Efficient neocentromere formation is suppressed by gene conversion to maintain centromere function at native physical chromosomal loci in Candida albicans

CENPA/Cse4 assembles centromeric chromatin on diverse DNA. CENPA chromatin is epigenetically propagated on unique and different centromere DNA sequences in a pathogenic yeast Candida albicans. Formation of neocentromeres on DNA, nonhomologous to native centromeres, indicates a role of non-DNA sequence determinants in CENPA deposition. Neocentromeres have been shown to form at multiple loci in C. albicans when a native centromere was deleted. However, the process of site selection for CENPA deposition on native or neocentromeres in the absence of defined DNA sequences remains elusive. By systematic deletion of CENPA chromatin-containing regions of variable length of different chromosomes, followed by mapping of neocentromere loci in C. albicans and its related species Candida dubliniensis, which share similar centromere properties, we demonstrate that the chromosomal location is an evolutionarily conserved primary determinant of CENPA deposition. Neocentromeres on the altered chromosome are always formed close to the site which was once occupied by the native centromere. Interestingly, repositioning of CENPA chromatin from the neocentromere to the native centromere occurs by gene conversion in C. albicans

Centromeric DNA sequences in the pathogenic yeast Candida albicans are all different and unique

In an approach to clone and characterize centromeric DNA sequences of Candida albicans by chromatin immunoprecipitation, we have used antibodies directed against an evolutionarily conserved histone H3-like protein, CaCse4p (CENP-A homolog). Sequence analysis of clones obtained by this procedure reveals that only eight relatively small regions (≈3 kb each) of the Can. albicans genome are selectively enriched. These CaCse4-bound sequences are located within 4- to 18-kb regions lacking ORFs and occur once in each of the eight chromosomes of Can. albicans. Binding of another evolutionarily conserved kinetochore protein, CaMif2p (CENP-C homolog), colocalizes with CaCse4p. Deletion of the CaCse4p-binding region of chromosome 7 results in a high rate of loss of the altered chromosome, confirming that CaCse4p, a centromeric histone in the CENP-A family, indeed identifies the functional centromeric DNA of Can. albicans. The CaCse4p-rich regions not only lack conserved DNA motifs of point (<400 bp) centromeres and repeated elements of regional (>40 kb) centromeres, but also each chromosome of Can. albicans contains a different and unique CaCse4p-rich centromeric DNA sequence, a centromeric property previously unobserved in other organisms

Functional characterization of the Saccharomyces cerevisiae protein Chl1 reveals the role of sister chromatid cohesion in the maintenance of spindle length during S-phase arrest

BACKGROUND: Metaphase cells have short spindles for efficient bi-orientation of chromosomes. The cohesin proteins hold sister chromatids together, creating Sister Chromatid Cohesion (SCC) that helps in the maintenance of short spindle lengths in metaphase. The budding yeast protein Chl1p, which has human homologs, is required for DNA damage repair, recombination, transcriptional silencing and aging. This protein is also needed to establish SCC between sister chromatids in S-phase. RESULTS: In the present study we have further characterized Chl1p for its role in the yeast Saccharomyces cerevisiae when cells are under replication stress. We show that when DNA replication is arrested by hydroxyurea (HU), the chl1 mutation causes growth deficiency and a mild loss in cell viability. Although both mutant and wild-type cells remained arrested with undivided nuclei, mutant cells had mitotic spindles, which were about 60-80% longer than wild-type spindles. Spindle extension occurred in S-phase in the presence of an active S-phase checkpoint pathway. Further, the chl1 mutant did not show any kinetochore-related defect that could have caused spindle extension. These cells were affected in the retention of SCC in that they had only about one-fourth of the normal levels of the cohesin subunit Scc1p at centromeres, which was sufficient to bi-orient the chromosomes. The mutant cells showed defects in SCC, both during its establishment in S-phase and in its maintenance in G2. Mutants with partial and pericentromeric cohesion defects also showed spindle elongation when arrested in S-phase by HU. CONCLUSIONS: Our work shows that Chl1p is required for normal growth and cell viability in the presence of the replication block caused by HU. The absence of this protein does not, however, compromize the replication checkpoint pathway. Even though the chl1 mutation gives synthetic lethal interactions with kinetochore mutations, its absence does not affect kinetochore function; kinetochore-microtubule interactions remain unperturbed. Further, chl1 cells were found to lose SCC at centromeres in both S- and G2 phases, showing the requirement of Chl1p for the maintenance of cohesion in G2 phase of these cells. This work documents for the first time that SCC is an important determinant of spindle size in the yeast Saccharomyces cerevisiae when genotoxic agents cause S-phase arrest of cells

Rad51-Rad52 mediated maintenance of centromeric chromatin in candida albicans

La ubicación específica de un centrómero en la mayoría de los eucariotas no depende únicamente de la secuencia de ADN. Sin embargo, los determinantes no genéticos de identidad de un centrómero no están claramente definidos. Aunque varios mecanismos, de forma individual o en conjunto, pueden especificar centrómeros epigenéticos, la mayoría de los estudios en este área se centran en un factor universal, un centromerospecific histona H3 variante CENP-A, a menudo considerado como el determinante de la identidad epigenética del centrómero. A pesar de la sincronización variable de su carga en centrómeros través de las especies, una replicación junto a una deposición en fase temprana S de CENP-A se encuentra en la mayoría de los centrómeros de levadura. Centrómeros son las regiones más tempranas de replicación cromosómica en una levadura en ciernes patógeno Candida albicans. Al aplicar un ensayo de electroforesis en gel de agarosa de dos dimensiones se identifican los orígenes de replicación (ORI7-LI y ORI7-RI) proxima a un centrómero temprano replicante (CEN7) en C. albicans. Se demuestra que las horquillas de replicación se estancan en CEN7 de una manera dependiente del cinetocoro y el estancamiento tenedor se reduce en ausencia de la recombinación homóloga (HR) proteínas Rad51 y Rad52. La supresión de ORI7-RI provoca una reducción significativa en la señal de tenedor estancado y una mayor tasa de pérdida del cromosoma alterado 7. Las proteínas de recursos humanos, Rad51 y Rad52, han demostrado que desempeñan un papel en el reinicio del tenedor. La microscopía confocal muestra cinetocoros declustered en rad51 y rad52 mutantes, que son evidencia de la disrupción cinetocoro. Los niveles de CENP-ACaCse4 en centrómeros, como se determina por los experimentos de inmunoprecipitación de la cromatina (ChIP), se reducen en ausencia de Rad51 / Rad52 que resulta en la interrupción de la estructura cinetocoro. Además, el análisis de transferencia Western revela que las moléculas de CENP-A deslocalizados en mutantes de recursos humanos se degradan de un modo similar como en otros mutantes kinetochore descritos antes. Finalmente, los ensayos de co-inmunoprecipitación indican que Rad51 y Rad52 interactuan físicamente con CENPA CaCse4 in vivo. Por lo tanto, las proteínas Rad51 y Rad52 de recursos epigenéticos humanos mantienen el funcionamiento del centrómero mediante la regulación de los niveles de CENPA CaCse4 en los sitios de parada programados en los principios centrómeros a replicar.Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromerespecific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENPA CaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENPA CaCse4 levels at the programmed stall sites of early replicating centromeresK. Sanyal y D. D. Dubey han recibido financiación del Government of India. Department of Biotechnology S. Mitra fue Senior Research Fellowship financiado por Council of Scientific and Industrial Research K Sanyal recibió ayuda de Jawaharlal Nehru Centre for Advanced Scientific Research G. Larriba fue financiado por Junta de Extremadura, Ayuda a grupos CCV014, Fondos FEDER; y por el Ministerio de Ciencia e Innovación, SAF2010-19848peerReviewe

A stable hybrid containing haploid genomes of two obligate diploid Candida species

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future

A comprehensive model to predict mitotic division in budding yeasts

High-fidelity chromosome segregation during cell division depends on a series of concerted interdependent interactions. Using a systems biology approach, we built a robust minimal computational model to comprehend mitotic events in dividing budding yeasts of two major phyla: Ascomycota and Basidiomycota. This model accurately reproduces experimental observations related to spindle alignment, nuclear migration, and microtubule (MT) dynamics during cell division in these yeasts. The model converges to the conclusion that biased nucleation of cytoplasmic microtubules (cMTs) is essential for directional nuclear migration. Two distinct pathways, based on the population of cMTs and cortical dyneins, differentiate nuclear migration and spindle orientation in these two phyla. In addition, the model accurately predicts the contribution of specific classes of MTs in chromosome segregation. Thus we present a model that offers a wider applicability to simulate the effects of perturbation of an event on the concerted process of the mitotic cell division

Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans

Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes

How Do Microbial Pathogens Make CENs?

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Structural organization of centromeres in various microbial pathogens.

<p>(A) A schematic showing relatedness of various microbial pathogens. (B) A table summarizing essential features of different types of centromeres identified in microbial pathogens.</p

Sad1 Spatiotemporally Regulates Kinetochore Clustering To Ensure High-Fidelity Chromosome Segregation in the Human Fungal Pathogen Cryptococcus neoformans

The linker of nucleoskeleton and cytoskeleton (LINC) complex is present in fungi, animals, and plants. It performs diverse functions in animals, and its role(s) have recently been explored in plants. In ascomycetous yeast species, the role of the LINC complex in spindle pole body function and telomere clustering during meiosis has been determined. However, nothing is known about the LINC complex in the fungal phylum of Basidiomycota. In this study, we identified the role of the LINC complex in kinetochore dynamics as well as in nuclear migration in a basidiomycetous yeast, Cryptococcus neoformans, a human pathogen. Unlike most other yeast species, kinetochores remain unclustered during interphase but gradually cluster during mitosis in C. neoformans. We report that the LINC complex is required for timely onset of kinetochore clustering and high-fidelity chromosome segregation in C. neoformans. Thus, our study identifies a novel factor required for kinetochore clustering during mitosis in yeast species.Kinetochore clustering, frequently observed in yeasts, plays a key role in genome organization and chromosome segregation. In the absence of the metaphase plate arrangement, kinetochore clustering in yeast species is believed to facilitate timely kinetochore-microtubule interactions to achieve bivalent attachments of chromosomes during metaphase. The factors determining the dynamics of kinetochore clustering remain largely unknown. We previously reported that kinetochores oscillate between an unclustered and a clustered state during the mitotic cell cycle in the basidiomycetous yeast Cryptococcus neoformans. Based on tubulin localization patterns, while kinetochore clustering appears to be microtubule dependent, an indirect interaction of microtubules with kinetochores is expected in C. neoformans. In this study, we sought to examine possible roles of the SUN-KASH protein complex, known to form a bridge across the nuclear envelope, in regulating kinetochore clustering in C. neoformans. We show that the SUN domain protein Sad1 localizes close to kinetochores in interphase as well as in mitotic cells. Sad1 is nonessential for viability in C. neoformans but is required for proper growth and high-fidelity chromosome segregation. Further, we demonstrate that the onset of kinetochore clustering is significantly delayed in cells lacking Sad1 compared to wild-type cells. Taken together, this study identifies a novel role of the SUN domain protein Sad1 in spatiotemporal regulation of kinetochore clustering during the mitotic cell cycle in C. neoformans