocentromere recovery provide an additional perturbation test, but establishment after deletion is different from ordinary meiotic inheritance.^[ref]