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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-014-0493-6) contains supplementary material, which is available to authorized users.

SummaryA common assumption is that human chromosomes carry equal chances of mis-segregation during compromised cell division. Human chromosomes vary in multiple parameters that might generate bias, but technological limitations have precluded a comprehensive analysis of chromosome-specific aneuploidy. Here, by imaging specific centromeres coupled with high-throughput single-cell analysis as well as single-cell sequencing, we show that aneuploidy occurs non-randomly following common treatments to elevate chromosome mis-segregation. Temporary spindle disruption leads to elevated mis-segregation and aneuploidy of a subset of chromosomes, particularly affecting chromosomes 1 and 2. Unexpectedly, we find that a period of mitotic delay weakens centromeric cohesion and promotes chromosome mis-segregation and that chromosomes 1 and 2 are particularly prone to suffer cohesion fatigue. Our findings demonstrate that inherent properties of individual chromosomes can bias chromosome mis-segregation and aneuploidy rates, with implications for studies on aneuploidy in human disease.

Summary: Recurrent patterns of chromosomal changes (aneuploidy) are widespread in cancer. These patterns are mainly attributed to selection processes due to an assumption that human chromosomes carry equal chance of being mis-segregated into daughter cells when fidelity of cell division is compromised. Human chromosomes vary widely in size, gene density and other parameters that might generate bias in mis-segregation rates, however technological limitations have precluded a systematic and high throughput analysis of chromosome-specific aneuploidy. Here, using fluorescence In-Situ hybridization (FISH) imaging of specific centromeres coupled with high-throughput single cell analysis, as well as single-cell sequencing we show that human chromosome mis-segregation is non-random.Merotelic kinetochore attachment induced by nocodazole washout leads to elevated aneuploidy of a subset of chromosomes, and high rates of anaphase lagging of chromosomes 1 and 2. Mechanistically, we show that these chromosomes are prone to cohesion fatigue that results in anaphase lagging upon release from nocodazole or Eg5 inhibition. Our findings suggest that inherent properties of specific chromosomes can influence chromosome mis-segregation and aneuploidy, with implications for studies on aneuploidy in human disease.

BackgroundIn 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.ResultsIn 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.ConclusionsSequence 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0281-1) contains supplementary material, which is available to authorized users.

BackgroundThe 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.ResultsIn 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-016-0242-z) contains supplementary material, which is available to authorized users.