Functional and Comparative Analysis of Centromeres Reveals Clade-Specific Genome Rearrangements in Candida auris and a Chromosome Number Change in Related Species

ABSTRACT The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 causing systemic infections worldwide and simultaneously evolved in different geographical zones. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional GC-poor centromere landscape lacking pericentromeres or repeats. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Interspecies genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris. Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug-susceptible isolates of C. auris. IMPORTANCE Candida auris, the killer fungus, emerged as different geographical clades, exhibiting multidrug resistance and high karyotype plasticity. Chromosomal rearrangements are known to play key roles in the emergence of new species, virulence, and drug resistance in pathogenic fungi. Centromeres, the genomic loci where microtubules attach to separate the sister chromatids during cell division, are known to be hot spots of breaks and downstream rearrangements. We identified the centromeres in C. auris and related species to study their involvement in the evolution and karyotype diversity reported in C. auris. We report conserved centromere features in 10 related species and trace the events that occurred at the centromeres during evolution. We reveal a centromere inactivation-mediated chromosome number change in these closely related species. We also observe that one of the geographical clades, the East Asian clade, evolved along a unique trajectory, compared to the other clades and related species.

Functional and comparative genome analysis reveals clade-specific genome innovations in the killer fungus Candida auris

The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 and simultaneously evolved in different geographical zones worldwide, causing superficial as well as systemic infections. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional centromere landscape lacking pericentromeres. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Inter-species genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris. Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug susceptible isolates of C. auris.

Loss of centromere function drives karyotype evolution in closely related Malassezia species

Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3–5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome–chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.

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.

Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3

Centromeres are characterized by the centromere-specific H3 variant CENP-A, which is embedded in chromatin with a pattern characteristic of active transcription that is required for centromere identity. It is unclear how centromeres remain transcriptionally active despite being flanked by repressive pericentric heterochromatin. To further understand centrochromatin’s response to repressive signals, we nucleated a Polycomb-like chromatin state within the centromere of a human artificial chromosome (HAC) by tethering the methyltransferase EZH2. This led to deposition of the H3K27me3 mark and PRC1 repressor binding. Surprisingly, this state did not abolish HAC centromere function or transcription, and this apparent resistance was not observed on a noncentromeric locus, where transcription was silenced. Directly tethering the reader/repressor PRC1 bypassed this resistance, inactivating the centromere. We observed analogous responses when tethering the heterochromatin Editor Suv39h1-methyltransferase domain (centromere resistance) or reader HP1α (centromere inactivation), respectively. Our results reveal that the HAC centromere can resist repressive pathways driven by H3K9me3/H3K27me3 and may help to explain how centromeres are able to resist inactivation by flanking heterochromatin.

Point mutation impairs centromeric CENH3 loading and induces haploid plants

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.