Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras.

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus 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 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. 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 11 chromosomes, whose primary constrictions seemed to be associated with 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.

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.

Molecular Dynamics and Evolution of Centromeres in the Genus Equus.

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.

Telomeric-Like Repeats Flanked by Sequences Retrotranscribed from the Telomerase RNA Inserted at DNA Double-Strand Break Sites during Vertebrate Genome Evolution.

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.

Insertion of Telomeric Repeats in the Human and Horse Genomes: An Evolutionary Perspective.

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.

Centromere sliding on a mammalian chromosome.

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression.

BACKGROUND: In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. RESULTS: In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. CONCLUSIONS: Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter.

Discovery and comparative analysis of a novel satellite, EC137, in horses and other equids.

Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37 cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37 cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.

Uncoupling of satellite DNA and centromeric function in the genus Equus.

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

The localization of centromere protein A is conserved among tissues.

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development.

Polymorphic organization of constitutive heterochromatin in Equus asinus (2n = 62) chromosome 1.

In the karyotype of Equus asinus (domestic donkey, 2n = 62), non-centromeric heterochromatic bands have been described in subcentromeric and telomeric positions. In particular, chromosome 1 is characterised by heterochromatic bands in the proximal region of the long arm and in the short arm; it has been shown that these regions are polymorphic in size. Here we investigated the variation in the intensity and distribution of fluorescence signals observed on donkey chromosome 1 after in situ hybridization with two DNA probes containing fragments from the two major equine satellite DNA families. Our results show that, in Equus asinus chromosome 1, the amount and distribution of large clusters of satellite DNA can define at least nine polymorphic variants of the constitutive heterochromatin that cannot be detected by C-banding alone.

Chromosomal assignment of six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) in four species of the genus Equus.

We mapped six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) on the chromosomes of Equus caballus, Equus asinus, Equus grevyi, and Equus burchelli by fluorescence in situ hybridization. Our results add six type I markers to the cytogenetic map of these species and provide new information on the comparative genomics of the genus Equus.

Satellite DNA at the Centromere is Dispensable for Segregation Fidelity.

The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus Equus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long stretches of satellite DNA. The mitotic stability of the two chromosomes was compared under normal conditions and under mitotic stress induced by the spindle inhibitor, nocodazole. Two independent molecular-cytogenetic approaches were used-the interphase aneuploidy analysis and the cytokinesis-block micronucleus assay. Both assays were coupled to fluorescence in situ hybridization with chromosome specific probes in order to identify chromosome 11 and chromosome 13, respectively. In addition, we tested if the lack of centromeric satellite DNA affected chromatid cohesion under normal and stress conditions. We demonstrated that, in our system, the segregation fidelity of a chromosome is not influenced by the presence of long stretches of tandem repeats at its centromere. To our knowledge, the present study is the first analysis of the mitotic behavior of a natural satellite-free centromere.

CENP-A binding domains and recombination patterns in horse spermatocytes.

Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the satellite-less centromere of chromosome 11 causes crossover suppression, similarly to satellite-based centromeres. These results suggest that the centromere effect does not depend on satellite DNA. During this analysis, we observed a peculiar phenomenon: while, as expected, the centromere of the majority of meiotic bivalent chromosomes was labelled with a single immunofluorescence centromeric signal, double-spotted or extended signals were also detected. Their number varied from 0 to 7 in different cells. This observation can be explained by positional variation of the centromeric domain on the two homologs and/or misalignment of pericentromeric satellite DNA arrays during homolog pairing confirming the great plasticity of equine centromeres.

Characterization of the equine glycogen debranching enzyme gene (AGL): Genomic and cDNA structure, localization, polymorphism and expression.

Glycogen debranching enzyme (AGL) is a multifunctional enzyme acting in the glycogen degradation pathway. In humans, the AGL activity deficiency causes a type III glycogen storage disease (Cori-Forbes disease). One particularity of AGL gene expression lies in the multiple alternative splicing in its 5' region. The AGL gene was localized on ECA5q14-q15. The sequence of the equine cDNA was determined to be 7.5 kb in length with an open reading frame of 4602 bp. The gene is 69 kb long and contains 35 exons. The equine AGL gene has an ubiquitous expression and presents five tissue-dependent cDNA variants arising from alternative splicing of the first exons. The equine skeletal muscle and heart contain four out of six variants previously described in humans and the equine liver express three of these four human variants. We identified a new alternative splicing variant expressed in equine skeletal and heart muscles. All these mRNA variants most probably encode only two different protein isoforms of 1533 and 1377 amino-acids. Four SNPs were detected in the mRNA. The equine in silico promoter sequence reveals a structure similar to those of other mammalian species. The disposition of the transcription factor biding sites does not correlate to the transcription start sites of tissue-specific variants.

The major horse satellite DNA family is associated with centromere competence.

BACKGROUND: The centromere is the specialized locus required for correct chromosome segregation during cell division. The DNA of most eukaryotic centromeres is composed of extended arrays of tandem repeats (satellite DNA). In the horse, we previously showed that, although the centromere of chromosome 11 is completely devoid of tandem repeat arrays, all other centromeres are characterized by the presence of satellite DNA. We isolated three horse satellite DNA sequences (37cen, 2P1 and EC137) and described their chromosomal localization in four species of the genus Equus. RESULTS: In the work presented here, using the ChIP-seq methodology, we showed that, in the horse, the 37cen satellite binds CENP-A, the centromere-specific histone-H3 variant. The 37cen sequence bound by CENP-A is GC-rich with 221 bp units organized in a head-to-tail fashion. The physical interaction of CENP-A with 37cen was confirmed through slot blot experiments. Immuno-FISH on stretched chromosomes and chromatin fibres demonstrated that the extension of satellite DNA stretches is variable and is not related to the organization of CENP-A binding domains. Finally, we proved that the centromeric satellite 37cen is transcriptionally active. CONCLUSIONS: Our data offer new insights into the organization of horse centromeres. Although three different satellite DNA families are cytogenetically located at centromeres, only the 37cen family is associated to the centromeric function. Moreover, similarly to other species, CENP-A binding domains are variable in size. The transcriptional competence of the 37cen satellite that we observed adds new evidence to the hypothesis that centromeric transcripts may be required for centromere function.