The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

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.

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Chromosome segregation during the cell cycle is an evolutionarily conserved, fundamental biological process. Dynamic interaction between spindle microtubules and the kinetochore complex that assembles on centromere DNA is required for faithful chromosome segregation. The first artificial minichromosome was constructed by cloning the centromere DNA of the budding yeast Saccharomyces cerevisiae. Since then, centromeres have been identified in >60 fungal species. The DNA sequence and organization of the sequence elements are highly diverse across these fungal centromeres. In this article, we provide a comprehensive view of the evolution of fungal centromeres. Studies of this process facilitated the identification of factors influencing centromere specification, maintenance, and propagation through many generations. Additionally, we discuss the unique features and plasticity of centromeric chromatin and the involvement of centromeres in karyotype evolution. Finally, we discuss the implications of recurrent loss of RNA interference (RNAi) and/or heterochromatin components on the trajectory of the evolution of fungal centromeres and propose the centromere structure of the last common ancestor of three major fungal phyla—Ascomycota, Basidiomycota, and Mucoromycota. Expected final online publication date for the Annual Review of Microbiology, Volume 74 is September 8, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

50Aneuploidy is associated with drug resistance in fungal pathogens. In tropical 51 countries, Candida tropicalis is the most frequently isolated Candida species from 52 patients. To facilitate the study of genomic rearrangements in C. tropicalis, we 53 assembled its genome in seven gapless chromosomes by combining next-54 generation sequencing (NGS) technologies with chromosome conformation capture 55 sequencing (3C-seq). Our 3C-seq data revealed interchromosomal centromeric and 56 telomeric interactions in C. tropicalis, similar to a closely related fungal pathogen 57 Candida albicans. By performing a genome-wide synteny analysis between C. 58 tropicalis and C. albicans, we identified 39 interchromosomal synteny breakpoints 59 (ICSBs), which are relics of ancient translocations. Majority of ICSBs are mapped 60 within 100 kb of homogenized inverted repeat-associated (HIR) centromeres (17/39) 61 or telomere-proximal regions (7/39) in C. tropicalis. Further, we developed a genome 62 assembly of Candida sojae and used the available genome assembly of Candida 63 viswanathii, two closely related species of C. tropicalis, to identify the putative 64 centromeres. In both species, we identified the putative centromeres as HIR-65 associated loci, syntenic to the centromeres of C. tropicalis. Strikingly, a centromere-66 specific motif is conserved in these three species. Presence of similar HIR-67 associated putative centromeres in early-diverging Candida parapsilosis indicated 68 that the ancestral CUG-Ser1 clade species possessed HIR-associated centromeres. 69We propose that homology and spatial proximity-aided translocations among the 70 ancestral centromeres and loss of HIR-associated centromere DNA sequences led 71 to the emergence of evolutionary new centromeres (ENCs) on unique DNA 72 sequences. These events might have facilitated karyotype evolution and centromere-73 type transition in closely-related CUG-Ser1 clade species. 74 75 76 Significance Statement 77We assembled the genome of Candida tropicalis, a frequently isolated fungal 78 pathogen from patients in tropical countries, in seven complete chromosomes. 79 Comparative analysis of the CUG-Ser1 clade members suggests chromosomal 80 rearrangements are mediated by homogenized inverted repeat (HIR)-associated 81 centromeres present in close proximity in the nucleus as revealed by chromosome 82 conformation capture. These translocation events facilitated loss of ancestral HIR-83 associated centromeres and seeding of evolutionary new centromeres on unique 84 DNA sequences. Such karyotypic rearrangements can be a major source of genetic 85 variability in the otherwise majorly clonally propagated human fungal pathogens of 86 the CUG-Ser1 clade. The improved genome assembly will facilitate studies related to 87 aneuploidy-induced drug resistance in C. tropicalis. 88 89 Introduction 90 91 The efficient maintenance of the genetic material and its propagation to subsequent 92 generations determine the fitness of an organism. Genomic rearrangements are 93 often associat...