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.

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.

In mammals, centromeres are epigenetically specified by the histone H3 variant CENP-A and are typically associated with satellite DNA. We previously described the first example of a natural satellite-free centromere on Equus caballus chromosome 11 (ECA11) and, subsequently, on several chromosomes in other species of the genus Equus. We discovered that these satellite-free neocentromeres arose recently during evolution through centromere repositioning and/or chromosomal fusion, after inactivation of the ancestral centromere, where, in many cases, blocks of satellite sequences were maintained. Here, we investigated by FISH the chromosomal distribution of satellite DNA families in Equus przewalskii (EPR), demonstrating a good degree of conservation of the localization of the major horse satellite families 37cen and 2PI with the domestic horse. Moreover, we demonstrated, by ChIP-seq, that 37cen is the satellite bound by CENP-A and that the centromere of EPR10, the ortholog of ECA11, is devoid of satellite sequences. Our results confirm that these two species are closely related and that the event of centromere repositioning which gave rise to EPR10/ECA11 centromeres occurred in the common ancestor, before the separation of the two horse lineages.

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.