A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere

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Abstract
Centromere protein A (CENP‐A) is an essential centromere‐specific histone H3 homologue. Using combined chromatin immunoprecipitation and DNA array analysis, we have defined a 330 kb CENP‐A binding domain of a 10q25.3 neocentromere found on the human marker chromosome mardel(10). This domain is situated adjacent to the 80 kb region identified previously as the neocentromere site through lower‐resolution immunofluorescence/FISH analysis of metaphase chromosomes. The 330 kb CENP‐A binding domain shows a depletion of histone H3, providing evidence for the replacement of histone H3 by CENP‐A within centromere‐specific nucleosomes. The DNA within this domain has a high AT‐content comparable to that of α‐satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surrounding region. FISH analysis indicates that the normal 10q25.3 genomic region replicates around mid‐S phase. Neocentromere formation is accompanied by a replication time lag around but not within the CENP‐A binding region, with this lag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful model for dissecting the functional domains of complex higher eukaryotic centromeres.