Comparative cytogenomics reveals genome reshuffling and centromere repositioning in the legume tribe Phaseoleae

The tribe Phaseoleae (Leguminosae; Papilionoideae) includes several legume crops with assembled genomes. Comparative genomic studies indicated the preservation of large genomic blocks in legumes. However, the chromosome dynamics along its evolution was not investigated in the tribe. We conducted a comparative genomic analysis using CoGe Synmap platform to define a useful genomic block (GB) system and to reconstruct the ancestral Phaseoleae karyotype (APK). We defined the GBs based on orthologous genes between Phaseolus vulgaris and Vigna unguiculata genomes (n = 11), then searched for these GBs in different genome species belonging to the Phaseolinae (P. lunatus, n = 11) and Glycininae (Amphicarpaea edgeworthii, n = 11 and Spatholobus suberectus, n = 9) subtribes, and in the outgroup (Medicago truncaluta, n = 8). To support our in silico analysis, we used oligo-FISH probes of P. vulgaris chromosomes 2 and 3 to paint the orthologous chromosomes of the non-sequenced Phaseolinae species (Macroptilium atropurpureum and Lablab purpureusi, n = 11). We inferred the APK with n = 11, 22 GBs (A to V) and 60 sub-GBs. We hypothesized that the main rearrangements within Phaseolinae involved nine APK chromosomes, with extensive centromere repositioning resulting from evolutionary new centromeres (ENC) in the Phaseolus lineage. We demonstrated that the A. edgeworthii genome is more reshuffled than the dysploid S. suberectus genome, in which we could reconstructed the main events responsible for the chromosome number reduction. The development of the GB system and the proposed APK provide useful tools for future comparative genomic analyses of legume species.

Centromere formation remodels chromatin fibre structure

Human centromeric chromatin is assembled from CENP-A nucleosomes and repetitive α-satellite DNA sequences and provides a foundation for kinetochore assembly in mitosis. Biophysical experiments led to a hypothesis that the repetitive DNA sequences form a highly folded chromatin scaffold necessary for function, but this idea was revised when fully functional evolutionary new centromeres (ENCs) or neocentromeres were found to form on non-repetitive DNA. To understand if centromeres have a special chromatin structure we have genetically isolated a single human chromosome harbouring a neocentromere and investigated its organisation. The centromere core is enriched in RNA pol II, active epigenetic marks and remodelled by transcription to form a negatively supercoiled open chromatin domain. In contrast, centromerisation causes a spreading of repressive epigenetic marks to flanking regions, delimited by H3K27me3 polycomb boundaries and divergent genes. The flanking domain is partially remodelled to form compact chromatin, with characteristics similar to satellite-containing pericentromeric chromatin, but exhibits low level genomic instability. We provide a model for centromere chromatin structure and suggest that open chromatin provides a foundation for a stable kinetochore whilst pericentromeric heterochromatin generates surrounding mechanical rigidity.

Spatial inter-centromeric interactions facilitated the emergence of evolutionary new centromeres

Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

Spatial inter-centromeric interactions facilitated the emergence of evolutionary new centromeres

Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere-type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

Centromere repositioning causes inversion of meiosis and generates a reproductive barrier

The chromosomal position of each centromere is determined epigenetically and is highly stable, whereas incremental cases have supported the occurrence of centromere repositioning on an evolutionary time scale (evolutionary new centromeres, ENCs), which is thought to be important in speciation. The mechanisms underlying the high stability of centromeres and its functional significance largely remain an enigma. Here, in the fission yeast Schizosaccharomyces pombe, we identify a feedback mechanism: The kinetochore, whose assembly is guided by the centromere, in turn, enforces centromere stability. Upon going through meiosis, specific inner kinetochore mutations induce centromere repositioning—inactivation of the original centromere and formation of a new centromere elsewhere—in 1 of the 3 chromosomes at random. Repositioned centromeres reside asymmetrically in the pericentromeric regions and cells carrying them are competent in mitosis and homozygotic meiosis. However, when cells carrying a repositioned centromere are crossed with those carrying the original centromere, the progeny suffer severe lethality due to defects in meiotic chromosome segregation. Thus, repositioned centromeres constitute a reproductive barrier that could initiate genetic divergence between 2 populations with mismatched centromeres, documenting a functional role of ENCs in speciation. Surprisingly, homozygotic repositioned centromeres tend to undergo meiosis in an inverted order—that is, sister chromatids segregate first, and homologous chromosomes separate second—whereas the original centromeres on other chromosomes in the same cell undergo meiosis in the canonical order, revealing hidden flexibility in the perceived rigid process of meiosis.

Centromere repositioning induced by inner kinetochore impairment generates a meiosis barrier

Centromeres dictate the sites for kinetochore assembly on chromosomes, while their own position on each chromosome is determined epigenetically by a specific histone H3 variant CENP-A. For all eukaryotic species, the chromosomal position of each centromere is distinct and inherited with high fidelity, although the mechanisms underlying the epigenetic stability and its functional significance remain largely unknown. Here in the fission yeast Schizosaccharomyces pombe, we show that mutations in inner kinetochore components influence centromeric chromatin organization to various levels. In extreme cases, a single deletion of wip1, mhf1 and mhf2 (the conserved CENP-T-W-S-X complex subunits) or double deletions of cnp3 (a homologue of mammalian CENP-C) and fta6 (a pombe specific component) induce centromere repositioning - inactivation of the original centromere and formation of a neocentromere - in one of the three chromosomes at random. Neocentromeres tend to locate in pericentromeric heterochromatin regions, although heterochromatin is not required for centromere inactivation. Cells carrying a neocentromere are competent in mitosis and in meiosis of homozygotes. However, when these cells are crossed to cells carrying the original centromere, the progeny suffers severe lethality due to defects in meiotic chromosome segregation. These results recapitulate a meiosis barrier that could initiate genetic divergence between two populations with mismatched centromeres, documenting a potential role of the Evolutionary New Centromeres (ENCs) in speciation.Significance StatementIn eukaryotes, centromeres are chromosomal regions where kinetochores are assembled and the positions of centromeres are accurately inherited. While the centromere and kinetochore assembly are extensively studied, the mechanisms that each centromere maintain its identity on chromosomes are still not well understood. In this study, we demonstrated that the inner kinetochore is required for the normal centromere identity as single depletion of the inner kinetochore CENP-T-W-S-X complex or double deletions of cnp3/CENP-C and fta6 induce centromere repositioning. We further showed cells carrying a neocentromere are reproductively isolated from the wildtype population carrying the original centromere. Taken together, these results suggest that induced centromere repositioning mimics the evolutionary new centromeres and is sufficient to cause reproductive isolation.