The 10q25 neocentromere and its inactive progenitor have identical primary nucleotide sequence: further evidence for epigenetic modification

Abstract

We have previously localized the core centromere protein-binding domain of a 10q25.2-derived neocentromere to an 80-kb genomic region. Detailed analysis has indicated that the 80-kb neocentromere (NC) DNA has a similar overall organization to the corresponding region on a normal chromosome 10 (HC) DNA, derived from a genetically unrelated CEPH individual. Here we report sequencing of the HC DNA and its comparison to the NC sequence. Single-base differences were observed at a maximum rate of 4.6 per kb; however, no deletions, insertions, or other structural rearrangements were detected. To investigate whether the observed changes, or subsets of these, might be de novo mutations involved in neocentromerization (i.e., in committing a region of a chromosome to neocentromere formation), the progenitor DNA (PnC) from which the NC DNA descended, was cloned and sequenced. Direct comparison of the PnC and NC sequences revealed 100% identity, suggesting that the differences between NC and HC DNA are single nucleotide polymorphisms (SNPs) and that formation of the 10q25.2 NC did not involve a change in DNA sequence in the core centromere protein-binding NC region. This is the first study in which a cloned NC DNA has been compared directly with its inactive progenitor DNA at the primary sequence level. The results form the basis for future sequence comparison outside the core protein-binding domain, and provide direct support for the involvement of an epigenetic mechanism in neocentromerization.

Figure 1

Differences between the ∼80-kb HC and NCII sequences. Vertical bars represent the positions of single nucleotide differences between the two sequences. The numbers below represent the number of single nucleotide differences (where there is more than one difference) over a 1-kb region. Numbers with asterisks represent clusters of differences.

Figure 2

TAR cloning and sequencing of PnC DNA. The shaded area represents the region corresponding to the ∼80-kb 10q25.5 NC DNA (du Sart et al. 1997). (A) Sequenced regions of the HC DNA (derived from a CEPH library YAC clone) and NC DNA [derived from the mardel(10) neocentromere]. Total number of nucleotides sequenced is shown in brackets. (B) Structure of the HC/NC region and flanking DNA. Solid boxes represent STSs used in the identification and cloning of the DNA. AFM259xg5 is a (CA)n microsatellite located ∼150 kb (represented by the broken line) from the core region (Cancilla et al. 1998). AT28 (Barry et al. 1999) is a polymorphic VNTR used to identify the progenitor allele. C3-F2 is a 1.4-kb EcoRI fragment that served as the specific TAR “hook”(Cancilla et al. 1998). Small arrows indicate oligonucleotides used in PCR of the STSs. p′ and q′ refer to the short and long arms of mardel(10), respectively. (C) Radial TAR strategy using the vector pVC39-Alu/C3-F2(+) for the direct cloning of the progenitor DNA from the total genomic DNA of CE. The hatched box indicates the position of the Alu consensus sequence hook. Crosses denote the sites of recombination between the TAR vector pVC39-Alu/C3-F2( +) and CE genomic DNA at the C3-F2 and Alu hooks during cloning. The resulting circular YAC, CE-4–27, was shown by the AT28 polymorphism (see Fig. 3) to contain the PnC DNA from the progenitor chromosome 10. (D) The ∼69-kb sequenced portion of PnC DNA, represented by the bar.

Figure 3

Identification and confirmation of the PnC DNA clone using the AT28 polymorphism. AT28 was amplified by PCR as described in Methods. PCR products were purified and digested with RsaI and electrophoresed on 3% (wt/vol) agarose. (BE) Cell line from mardel(10) patient; (BE2C1–18–5f) a somatic cell hybrid line containing mardel(10) chromosome but not the normal chromosome 10; (5f-52-E8) a BAC clone containing the NC region derived from the BE2C1–18–5f somatic cell hybrid; (CE-4–27) a circular YAC containing the PnC DNA; (CE) a cell line from patient BE's father; (PAC4) a PAC clone containing the HC DNA derived from the normal chromosome 10 of a CEPH individual (du Sart et al. 1997; Cancilla et al. 1998). Note the identical fingerprints of CE-4–27, BE2C1–18–5f and 5f-52-E8.