Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast
- Center for Cell Dynamics, Johns Hopkins University School of Medicine, United States
- Johns Hopkins University School of Medicine, United States
- Stowers Institute for Medical Research, United States
- Whiting School of Engineering, Johns Hopkins University, United States
Figures
Figure 1 with 4 supplements
Aneuploid yeast strains show defective silencing at HML, subtelomeric, and rDNA chromatin regions.
(A) The design of a microscopy-based screen to isolate karyotypically stable aneuploid strains, generated by inducing triploid meiosis, that exhibit defective silencing of the HML locus. (B) Representative fluorescence images show HML::YFP reporter expression in euploid and aneuploid cells of various karyotypes, as indicated. YFP expression from the HML locus is not detectable in the parental haploid, diploid and triploid strains; YFP fluorescence is heterogeneous within Δsir1 and aneuploid cell populations suggests defective silencing at the HML locus. Scale bar, 4 µm. (C) The bar plot shows the number of times each of the sixteen yeast chromosomes was found to be aneuploid (chromosome number different from the basal ploidy) in 24 strains with defective silencing. Aneuploidies of Chr III and Chr X are significantly overrepresented in strains with defective silencing compared with other 38 stable aneuploids isolated by the same method (Pavelka et al., 2010). *p<0.05 for Chr III and Chr X; p=0.09 for Chr XII calculated using an exact binomial test. (D) The box plot shows mean YFP intensities, determined by microscopy as in Figure 1B, for 175 individual cells per strain. The karyotype of each aneuploid strain is indicated; WT and Δsir1 cells are haploid. The box spans the first through third quartile values, the line inside each box indicates the median, the solid black square designates the mean, and the whiskers mark the 90/10 percentile range. *p<0.01, **p<0.001, ***p<0.0001 compared with WT haploid; calculated using a Mann–Whitney U test. (E) The bar plot depicts the expression, measured by quantitative RT-PCR, of several normally silenced genes: YFP inserted into the endogenous HML locus; subtelomeric genes YFR057W (Chr III), COS12 (Chr XII), AAD15 (Chr XV), and PAU4 (Chr XII); and rDNA gene NTS1-2 (Chr XII). Transcriptional levels are plotted as fold expression relative to the WT haploid strain. Error bars represent the standard deviation (SD) of three biological replicates. *p<0.05, **p<0.01, ***p<0.005 compared with WT haploid; calculated using a two-tailed t-test.
Figure 1—figure supplement 1
Heterogeneous expression pattern of YFP+ and YFP- signals within the aneuploid population was not due to the karyotypic variations.
(A) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. (B) FACS sorting of YFP- vs YFP+ cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP- and YFP+ cells sorted for the qPCR analysis in C and D, respectively. (C–D) qPCR karyotyping of YFP-(C) and YFP+ (D) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. (E–G) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: (E) Gain of III, III, X; (F) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and (G) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.
Figure 1—Video 1
Transitions between repression and derepression of the HML locus in proliferating cell lineages with the following karyotypes: Gain of III, III, X.
https://doi.org/10.7554/eLife.27991.004
Figure 1—Video 2
Transitions between repression and derepression of the HML locus in proliferating cell lineages with the following karyotypes: Loss of I, V, VII, VIII, XI (basal ploidy, 2N).
https://doi.org/10.7554/eLife.27991.005
Figure 1-—Video 3
Transitions between repression and derepression of the HML locus in proliferating cell lineages with the following karyotypes: Gain of I, X, XII, XIII.
https://doi.org/10.7554/eLife.27991.006
Figure 2
Gain of Chr X is sufficient to disrupt silencing.
(A) The box plot shows mean YFP intensities, determined by microscopy of 125 individual cells for each of the following strains: WT haploid, Δsir1, and two parental aneuploid strains (Gain of III, III, X and Gain of I, X, XII, XIII) and their segregants (Gain of III; Gain of III, III; and Gain of I, XII, XIII). The box spans the first through third quartile values, the line inside each box indicates the median, the solid black square designates the mean, and the whiskers mark the 90/10 percentile range. *p<0.01, **p<0.001, ***p<0.0001 compared with WT haploid unless indicated by brackets; calculated using a Mann–Whitney U test. (B) The bar plot depicts the expression, measured by quantitative RT-PCR, of several normally silenced genes in haploid and aneuploid cells with two extra copies of Chr III. These genes are YFP inserted into the endogenous HML locus; subtelomeric genes YFR057W (Chr III), COS12 (Chr XII), AAD15 (Chr XV), and PAU4 (Chr XII); and rDNA gene NTS1-2 (Chr XII). Transcription levels are plotted as fold expression relative to the WT haploid strain and not significantly different in Gain III, III strain compared to WT haploid (p<0.05, calculated using a two-tailed t-test). Error bars represent SD of three biological replicates. (C) The box plots show mean YFP intensities, determined by microscopy of 125 individual cells for each of the indicated WT haploid or aneuploid strains. The GAL1 promoter (Pgal1) was integrated into Chr X either directly adjacent to (Pgal1-CEN-X) or far from (Chr X::Pgal1) consensus centromere sequences. The box plot presentation and statistical analysis are performed as described in Figure 2A. (D) The box plots show mean YFP intensities, determined by microscopy of 125 individual cells for each of the following strains: WT haploid, Δsir1, disome X/SIR1 and disome X/Δsir1 double mutant. The box plot presentation and statistical analysis are performed as described in Figure 2A.
Figure 3
Cells with a gain of Chromosome X show abnormal growth arrest in response to α-factor.
(A) The plots show FACS-based DNA content analysis, indicating cell cycle stage, in MATa WT haploid, Δsir1, and disome X strains. Left panels represent untreated cells; right panels represent strains treated with 2 µg/ml α-factor for 90 min. Peaks overlapping with the red dotted line represent cells in the G1 phase with a haploid genome content (1N). Peaks overlapping with the blue dotted line represent cells in the G2 phase with a diploid genome content (2N). Percentages are the fraction of total cells in G2,±SD. *p<0.001 compared with WT haploid; calculated using a two-tailed t-test. (B) Images depict a pheromone sensitivity assay conducted by applying filter discs carrying α-factor (15 µl of 2 µg/ml) to lawns of WT haploid, Δsir1, or disome X MATa strains. The images shown were used to calculate the size of the zone devoid of cell growth (the region between the rim of the disc and the dashed circle); these areas, indicative of cellular sensitivity to α-factor, were normalized to the WT haploid strain and plotted. The plot shows the mean and SD from three replicates per strain. *p<0.001 compared with WT haploid; calculated using a two-tailed t-test.
Figure 4
HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci.
(A–B) Bottom: The plots show levels of H4K16 acetylation across the HML (A) and HMR (B) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p<0.05, **p<0.01, ***p<0.005 calculated using two-tailed t-test and indicate statistically significant difference in H4K16 acetylation level at corresponding genomic locations in Δsir1 and disome X strains compared to WT haploid. (C–D) The plots indicate Sir2 occupancy across the HML (C) and HMR (D) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-Sir2::HA ChIP followed by qPCR analysis with the same primer sets depicted in (A) and (B) for (C) and (D), respectively. Plots show the mean and SD from three biological replicates. *p<0.01, **p<0.005 compared with WT haploid; calculated using t-test and indicate a statistically significant difference in Sir2 occupancy at corresponding genomic locations across HM loci in Δsir1 and disome X strains compared to WT haploid.
Figure 5 with 1 supplement
Disome X cells display abnormal Sir2 protein localizations and lack proper perinuclear positioning of silenced chromatin region.
(A) Representative fluorescent images are shown for Sir2-mTurq and the HML::YFP reporter in WT haploid and disome X strains. White boxes in the top panels display magnified images (insets) of representative Sir2 foci. Scale bar, 4 µm. (B) The scatter plots show, for each WT haploid or disome X cell, the coefficient of variation (CV) of Sir2-mTurq fluorescence plotted against the mean YFP pixel intensity. The CV was calculated as the ratio of the standard deviation to the mean pixel intensity of Sir2-mTurq fluorescence over the total area of the sum projection of each cell. The CV in the disome X strain is significantly reduced compared with haploid cells (p<0.001, one-tailed t-test), indicating a more diffusive distribution of Sir2 in aneuploid cells. Additionally, 35% of disome X cells (determined using Tukey's outlier test on the WT strain) have significantly higher (p<0.01) mean YFP intensities than the WT haploid cell population; these disome X cells also show significantly (p<0.05) reduced CV compared with haploid controls. (C) Left: Representative images show the position of the HML locus, tagged with LacO array and bound by LacI-GFP, relative to the nuclear envelope (NE), marked by Nup60-mCherry. Top right: The illustration shows the three concentric zones of equal area used to map the location of the HML locus. Bottom right: The bar graph shows the percentage of WT haploid or disome X cells with GFP puncta located in each of the three zones (n = 100 cells per strain). Confidence values (p) are shown for a χ2 analysis comparing random (33% in each zone) and test distributions. *: Value significantly differs from a random distribution (p<0.005, Chi-square test for independence). Scale bar, 1 µm.
Figure 5—figure supplement 1
SIR2 is not haploinsufficient for HML silencing
(A–B) Western blot analysis of total Sir2 protein by using anti-Sir2 and anti-α-PGK (loading control) antibodies for the following strains: WT haploid, Δsir1, disome X, and Δsir2/SIR2. The total Sir2 protein levels were quantified using densitometric analysis, normalized to the WT haploid strain and plotted in (B). (C) The box plot shows mean Sir2-mTurq intensities, determined by microscopy of 68 individual cells for WT haploid and disome X strains. The box spans the first through third quartile values, the line inside each box indicates the median, the solid black square designates the mean, and the whiskers mark the 90/10 percentile range. *p<0.001 compared with WT haploid; calculated using a Mann–Whitney U test. (D) The box plots show mean YFP intensities, determined by microscopy of 100 individual cells for each of the following strains: WT haploid, Δsir1, disome X and Δsir2/SIR2, all with YFP inserted into HML. The box plot presentation and statistical analysis are performed as described in (C); *p<0.01, **p<0.001.
Figure 6 with 1 supplement
Genome-wide analysis shows that disome X cells upregulate histone modifications and transcription of typically silenced genes.
(A–B) Gene expression changes determined by RNA-seq are plotted on the X-axis as log2 fold change (disome X/WT haploid), and H3K4me3 (A) and H3K79me3 (B) histone modification enrichments determined by ChIP-seq are plotted on the Y-axis as the difference in Z-scores (disome X - WT haploid), with each dot representing an individual gene. Genes that were expressed (RPKM >1) and enriched for a given histone modification in both haploid and disome X strains were included in this analysis and categorized into four groups: (I) Genome-wide, representing all of the included genes (grey dots); (II and III) Subsets of genes from category I that were significantly (1.5- fold change, p<0.01) upregulated (blue dots) or down-regulated (green dots) in disome X cells compared with haploid controls; and (IV) subtelomeric genes (red dots). Note that identities of subtelomeric genes in A and B are different because the genes with occupancy of the two histone markers (K4me and K79me) were not the same and hence are at different points along the x-axis of A and B. More detailed information about the genes in these categories is listed in Supplementary file 2. (C) Transcriptional levels of COS12 (Chr XII), IMD2 (Chr VIII), and YIR042C (Chr IX) genes were measured by RNA-seq and plotted as fold change (disome X/WT haploid). Each bar depicts the mean and SD of three biological replicates. *p<0.001 compared with WT haploid; calculated using two-tailed t-test. (D) Enrichment profiles of H3K4me3 and H3K79me3 were determined by ChIP-seq and plotted as reads per million per nucleotide (RPM) for the indicated gene ORFs (black arrows), with 500 bp of flanking sequence on both sides. Each plot shows the enrichment profiles for two biological replicates per WT haploid or disome X strain. Both epigenetic marks are enriched at all three genomic loci in disome X cells compared with the haploid controls.
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Figure 6—source data 1
Source data for genome-wide analysis performed in Figure 6.
- https://doi.org/10.7554/eLife.27991.014
Figure 6—figure supplement 1
No difference in gene expression between disome X and haploid populations for sets of genes that had H3K4me3 and H3K79me3 modifications only in one strain or the other.
(A) Expression levels determined by RNA-seq experiments are plotted as log10 of the RPKM values on the X-axis, and H3K4me3 enrichment determined by ChIP-seq analysis is plotted as log10 of the Z-scores on the Y-axis for individual genes in disome X (red) and WT haploid (green) strains. (B–E) The violin plots show gene expression levels, plotted as log2 of the RPKM values, in disome X and WT haploid strains for individual genes carrying: (B) H3K4me3 enrichment in WT haploid but not disome X cells; (C) H3K4me3 enrichment in disome X but not haploid cells; (D) H3K79me3 enrichment in haploid but not disome X cells; and (E) H3K79me3 enrichment in disome X but not haploid cells. In these plots, the width of the violin shape at a given log2(RPKM) value indicates the number of genes expressed at that level. The white dot represents the geometric mean RPKM value for all the genes, and the thick and thin lines are box plots that show the first and third quartiles and 1.5 times interquartile ranges (IQR), respectively.
Figure 7
The combined increase in copy number of at least four genes on Chr X causes HML silencing defects.
(A) Representative images of YFP fluorescence from the HML::YFP reporter in Δsir1, disome X, and WT haploid cells with a single extra copy of the indicated genes, where relevant. Scale bar, 4 µm. (B) The box plot shows the mean YFP intensity of 125 cells for each strain shown in Figure 7A. The box spans the first through third quartile values, the line inside each box indicates the median, the solid black square designates the mean, and the whiskers mark the 90/10 percentile range. *p<0.01, **p<0.001, ***p<0.0001 compared with WT haploid; calculated using a Mann–Whitney U test.
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Figure 7—source data 1
Source data for gain-of-funtion screen.
- https://doi.org/10.7554/eLife.27991.016
Additional files
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Supplementary file 1
The karyotypes of stable aneuploid strains that exhibit defective silencing of YFP at the HML locus obtained from a microscopy-based screen are listed.
- https://doi.org/10.7554/eLife.27991.017
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Supplementary file 2
The number of genes plotted for each category in Figure 6A–B and Figure 6—figure supplement 1 is listed.
- https://doi.org/10.7554/eLife.27991.018
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Supplementary file 3
A list of fifteen Chr X genes that cause the strongest silencing defects as a result of increased copy number.
Genes leading to the loss of HML::YFP silencing when copy number is increased are listed with a functional description and a desilencing score. The desilencing score was calculated as the average YFP intensity in WT haploid strains carrying individual candidate genes on a low-copy (centromeric) plasmid, relative to the average YFP fluorescence in the disome X strain. Average YFP intensities were calculated using three biological replicates per strain.
- https://doi.org/10.7554/eLife.27991.019
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Supplementary file 4
List of yeast strains used in this study, and not listed in Supplementary file 1.
- https://doi.org/10.7554/eLife.27991.020
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Supplementary file 5
List of plasmids used in this study.
- https://doi.org/10.7554/eLife.27991.021
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Supplementary file 6
List of oligos used in this study.
- https://doi.org/10.7554/eLife.27991.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.27991.023
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Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast
eLife 6:e27991.
https://doi.org/10.7554/eLife.27991
