Human centromeric alphoid domains are periodically homogenized so that they vary substantially between homologues. Mechanism and implications for centromere functioning

Abstract

Sequence analysis of alphoid repeats from human chromosomes 17, 21 and 13 reveals recurrent diagnostic variant nucleotides. Their combinations define haplotypes, with higher order repeats (HORs) containing identical or closely-related haplotypes tandemly arranged into separate domains. The haplotypes found on homologues can be totally different, while HORs remain 99.8% homogeneous both intrachromosomally and between homologues. These results support the hypothesis, never before demonstrated, that unequal crossovers between sister chromatids accumulate to produce homogenization and amplification into tandem alphoid repeats. I propose that the molecular basis of this involves the diagnostic variant nucleotides, which enable pairing between HORs with identical or closely-related haplotypes. Domains are thus periodically renewed to maintain high intrachromosomal and interhomologue homogeneity. The capacity of a domain to form an active centromere is maintained as long as neither retrotransposons nor significant numbers of mutations affect it. In the presented model, a chromosome with an altered centromere can be transiently rescued by forming a neocentromere, until a restored, fully-competent domain is amplified de novo or rehomogenized through the accumulation of unequal crossovers.

Figure 1

(A) The chromosome 17 satellite alpha HOR is 2712 bp long. The primers chosen for PCR are indicated in the Materials and Methods section. On PCR amplification with 17-1A/17-2A, a DNA fragment of 1886 bp is generated when the HOR is a 16mer, or diminished by one, two or three… integral basic repeat units (171 bp). This figure represents the deletion by three basic repeat units leading to the 13mer HOR. (B) PCR DNA fragments obtained with 17-1A/17-2A with the two hybrid cell lines GM10498 and GM10321 analysed in this study. (C) The PCR products with 17-α1/17-α2 and 17-1A/17-2A are shown together with the positions () of the diagnostic variant nucleotides and the alternative nucleotides they exhibit.

Figure 2

Haplotypes obtained with 17-α1/17-α2 in the five DNA fragments (F1–F5) generated upon SacI digestion of GM10498 DNA prepared in agarose plugs. Pulsed field gel electrophoresis (data not shown) allowed an estimation of the DNA fragment lengths as about 100, 200, 250, 450 kb, and more than 1 Mb, for F1 to F5, respectively.

Figure 3

The haplotypes of the 87 sequenced clones of GM10498 DNA are represented with the alternative nucleotides found at each diagnostic position of the 17-3A / 17-4A portion of the 16mer HOR (Figure 1). The percentage of sporadic mutations of each clone is also indicated, averaging 0.18 ± 0.22%. Clones 1.3, 2.31, 2.23, 1.30, 1.29 and 2.1 have been arbitrarily excluded from the calculation because they exhibited more than 1% mutation.

Figure 4

Representation of the 71 haplotypes detected in the nine chromosomes 17 of this study (one for the hybrid cell lines GM10498, GM10321 and GM12506; two for individuals 103 and TOU, and hybrid cell line GM08729). They have been ordered by the multiple sequence alignment program clustalW (47). The number of clones exhibiting a given haplotype is indicated for each sample.

Figure 5

Representation of the haplotypes detected in the six chromosomes 21 of this study. For clarity, different haplotypes that are identical except for one diagnostic variant nucleotide have been grouped so that their number is reduced from the original 78 to 50 ordered by the multiple sequence alignment program clustalW.

Figure 6

Representation of the haplotypes detected in the two chromosomes 13 of the two hybrid cell lines GM11767 and GM11689.

Figure 7

A fully functional centromere is normally formed within an alphoid array when a domain that is both highly homogeneous and devoid of retrotransposons is present. Given the large number of alphoid sequences, several domains can fulfill these conditions. Here, domain C is supposed to be the centromere locus. With time, a number of undermining events can occur, such as L1 retrotransposition and accumulation of nucleotide changes, so that the centromere can become altered in its functionality. The chromosome could then be either lost or rescued by the formation of a neocentromere, which would transiently substitute for the altered centromere until a fully functional centromere is again formed through the accumulation of unequal crossovers.