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) …
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 …
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, …
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 …
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 …
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 …
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 …
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 …
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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 …
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)…
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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
