Acquisition and metastability of centromere identity and function: sequence analysis of a human neocentromere.

In this issue, Barry and colleagues (Barry et al. 2000) report the sequence of an 80 kb region of euchromatin from human chromosome 10 that can acquire centromeric activity. This new centromere, or neocentromere, drives stable mitotic inheritance once established. Approximately 40 neocentromeres have so far been identified in humans (Warburton et al. 2000). Patients with such neocentromere-containing rearranged chromosomes are heterozygous for the chromosome aberration [marker deletion, or mardel(10)], and so contain homologous loci that are independently inert or fully functional for centromere activity (Voullaire et al. 1993). This study completes a sequence analysis of the neocentromere region (Barry et al. 1999) and investigates what sequence polymorphisms, if any, occur when acquiring neocentromeric activity (Barry and colleagues 2000). We find no evidence for any sequence change, data that strongly support an epigenetic mechanism for neocentromere identity and regulation. DNA associated with neocentromere activity in mardel(10) (NC DNA) was previously identified by examining the distribution of centromere proteins (primarily centromere proteins CENPs A and C) on stretched chromosomes, relative to the location of regions identified by fluorescence in situ hybridization (du Sart et al. 1997). The restriction map of this 80 kb region of NC DNA was compared to that of homologous nonneocentromeric (HC) DNA from a nonparental source, which demonstrated that no substantial polymorphisms exist between the neocentromere and wildtype genomic library clones (Barry et al. 1999). However, these results are open to the caveat that small changes in primary DNA structure can be causative in centromeric activity, and that these changes are below the resolution of restriction mapping. Additionally, the entire NC sequence had been determined and analyzed for motifs or presence of repeat DNAs, some of which have been weakly correlated with centromeric activity. The NC sequence was not significantly different from random sequence in regard to satellite DNAs; however, a notable motif of unknown function, AT28, was discovered, and Koch (2000) has discussed its potential contribution to centromeric activity. A superficial structural similarity to alphoid DNA and the centromere of Saccharomyces cerevisiae were enough to implicate AT28 as a potential centromere seed; however, it was not known whether AT28 was unique to the NC DNA, or also present when centromere activity was absent. Given the small size of the Saccharomyces centromere and the ability of single nucleotide mutations to completely disrupt centromere function (Hyman and Sorger 1995), it is not unreasonable to argue that a small region (∼600 bp) can account for the centromere activity in NC. Barry and colleagues (2000) have put this issue to rest by sequencing two additional sources of this same DNA: loci from an unrelated subject (HC DNA) and from the paternal progenitor chromosome (PnC DNA), both of which are inert with respect to centromeric activity. Sequence comparison between the NC DNA and HC DNA showed 370 single nucleotide polymorphisms (SNPs), leading to the possibility that any subset of these SNPs could be correlated with neocentromere activity. However, the sequence of the centromere-inactive PnC progenitor was identical to the NC DNA, including the AT28 region. This clearly and simply rules out any notion that neocentromeric activity relies on these polymorphisms. This study presents unequivocal evidence for epigenetic regulation of neocentromere activity on mardel(10). Centromere activity clearly maps to this 80 kb region; yet not a single nucleotide differs between it and parental sequence, which shows no neocentromere activity (as assayed by chromosome segregation and localization of twenty centromere-specific factors (Depinet et al. 1997, Saffery et al. 2000). Something other than DNA sequence, such as chromatin structure, must differ between chromosomes of father (PnC) and son (NC), and must be responsible for distinguishing between centromere-on and centromere-off states (Karpen and Allshire 1997; Murphy and Karpen 1998). The persistence of mardel(10)’s centromere, and the absence of centromere activity on normal 10q, shows that the state of centromere activity is stably propagated through the entire cell cycle once it is established. The neocentromere, then, must remain marked throughout the cell cycle, and the mark must be accurately templated to newly synthesized DNA prior to the next Sphase. If we liken the centromere to any other example of epigenetic inheritance, we are left with a wealth of speculative models underlying a potential mark. At one time or another, structural RNA (Clemson et al. 1996), protein localization (Cavalli and Paro 1998), localized Corresponding author. E-MAIL karpen@salk.edu; FAX 858–622–0417. Insight/Outlook