Neocentromere formation through Robertsonian fusion and centromere repositioning during the evolution of zebras

Abstract

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated to highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and E. asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras E. burchelli (15 of 22) and E. grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine neocentromeres are shared between the two species in agreement with their recent separation. We de novo assembled all neocentromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a neocentromere was seeded on an inversion breakpoint. At eleven chromosomes, whose primary constrictions seemed to be associated to satellite repeats by cytogenetic analysis, neocentromeres were instead located near the ancestral inactivated satellite-based centromeres, therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.

CENP-A/CENP-B uncoupling in the evolutionary reshuffling of centromeres

“…It is important to notice that, in all the four species, the 2PI satellite, which is not enriched in CENP-A chromatin, is one of the most abundant satellite families. This satellite is found at most horse primary constrictions, at numerous donkey and Grevy's zebra non centromeric termini and noncentromeric interstitial or terminal positions of Burchell's zebra (36,44). This distribution suggests that the 2PI observed now may be the relics of the oldest equid centromeric satellite that was progressively dissociated from the centromeric function following the expansion of CENPB-sat and 37cen satellites in a lineage-specific manner.…”

“…Paired-end sequencing was performed with Illumina HiSeq2000 and Illumina HiSeq2500 platforms by IGA Technology Services (Udine, Italy). Reads from ChIP-seq experiments with the anti-CENP-A antibody were previously described (41,44) and deposited in NCBI SRA Archive (SRR27325169, SRR27325168, SRR5515973, SRR5515972, SRR17956804, SRR17956803, SRR17956806, SRR17956805). The details of each dataset are reported in Additional file 1: Table S11.…”

“…In order to shed light on the role of this elusive protein, we investigated CENP-B in the genus Equus (horses, asses and zebras). The rapid evolution of these species is marked by exceptionally frequent centromere repositioning events and chromosomal fusions that gave rise to satellite-free centromeres (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). In addition, blocks of satellite DNA are often present at non-centromeric chromosome ends, representing relics of ancestral inactivated centromeres or traces of satellite loci exchange (36,44).…”

“…In these two species, satellite-free centromeres derive from repositioning, that is the movement of the centromeric function without DNA sequence modification (48). A high number of chromosomal fusion events led to the karyotypes of the Grevy's zebra (2n = 46) and the Burchell's zebra (2n = 44), where 13 and 15 satellite-free centromeres, respectively, were identified (44). However, in the Grevy's zebra, the majority of satellite DNA loci are found at non centromeric chromosomal termini, while in the Burchell's zebra satellite DNA is mainly present at satellite-based centromeres or at fusion sites (36,44).…”

“…A high number of chromosomal fusion events led to the karyotypes of the Grevy's zebra (2n = 46) and the Burchell's zebra (2n = 44), where 13 and 15 satellite-free centromeres, respectively, were identified (44). However, in the Grevy's zebra, the majority of satellite DNA loci are found at non centromeric chromosomal termini, while in the Burchell's zebra satellite DNA is mainly present at satellite-based centromeres or at fusion sites (36,44). Thus, the karyotypes of these species represent four different scenarios, providing the opportunity to evaluate the association between CENP-B, centromeres and satellites.…”