Fig 1.
C. albicans encodes 2 alleles of histone H2A that differ in C-terminal amino acid sequence.
(A) Schematic of Bub1–H2A.1–Sgo1 interactions in most eukaryotes. C. albicans retains orthologs of Bub1 (orf19.2678) and Sgo1 (orf19.3550). (B) C. albicans histone genes are distributed over 3 chromosomes. H2A.1 (orf19.1051), H2B.1 (orf19.1052), H4.1 (orf19.1061), and H3.1 (orf19.1059) occur on Chromosome 1; CSE4 (orf19.6163), H2A.Z (orf19.327), H2B.2 (orf19.6924), H2A.2 (orf19.6925), and H3.A (orf19.6791) are on Chromosome 3; and H4.2 (orf19.1854) and H3.2 (orf19.1853) are on Chromosome R, which also contains rRNA genes. (C) C. albicans, C. dubliniensis, and C. tropicalis evolved divergent alleles of H2A. Among these 3 yeasts, H2A.1 (blue shading) and H2A.2 (orange shading) are more similar to orthologs in the 2 other species than to the H2A paralog in their own species. Scale bar designates substitutions per amino acid. (D) Amino acid differences between the H2A paralogs of C. albicans, C. dubliniensis, and C. tropicalis (shown in S1 Fig) localize to the C terminus and include replacement of the highly conserved S/T121 residue with lysine (asterisk). (E) Deletion of H2A.2 but not H2A.1 produces a colony sectoring phenotype. WT and homozygous deletion mutants were propagated on YEPD medium and incubated at 30 °C. CPC, Chromosomal Passenger Complex; H2A, Histone 2A; PP2A, Protein Phosphatase 2A; Sgo1, Shugoshin 1; S/T121, serine or threonine at position 121; WT, wild type; YEPD, yeast extract peptone dextrose.
Fig 2.
A C. albicans mutant lacking H2A.2 phenocopies spindle-checkpoint mutants.
(A and B) Deletion of H2A.2, BUB1, or SGO1, or replacement of H2A.2 with an H2A.2 S121A mutation, results in similar colony sectoring phenotypes. Colony morphology (A) and percent of colony sectoring (B) are shown for WT C. albicans, the [11,11] strain that lacks H2A.2, the [22,22] strain that lacks H2A.1, knockouts of the spindle-checkpoint genes, BUB1 and SGO1, and [11,--], [11,--]+H2A.2WT, and [11,--]+H2A.2S121A strains. (C, D, E, and F) Mutants lacking H2A.2, BUB1, or SGO1 display increased aneuploidy. (C) Percent of colonies that have lost the ability to grow on arginine-deficient medium, relative to growth on YEPD. Strains were plated on arginine-deficient medium or YEPD after 4 days on presporulation medium. Because these ARG4/Δ strains contain the ARG4 gene on only 1 copy of Chromosome 7, loss of that copy renders cells auxotrophic for arginine. (D) Percent of colonies that grow on sorbose-containing medium, relative to growth on YEPD. Because cells with both copies of Chromosome 5 are killed by sorbose, this medium selects for cells that have lost 1 copy of the chromosome. (E, top panel) Superimposed fluorescence and phase microscopy of QMY85, which contains approximately 120 tandem copies of the tet operator on one of its 2 Chromosome 5 homologs, as well as the Tet repressor fused to YFP. A yellow spot marks the tagged copy of Chromosome 5 (white arrow), and the nucleus is stained with DAPI (blue arrow). (Bottom panel) Example of neighboring cells that contain 1 spot (normal) or 2 spots (indicating an extra copy of Chromosome 5). (F) Percent of cells with 2 spots, counting only unbudded cells. (G) Sensitivity to the microtubule poison nocodazole (50 μM) shown by serial dilution. Please see S1 Data for the numerical values summarized in (B, C, D, and F). prespo, presporulation; WT, wild type; YEPD, yeast extract peptone dextrose; YFP, Yellow Fluorescent Protein.
Fig 3.
H2A.1 and H2A.2 play opposing roles in chromosome stability in tetraploid cells and in the acquisition of tolerance to fluconazole.
(A) Cartoon of the C. albicans parasexual cycle, which consists of mating between diploid a and α cells to form tetraploid a/α cells, followed by ploidy reduction by concerted chromosome loss rather than meiosis, as occurs in other eukaryotes. (B) Viability of WT diploid, WT tetraploid, or tetraploid cells containing only H2A.1 ([1111,1111]) or H2A.2 ([2222,2222]) on YEPD (left) versus prespo medium (right). Plated cells were propagated for 4 days at 37 °C, and survival was calculated as the ratio of colonies on each medium to the estimated number of plated cells determined using a hemocytometer. Error bars represent standard deviation. Significant differences from the tetraploid WT strain were determined using one-way ANOVA with Dunnett’s correction for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001. (C) Chromosome stability of ARG4/arg4Δ WT diploid, WT tetraploid, and derivative strains on prespo medium after 4 days, as measured by growth on SC-Arg medium, indicative of the loss of 1 copy of Chromosome 7. (D) Chromosome stability of ARG4/arg4Δ WT diploid and derivative strains after 3 days of pregrowth in YEPD containing 1 μg/ml fluconazole. (E) Serial dilutions of C. albicans diploid strains (WT = [11,22], [11,11], [22,22], bub1, sgo1, h2a.2 = [11,--], h2a.2+H2A.2WT, and h2a.2+H2A.2S121A) plated onto YEPD or YEPD + fluconazole (10 μg/ml). Please see S1 Data for the numerical values summarized in (B, C, and D). Chr, Chromosome; Fluc, Fluconazole; H2A, Histone 2A; n.s., nonsignificant; prespo, presporulation; SC-Arg, Synthetic complete lacking arginine; WT, wild type; YEPD, yeast extract peptone dextrose.
Fig 4.
Impact of ploidy and environment on histone expression and deposition.
(A) RT-qPCR analysis of expression of H2A.1 and H2A.2 in the indicated genotypes and conditions. “D” represents a WT [11,22] diploid strain and “T” a WT [1111,2222] tetraploid strain. The average results for 3 biological replicates are presented after normalization to H3 mRNA, along with the standard deviation. There were no significant differences by one-way ANOVA. (B) Immunoblot of levels of H2A.2-FLAG in the indicated genotypes and conditions. Quantification using a LI-COR Odyssey imaging system is shown on the right. There were no significant differences by one-way ANOVA. (C) ChIP-seq analysis. Plots of bedgraphs of normalized read densities are shown for the indicated genotypes and conditions. Data represent the results of 1 replicate of each sample for CEN5. Data for all centromeres are shown in S6 Fig, and scatterplots of replicate data are shown in S7 Fig. (D and E) Boxplots showing normalized ChIP-seq enrichments for H2A.2 (D), H3 (D), and Cse4/CENP-A (E) (relative to the WCE sample; see Materials and methods) of nonoverlapping 5 kb tiles of the C. albicans genome separated into those overlapping with annotated centromeres versus the remainder of the genome. p-Values were determined using the Mann-Whitney U test. Cse4/CENP-A association with centromeres was significantly different in diploids versus tetraploids (p < 1 × 10−5), diploids propagated in YEPD versus YEPD plus fluconazole (p < 1 × 10−15), and tetraploids propagated in YEPD versus prespo (p < 1 × 10−13). Please see S1 Data for the numerical values summarized in (A). ChIP-seq data are available from the NCBI GEO website (accession number GSE122037). CENP-A, Centromere Protein A; ChIP-seq, chromatin immunoprecipitation with massively parallel DNA sequencing; Cse4, Centromeric Histone H3-like Protein; Fluc, fluconazole; GEO, Gene Expression Omnibus; H2A, Histone 2A; NCBI, National Center For Biotechnology Information; n.s., not significant; prespo, presporulation; RT-qPCR, reverse-transcription quantitative PCR; r.u., relative units; WCE, whole-cell extract; WT, wild type; YEPD, yeast extract peptone dextrose.