Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

Abstract

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.

Fig 1. Identification of kinetochore proteins in C. tropicalis.

(A) An illustration showing the kinetochore organization in yeasts. (B) Live cell fluorescence microscopic images of indicated proteins at two different stages of the cell cycle: interphase (unbudded) and mitotic (large-budded). Scale bar, 5 μm.

Fig 2. Depletion of conserved kinetochore proteins causes chromosome mis-segregation in C. tropicalis.

(A) CENP-A, CENP-C, Nuf2 and Dad1 are essential for viability in C. tropicalis. C. tropicalis conditional mutant strains expressing the only copy of the above mentioned genes under the GAL1 promoter were streaked on plates with galactose (permissive) or glucose (restrictive) as the sole carbon source and were photographed after 2 to 3 days of incubation at 30°C. The GAL1 promoter is induced in the presence of galactose but repressed in glucose containing media in C. tropicalis. (B) FACS analysis of the conditional mutant strains of CENP-A, CENP-C, Nuf2 and Dad1 grown in either permissive (galactose), or non-permissive (glucose) media. The x-axis and y-axis represent the DNA content and number of cells respectively. (C) The distribution of unbudded, small-budded and large-budded (G2/M stage) cells of indicated mutant strains grown in either permissive (+) or non-permissive (-) conditions. The nuclear morphology was visualized by DAPI staining after 6 h of growth in permissive or non-permissive condition and the cells exhibiting proper or improper chromosome segregation during the G2/M stage are counted (n = >250 cells). The y-axis represents the percentage cell population.

Fig 3. CENP-A and CENP-C ChIP-seq analyses identified seven centromeres in C. tropicalis.

(A) CENP-A and CENP-C ChIP-seq reads along the seven enriched supercontigs are shown. Here, x-axis and y-axis represent the coordinates of the chromosomal regions and the distribution of sequence reads of the specific supercontig respectively. The asterisk (*) denotes the peak observed in input library. (B) Enrichment of indicated proteins at the centromeres on different supercontigs. ChIP DNA fractions of the indicated proteins were analyzed by PCR using a primer-pair unique to each supercontig (see S4 Table for primer sequences). CtLEU2, a non-centromeric locus, was used as negative control. ‘T’, Total DNA, ‘+’, IP DNA with antibodies and ‘-’, beads only control.

Fig 4. Repeat-associated centromere organization in C. tropicalis.

(A) ChIP-seq analysis revealed that CENP-A and CENP-C bind to the mid core region in C. tropicalis. Here, the x-axis represents the structural components of a centromere in C. tropicalis and the y-axis represents the distribution of sequence reads of CENP-A (red) or CENP-C (green) ChIP DNA of the respective supercontig. Schematic representations of structural components of a centromere in C. tropicalis are shown below each ChIP-seq reads. Black boxes represent the mid regions, blue arrows indicate inverted repeats, the left repeat (LR) and the right repeat (RR). Scale bar, 2 kb. (B) ChIP-qPCR assays confirm the binding of kinetochore proteins across the centromere in Scnt 8. The x-axis represents coordinates on the supercontigs and the y-axis denotes the qPCR value as a percentage of the total chromatin input with standard error mean (SEM). Scale bar, 2 kb (C) Sequence conservation between the mid core regions (mids) and inverted repeats (IRs) from different centromeres in C. tropicalis. Homology is calculated as the percentage of aligned nucleotides in a pair-wise alignment, measured from the shorter sequence. On the other hand, identity is the percentage of aligned and conserved nucleotides in the pair-wise alignment, again measured from the shorter sequence. Averages are calculated from all pair-wise alignments, weighted by length. IR* and IR# denote average value with respect to either others or self respectively.

Fig 5. Inter-chromosomal rearrangements at the centromeres of Candida species.

Orthologous genes are plotted on the x-axis as per C. tropicalis candidates (start to end on that supercontig), and on the y-axis as per C. albicans coordinates for the respective chromosome, and colour-coded according to the C. albicans chromosome. The vertical grey bar indicates the position of the centromere on the C. tropicalis supercontig. Continuous segments of lines indicate rows of syntenous genes.

Fig 6. Pericentric inverted repeats provide a structural signature in de novo CENP-A recruitment in C. tropicalis.

(A) Schematic of plasmids used in this study. The replicative plasmid pARS2 harbors CaARS2 and CaURA3 sequences. pmid8 has only the mid core region of CEN8, pCEN8 carries the full length centromere (CEN8), and pARS2-λ harbors a ~10 kb lambda DNA. pCEN801 carries LR8 in a direct orientation with respect to RR8. On the other hand, pCEN802 harbors CaLR5 and CaRR5 of chromosome 5 of C. albicans. The size of each of these plasmids is also mentioned. (B) The relative mitotic stability of various plasmids in C. tropicalis. The mitotic stability of each of the plasmids is normalized to that of the average mitotic stability of the replicative plasmid (pARS2). The mitotic stability for each class of plasmids was calculated for five independent transformants (n = 5). One way ANOVA and Bonferroni post tests were performed to determine statistical significance. Errors bars represent standard error mean (SEM). (C) CENP-A-ChIP assays were performed in the C. tropicalis strain CtKS102 (CSE4/CSE4-TAP) transformed with pmid8, pCEN8, pCEN801 and pCEN802. Immunoprecipitated (IP) DNA fractions were analyzed by qPCR with primer-pairs (see S4 Table) specific to each cloned insert to determine the extent of de novo CENP-A recruitment on the centromere DNA sequence on the plasmid exclusively. The enrichment of CENP-A on these exogenously introduced centromere sequences are represented as a percentage of the total chromatin input with standard error mean (SEM) and validated with three independent biological replicates (n = 3). The relative enrichment was calculated using the formula: (pCEN-LEU2)/ (nCEN-LEU2), where nCEN and pCEN indicate the percent input values of CENP-A enrichment at the native centromere (Scnt 8) and on the plasmid centromere sequence respectively. LEU2 is used as a non-centromeric negative control. Similarly, one way ANOVA and Bonferroni post tests were performed to determine statistical significance.

Fig 7. Evolution of centromere organization in ascomycetous fungi.

(A) Phylogeny of ascomycetous fungi showing the diverse nature of centromere structure. An unrooted phylogenetic tree was constructed using 573 uniformly evolving orthologous gene families (see methods). The phylogenetic relationship of the species with different types of centromeres is illustrated with colored shadows. The species names shown in white letters designate those with uncharacterized centromeres. The centromeres are mostly point or regional in nature in Ascomycota. However, the centromere of Y. lipolytica is an example of an unconventional intermediate centromere, which shares properties of both point and regional centromere. The centromeres of Y. lipolytica are small (<200 bp in size) and mutations in the partial palindrome lead to centromere dysfunction [96]. These are the characteristics of a point centromere. On the other hand, YlCENs lack the conserved DNA elements (CDEs). Y. lipolytica also does not code for the point centromere specific protein complex (the CBF3 complex) [4]. On the other hand, Y. lipolytica harbors Sim4 and Fta1 proteins, which are kinetochore proteins associated with regional centromere only. Moreover, it should also be noted that the recently identified centromeres of Naumovozyma castellii represent an unconventional class of point centromere with unique centromere DNA elements [97]. See text for the detailed information about the classification of the centromere. (B) Schematic shows a possible route of evolution of structural components of the centromere in ascomycetous yeasts. The length of the centromeres is also mentioned. However, it should be noted that the size of the centromeres in C. albicans, C. dubliniensis and C. lusitaniae is based on the length of the CENP-A binding domain. The inverted repeats of S. pombe and C. tropicalis centromeres aid in CENP-A recruitment de novo. It was also evident from the study in S. pombe that inverted repeats are essential in the establishment of the centromere, but is no longer required for the maintenance of an already established centromere. On the other hand, the centromeres mostly lack pericentric repeats in C. albicans where the role of DNA elements in de novo CENP-A recruitment is unknown. From these lines of evidence, we propose that the pericentric repeats would have been gradually lost in C. albicans and C. dubliniensis. It should also be noted that point centromeres might have originated from the regional ones [98].