Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres 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 satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric 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 satellite-free centromere was seeded on an inversion breakpoint. At eleven chromosomes, whose primary constrictions seemed to be associated to satellite repeats by cytogenetic analysis, satellite-free 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.

A Satellite-Free Centromere in Equus przewalskii Chromosome 10

“…We used the EquCab2.0 reference where the satellite-free centromeric region of horse chromosome 11 is well assembled [ 7 , 12 ]. Using the pipeline that we developed for the identification of satellite-free centromeres in other equids [ 7 , 14 ], a CENP-A binding domain was identified in the region comprised between 27.59 and 27.79 Mb of chromosome 11 in the reference genome ( Figure 4 A). In the Przewalski’s horse, this region is located on chromosome 10, which is orthologous to horse chromosome 11 ( Figure 4 A).…”

“…Therefore, at the centromere of ECA5 the 2PI satellite is either absent or undetectable by FISH suggesting that this satellite may have been lost during fusion of two ancestral acrocentric chromosomes corresponding to EPR23 and EPR24. The generation of ECA5, in the domestic horse lineage, from a centric fusion was previously proposed [ 57 ] and the loss of satellite repeats as a consequence of Robertsonian fusion was recently demonstrated by us in the zebras [ 14 ]. However, since the ECA5 synteny is maintained in all Ceratomorpha and disrupted in all equids with the exception of the domestic horse, the phylogenetic history of this chromosome remains controversial.…”

“…These sequences represent the most rapidly evolving DNA sequences in eukaryotic genomes [ 6 ] and their presence has so far hampered comprehensive molecular analysis of these intriguing loci. In this context, our discovery that the species of the genus Equus are characterized by an extraordinarily high number of centromeres completely devoid of satellite DNA made these species a good model system for studying the epigenetic control and evolution of the centromere function [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].…”

“…Later on, using an anti-CENP-A antibody in ChIP experiments on chromatin extracted from primary fibroblast cell lines, we proved at the molecular level that several equid centromeres are completely devoid of satellite repeats [ 7 , 9 , 12 , 13 , 14 , 17 ]. While only one satellite-free centromere was found in the horse, on chromosome 11 (ECA11) [ 12 ], 16 were described in the donkey [ 7 ], 15 in the Burchell’s zebra, and 13 in the Grevy’s zebra [ 14 ]. Interestingly, it was observed that these CENP-A binding domains can slide within a genomic region of a few hundred kbs, probably limited by epigenetic boundaries.…”

“…Interestingly, it was observed that these CENP-A binding domains can slide within a genomic region of a few hundred kbs, probably limited by epigenetic boundaries. These domains were defined as epialleles and the phenomenon was called centromere sliding [ 7 , 9 , 13 , 14 ]. Studies on families composed of donkeys, horses, and their hybrid offspring (mule/hinny) revealed that the epialleles are inherited as Mendelian traits but their position can slide in one generation [ 7 ] while it is stable during propagation in cell culture, suggesting that the sliding can presumably take place during meiosis [ 7 ].…”