Functional characterization of centromeres and of repetitive sequences in equids

The Equus represents the last extant genus in the Equidae family, encompassing eight species: two horses (E. caballus and E. przewalskii), three asses (E. asinus, E. hemionus, and E. kiang), and three zebras (E. grevyi, E. burchelli, and E. zebra hartmannae). Thanks to the extensive karyotypic rearrangement among Equus species, snapshots of centromere maturation in all stages were previously described by our group. Particularly relevant was the discovery over 80 satellite-free centromeres in different equine chromosomes. Moreover, two satellite DNA families (37cen and 2PI) were localized on Equus species centromeric and pericentromeric regions and can be uncoupled from the centromeric function. With the aim of characterizing centromeric domains cytogenetically, epigenetically, and at the molecular level, our group is currently using this genus as a powerful model system to study the functional and structural aspects of mammalian centromeres. Another research line in our laboratory demonstrated that insertion polymorphism of the equine repetitive element 1 (ERE-1) element and of the interstitial telomeric sequence (ITS) is frequent in the horse population. Additionally, it was shown that the insertion of these elements near the 5' end of horse genes could alter gene expression. Thanks to our collaboration with the Functional Annotation of ANimal Genome (FAANG) consortium, we have access to a large amount of data and resources for four thoroughbred horses (two mares and two stallions). This data enabled us to investigate the presence of ERE-1 and ITS polymorphic insertions in these four horses and to evaluate their possible effects on gene expression. During the Ph.D. period, my work covered three main areas utilizing various methodologies and techniques. One part involved investigating the centromeric domains of zebras and asses species utilizing species-specific reference genomes. Through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras, Equus burchelli (15 of 22) and Equus 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. We also described that, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. ChIP-seq experiments with an anti-CENP-A antibody were previously carried out on the three species of asses using the horse genome as a reference. Using a new assembly of the donkey genome as reference, we improved the sequence of the CENP-A binding domains which, in several cases, showed fewer gaps and more regular peak shapes. The goal of the second project was to study the centromeres of the wild horse, Equus przewalskii, both at cytogenetic and at the molecular level. We described the distribution of three satellite-DNA families; 37cen, 2PI, and CENP-B sat on E. przewalskii centromeres. Additionally, we showed that the centromere of EPR10 is completely devoid of satellite DNA sequences. Finally, we investigated the possible effect of specific ERE-1 and ITS insertions on the expression of horse protein-coding genes.