Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha
satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha
satellite DNA and permit analysis of centromeric
DNA and
chromatin organization. In this study, we present molecular cytogenetic and CENP-A
chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique
DNA and CENP-A
chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated
DNA. This is the first example of a neocentromere forming on repetitive
DNA, as all other mapped neocentromeres have formed on single copy
DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem
DNA in centromere structure. The second neocentromere was found on a neocentric
ring chromosome that contained the 8q21 tandemly repeated
DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the
ring chromosome. This neocentromere suggests that chromosomal rearrangement and
DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.