Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes.
ABSTRACT: Aneuploidy-the gain or loss of one or more whole chromosome-typically has an adverse impact on organismal fitness, manifest in conditions such as Down syndrome. A central question is whether aneuploid phenotypes are the consequence of copy number changes of a few especially harmful genes that may be present on the extra chromosome or are caused by copy number alterations of many genes that confer no observable phenotype when varied individually. We used the proliferation defect exhibited by budding yeast strains carrying single additional chromosomes (disomes) to distinguish between the "few critical genes" hypothesis and the "mass action of genes" hypothesis. Our results indicate that subtle changes in gene dosage across a chromosome can have significant phenotypic consequences. We conclude that phenotypic thresholds can be crossed by mass action of copy number changes that, on their own, are benign.
Project description:Aneuploidy, a state in which the chromosome number deviates from a multiple of the haploid count, significantly impacts human health. The phenotypic consequences of aneuploidy are believed to arise from gene expression changes associated with the altered copy number of genes on the aneuploid chromosomes. To dissect the mechanisms underlying altered gene expression in aneuploids, we used RNA-seq to measure transcript abundance in colonies of the haploid Saccharomyces cerevisiae strain F45 and two aneuploid derivatives harboring disomies of chromosomes XV and XVI. F45 colonies display complex "fluffy" morphologies, while the disomic colonies are smooth, resembling laboratory strains. Our two disomes displayed similar transcriptional profiles, a phenomenon not driven by their shared smooth colony morphology nor simply by their karyotype. Surprisingly, the environmental stress response (ESR) was induced in F45, relative to the two disomes. We also identified genes whose expression reflected a nonlinear interaction between the copy number of a transcriptional regulatory gene on chromosome XVI, DIG1, and the copy number of other chromosome XVI genes. DIG1 and the remaining chromosome XVI genes also demonstrated distinct contributions to the effect of the chromosome XVI disome on ESR gene expression. Expression changes in aneuploids appear to reflect a mixture of effects shared between different aneuploidies and effects unique to perturbing the copy number of particular chromosomes, including nonlinear copy number interactions between genes. The balance between these two phenomena is likely to be genotype- and environment-specific.
Project description:BACKGROUND: Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In Saccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication. RESULTS: The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, and ura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin gene TUB2. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene TUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes. CONCLUSION: This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect.
Project description:Purpose:To dissect the mechanisms underlying altered gene expression in aneuploids, we measured transcript abundance in colonies of haploid yeast strain F45 and derived strains, including strains disomic for chromosomes XV and XVI, using RNA-seq. F45 colonies display complex “fluffy” morphologies, while the disomic colonies are smooth, resembling laboratory strains Methods: RNA-seq analysis was carried out on RNA isolated from fully developed S. cerevisiae colonies, grown on solid medium for four days, either in triplicate or quadruplicate. Stranded, paired-end sequencing was carried out in two batches. In the first batch 2x51 bp sequencing was carried out on an Illumina Hiseq2000 and in the second batch 2x75 bp sequencing was carried out on an Illumina NextSeq. Readpairs were aligned using Bowtie2 (version 2.1.0)with the parameters [-N 1 -I 50 -X 450 -p 6 --reorder -x -S] and allowing 1 mismatch per read. Differential transcription was detected and quantified using EdgeR (v. 3.6.8) Results: Our two disomes displayed similar transcriptional profiles, a phenomenon not driven by their shared smooth colony morphology nor specified purely by the karyotype. Surprisingly, the environmental stress response (ESR) was induced in euploid F45, relative to the two disomes, rather than vice-versa. We also identified genes whose expression reflected a non-linear interaction between the copy number of a transcriptional regulatory gene on chromosome XVI, DIG1, and the copy number of other chromosome XVI genes. DIG1 and the remaining chromosome XVI genes also demonstrated distinct contributions to the effect of the chromosome XVI disome on ESR gene expression. Conclusions: Expression changes in aneuploids reflect a mixture of effects shared between different aneuploidies, including stress responses, and effects unique to perturbing the copy number of particular chromosomes, including non-linear copy number interactions between genes. The balance between these two phenomena is likely to be genotype and environment specific. Overall design: mRNA profiles of 4 day old haploid F45 colonies, and colonies derived from F45 were generated by deep sequencing, in triplicate or quadruplicate, using Illumina Hiseq2000 or Illumina Nextseq sequencing.
Project description:BACKGROUND: While changes in chromosome number that result in aneuploidy are associated with phenotypic consequences such as Down syndrome and cancer, the molecular causes of specific phenotypes and genome-wide expression changes that occur in aneuploids are still being elucidated. RESULTS: We employed a segmental aneuploid condition in maize to study phenotypic and gene expression changes associated with aneuploidy. Maize plants that are trisomic for 90% of the short arm of chromosome 5 and monosomic for a small distal portion of the short arm of chromosome 6 exhibited a phenotypic syndrome that includes reduced stature, tassel morphology changes and the presence of knots on the leaves. The knotted-like homeobox gene knox10, which is located on the short arm of chromosome 5, was shown to be ectopically expressed in developing leaves of the aneuploid plants. Expression profiling revealed that approximately 40% of the expressed genes in the trisomic region exhibited the expected 1.5 fold increased transcript levels while the remaining 60% of genes did not show altered expression even with increased gene dosage. CONCLUSION: We found that the majority of genes with altered expression levels were located within the chromosomal regions affected by the segmental aneuploidy and exhibits dosage-dependent expression changes. A small number of genes exhibit higher levels of expression change not predicted by the dosage, or display altered expression even though they are not located in the aneuploid regions.
Project description:Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression) genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets of autosomes, or 2X;4A). Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system "anticipates" the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal-independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components.
Project description:<h4>Background</h4>Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost.<h4>Methods</h4>Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids.<h4>Results</h4>In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region.<h4>Conclusion</h4>The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.
Project description:Chromosome instability and aneuploidy are hallmarks of cancer, but it is not clear how changes in the chromosomal content of a cell contribute to the malignant phenotype. Previously we have shown that we can readily isolate highly proliferative tumor cells and their revertants from highly invasive tumor cell populations, indicating how phenotypic shifting can contribute to malignant progression. Here we show that chromosome instability and changes in chromosome content occur with phenotypic switching. Further, we show that changes in the copy number of each chromosome quantitatively impose a proportional change in the chromosome transcriptome ratio. This correlation also applies to subchromosomal regions of derivative chromosomes. Importantly, we show that the changes in chromosome content and the transcriptome favor the expression of a large number of genes appropriate for the specific tumor phenotype. We conclude that chromosome instability generates the necessary chromosome diversity in the tumor cell populations and, therefore, the transcriptome diversity to allow for environment-facilitated clonal expansion and clonal evolution of tumor cell populations.
Project description:Transcriptome analysis to map transcriptomes of Mad2 p53null-driven aneuploid liver cancers and T-ALLs, to determine correlation between copy number changes and expression changes and to map the transcriptional response to CIN Chromosome instability (CIN) leads to aneuploidy and copy number variations (CNVs). Even though both are hallmarks of cancer cells, aneuploidy inhibits proliferation of untransformed cells, suggesting that cancer cells have adapted to cope with CIN. The spindle assembly checkpoint (SAC) prevents CIN by monitoring chromosome attachment and sister chromatid tension in mitosis. By conditionally inactivating Mad2, an essential SAC gene, we find that SAC inactivation in T-cells or hepatocytes is remarkably well tolerated and becomes tumorigenic when placed in a p53null or p53+/- predisposed background. The resulting T-ALLs and HCCs are highly aneuploid, exhibit clonal copy number changes that are tumor specific despite ongoing CIN, indicating that CIN is a powerful driver of tumor evolution. Overall design: Transcriptomes of liver tumors or T-ALL samples described in the accompanying manuscript were compared to the transcriptomes of healthy liver or thymus samples isolated from healthy mice
Project description:Aneuploidy, which leads to unpaired chromosomal axes during meiosis, is frequently accompanied by infertility. We previously showed, using three mouse models of Down syndrome, that it is an extra chromosome, but not extra gene dose, that is associated with male infertility and virtual absence of post-meiotic gem cells. Here, we test the hypothesis that aneuploid segments are differentially modified and expressed during meiosis, depending on whether they are present as an extra chromosome or not. In all three models examined, the trisomic region lacks a pairing partner, but in one case, spermatocytes have an extra (and unpaired) chromosome, while the two other models involve translocation of the trisomic region rather than an extra chromosome. An extra unpaired chromosome was always modified by phosphorylation of histone H2AX and lacked RNA PolII. But in the case of trisomic regions attached to a paired chromosome, assembly of these protein modifications was affected by the position of a trisomic region relative to a centromere and the physical extent of the unpaired chromatin. Analysis of gene expression in testes revealed that extra copy number alone was not sufficient for meiotic upregulation of genes in the trisomic interval. Additionally and unexpectedly, presence of meiotic gene silencing chromatin modifications was not sufficient for downregulation of genes in unpaired trisomic chromatin. Thus, the meiotic chromatin modifications that are cytologically visible are unlikely to be directly involved in sterility versus fertility of DS models. Finally, the presence of an extra unpaired chromosome, but not the presence of extra (trisomic) genes, caused global deregulation of transcription in spermatocytes. These results reveal mechanisms by which an extra chromosome, but not trisomic gene dose, impact on meiotic progress and infertility.
Project description:Chromosomal and segmental aneuploidies are usually lethal or common features of cancer cells and variety of disease, but little is known about how copy number relates to gene expression. Drosophila males have a single X chromosome and two sets of autosomes, but X chromosome and autosome transcripts are equally abundant. This 2-fold increase in X chromosome gene expression requires a dosage compensation complex (MSL) that acetylates histone 4 at lysine 16 (H4AcK16). To determine the contribution of general buffering and MSL to X chromosome dosage compensation, we analyzed genome-wide copy number, expression and histone modification pattern in male Drosophila S2 cells with and without MSL. Keywords: Gene Regulation Study, genomic analyses Overall design: RNA-seq and microarray were performed in Drosophila non-treated or RNAi treated S2 cells. Two biological replicates were used for expression profile. DNA-seq and CGH were performed (CGH data forthcoming) to check the copy number variation in S2 cells. Chromatin immunoprecipitations were performed in Drosophila non-treated or RNAi treated S2 cells with different histone modification antibodies. At least three biological replicates were performed for each antibody and treatment.