Molecular and evolutionary characteristics of the fraction of human alpha satellite DNA associated with CENP-A at the centromeres of chromosomes 1, 5, 19, and 21

Abstract

Background: The mode of evolution of the highly homogeneous Higher-Order-Repeat-containing alpha satellite arrays is still subject to discussion. This is also true of the CENP-A associated repeats where the centromere is formed.

Results: In this paper, we show that the molecular mechanisms by which these arrays evolve are identical in multiple chromosomes: i) accumulation of crossovers that homogenise and expand the arrays into different domains and subdomains that are mostly unshared between homologues and ii) sporadic mutations and conversion events that simultaneously differentiate them from one another. Individual arrays are affected by these mechanisms to different extents that presumably increase with time. Repeats associated with CENP-A, where the centromere is formed, are subjected to the same evolutionary mechanisms, but constitute minor subsets that exhibit subtle sequence differences from those of the bulk repeats. While the DNA sequence per se is not essential for centromere localisation along an array, it appears that certain sequences can be selected against. On chromosomes 1 and 19, which are more affected by the above evolutionary mechanisms than are chromosomes 21 and 5, CENP-A associated repeats were also recovered from a second homogeneous array present on each chromosome. This could be a way for chromosomes to sustain mitosis and meiosis when the normal centromere locus is ineluctably undermined by the above mechanisms.

Conclusion: We discuss, in light of these observations, possible scenarios for the normal evolutionary fates of human centromeric regions.

Figure 1

ClustalW alignment of the 55 complete HORs of BAC BX248407. Alignment was obtained using the ClustalW program (35). The HORs are ordered from 1 to 55 as they appear in the published DNA sequence. DVN positions are indicated above the alignment from 1 to 141 and from 142 to 281. The remaining positions are not shown, as they are identical in all repeats; the same holds true for the other figures. DVNs shared by subset 1 (repeats 1-30) are shaded in red, those shared by subset 2 (repeats 36-55) are shaded in green, and those specific for subset 3 (repeats 31-35) are shaded in yellow. When DVNs are of two types at the same nucleotide position, purple shading is also used. Repeats 32 and 33 have presumably been generated by an unequal crossover event. Shared nucleotides are coloured in green for A, blue for C, red for T, black for G and "-" is used for deletions.

Figure 2

The common portion of the repeats of both BX248407 and the corresponding locus of the GM 13139 homologue were ClustalW aligned simultaneously. In the first column, those from GM 13139 are shaded in yellow. Overall, this figure shows that the repeats of the two homologues are indeed quite different in their DVN distribution and combination into haplotypes.

Figure 3

ClustalW alignment of repeats from alphoid array D21Z1 of chromosome 21, E1-E3 (E4-E6 are shown in Additional file 1). The DVNs are shown in the upper line, with their positions along the 1866 bp long HOR. A minority of them are shared by the six homologues. 19 (E1), 11 (E2), 12 (E3) repeats exhibit no DVNs at all; they might, however, differ by a few sporadic mutations (less than 0.2% on average).

Figure 4

A) Schematic representation of the centromeric regions of chromosomes 1, 5 and 19. The three chromosomes share an alphoid array where the active centromere normally forms: D1Z7, D5Z2, and D19Z3 (15). As shown by Finelli et al (14), D1Z7 is embedded within D1Z5. Chromosomes 5 and 19 share a second alphoid array: D5Z1 and D19Z1. In situ hybridization with both pZ5.1 DNA (green signal) and BAC RP11-483B6 DNA (red signal) as probes on chromosomes in prometaphase (B, D) and nuclei (C).

Figure 5

ClustalW alignment of the sequenced repeats of D1Z7 from hybrid cell line GM 13139. ClustalW alignment was performed with all sequenced repeats, including those in which a DVN is only shared by two repeats. Those DVN positions are not included here for more clarity.

Figure 6

Alignment of the consensus sequences obtained from those determined in this study from the alpha satellite of D1Z7, D5Z2 (for the two homologues of this study) and D19Z3. Using FISH with oligonucleotides at positions where the consensus sequences were different, it was indeed possible to label specifically the chromosome with the specific consensus nucleotide (Toutirais, G, Witkowska, M, Piazza, A, Richard, F, Roizès, G and Escudé, C, submitted).

Figure 7

Simultaneous ClustalW alignment of the repeats recovered from loci D1Z7 and D19Z3 from hybrid cell lines GM 13139 (shaded in grey) and GM 10449 (shaded in purple). The clustalW alignment was performed with all sequenced repeats, including those in which a DVN is only shared by two repeats. Those DVN positions are not included here for more clarity. Vertical arrows point to positions which have been specifically homogenised within one or the other chromosome.

Figure 8

ClustalW alignment of locus D5Z2 from the chromosome 5 of the two hybrid cell lines GM10114 (A) and GM 11714 (B). Above each set of sequences the nucleotide positions where a significant subset of repeats exhibit the same DVNs are indicated.

Figure 9

ClustalW alignment of the IPE1-IPE3 (IPE4-IPE6 are shown in Additional file 3) CENP-A associated repeats recovered by immunoprecipitation from chromosome 21. The minority of DVNs that are shared between most of the six homologues are shown in the upper line, with their positions along the 1866 bp long HOR indicated. No DVNs at all were exhibited by 12 (IPE1), 15 (IPE2), or 29 (IPE3) repeats; they might, however, differ by a few sporadic mutations (less than 0.2% on average)

Figure 10

ClustalW alignment of the IP10114 (A) and IP11714 (B) CENP-A associated repeats recovered by immunoprecipitation from the two chromosome 5 homologues. Above each set of sequences the nucleotide positions where a significant subset of repeats exhibit the same DVNs are indicated.

Figure 11

PCR amplification of the DNA recovered by immunoprecipitation from chromatin with an anti-CENP-A antibody. Samples were from Chr 19 (GM 10449): 1 and 4; Chr 5 (GM 10114): 2 and 5; and Chr 5 (GM11714): 3 and 6. Amplification was performed with oligoprimers specific for D19Z3 and D5Z2, shared with Chr 1 for both chromosomes: 1, 2 and 3. The locus common to chromosomes 5 and 19 only was amplified with the appropriate oligoprimers: 4, 5 and 6. M = 100 bp marker.

Figure 12

Schematic representation of the simultaneous ClustalW alignments of the alphoid sequences obtained from D1Z7 (coloured in green), D5Z2 (yellow for GM10114 and blue for GM11714), and D19Z3 (purple). Top: CENP-A associated repeats; bottom: repeats from the bulk sequences. Each rectangle is strictly proportional to the number of repeats which were found clustered in the alignment.