Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Abstract

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) system to the newly synthesized megabase Sc2.0 chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by (1) removing most of the chromosome content or (2) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and specific chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic chromosome resource opens new paradigms in studying aneuploidy.

Keywords: SCRaMbLE; aneuploidy phenotypes; aneuploidy recovery approaches; growth rate; synVII; synthetic disomic yeast; synthetic genomics.

Graphical abstract

Figure 1

Functional dissection and repair of SynVII (A) Spot assay of synVII-R intermediate strains. The synVII-R is constructed in orientation from megachunk Y toward megachunk O. (B) Bug mapping by mating growth-defective query strain synVIIS and its parental strain synVIIT with yeast single-knockout nsr1Δ1 strain. (C) Transcriptome profiling of synVIIS compared with synVIIT reveals significant upregulation of NSR1 mRNA. Upregulated features are labeled in red, and downregulated features are labeled in green. (D) Quantitative real-time PCR (qPCR) validation of NSR1 mRNA expression in synVIIS and synVIIT strains. Error bars represent ±SD from three independent experiments. (E–G) Introduction of loxPsym (green) at the 5′ UTR of NSR1 led to a growth defect in both synVII intermediate strain synVIIS and wild-type strain BY4741. The corresponding phenotype by plating and protein expression level, quantified by multiple reaction monitoring mass spectrometry (MRM-MS) analysis, is shown for each constructed strain. Error bars indicate ±SD (n = 3). The white and orange blocks represent wild-type and synthetic PCRTags, respectively. Identified dysregulated genetic features at (F) transcriptome level and (G) proteome level of repaired synVII cells compared with BY4741. Total number of differentially expressed (p < 0.001) features in transcriptome and proteome are presented.

Figure 2

Construction and physiologic analysis of disomic yeasts (A) Schematic illustration of the construction of disome yeast strains YSy140 and YSy142. (B) 3D genome organization of native and synthetic chromosome VII in YSy142 strain in comparison with haploid BY4741 and YSy105. Each bead represents a 10-kb chromosome segment. Centromeres, telomeres, and wild-type chrVII and synVII are indicated with red, black, blue, and green beads, respectively. Other chromosomes are shown in gray. Two angles of view are shown for the YSy142 strain. (C) The corresponding DNA and mRNA levels track with gene copy number in both disomic yeast strains YSy140 and YSy142. A 60-kb tandem duplication in chromosome XIV is identified in YSy142 and all derived SCRaMbLEd strains. (D) Genome stability analysis of YSy140 and YSy142 through long-term growth assays across a time span of ∼220 generations. The number represents the average number of generations maintaining aneuploidy. Error bars indicate ±SD (n = 3). See also Figure S4.

Figure 3

SCRaMbLE of YSy142 disomic yeast (A) SCRaMbLE and analysis workflow. The auxotrophic marker of the YSy142 strain was swapped from HIS3 to MET15. See construction of aneuploid synVII strain section in the STAR Methods for details. (B) The fate of each segment flanked by two loxPsym sites in each strain is indicated as preserved (light blue) or deleted (white) by any SCRaMbLE event. The y axis shows the relative average phenotypic recovery rate of each SCRaMbLEd strain in comparison with that of YSy142 represented by the color scale (n ≥ 200). (C) The distribution of recombination events for all selected SCRaMbLEd aneuploid strains. The number of events per strain has a long tail with some strains having 22 and 25 distinct recombination events. (D) Correlation between chromosome retention rate and growth recovery in SCRaMbLEd strains with circular synVII and SCRaMbLEd strains with linear synVII. Each dot represents one SCRaMbLEd strain. R indicates Pearson’s correlation. Solid line: fitted curve (ggplot:geom_point), geom_smooth (method = lm, se = TRUE). Gray area: 95% confidence range for fitted curve.

Figure 4

Growth assay and ESR profiling of disomic yeast with circular SCRaMbLEd synVII (A) General improvement of fitness at varying degrees was observed for the top 18 SCRaMbLEd strains in five representative conditions. Each dot represents average growth recovery rate of one SCRaMbLEd strain calculated based on multiple single colonies (n ≥ 200). (B) Spot assay of two representative strains of the 18 SCRaMbLEd strains under various conditions, showing general improved fitness. (C) The ESR genes expression profile of the unSCRaMbLE strain YSy142 and two SCRaMbLEd strains (YSy151 and YSy150) after normalization with the reference wild-type strain BY4741 in three selected conditions. The fold change is represented by the color scale (yellow: upregulated; blue: downregulated). Conditions include: YPD at 30°C for 2 days; YPD + cycloheximide (Cyc; 0.01 μg/mL) at 30°C for 2 days; YPD + dl-dithiothreitol (DTT; 2.5 mM pretreatment for 1 h) for 2 days; YPD + hydroxyurea (HU; 100 mM) at 30°C for 4 days; YPD + methyl methanesulfonate (MMS; 0.01% v/v) at 30°C for 3 days. YPD, yeast extract peptone dextrose.

Figure 5

The improvement of synVII aneuploidy phenotypes by the deletion of a 20-kb region (del20K) on synVIIR is associated with the upregulation of protein biosynthesis (A) The proteomics analysis of five disomic strains with linear SCRaMbLEd synVII exhibited varying recovery rates grown on YPD and two stress conditions. (B) The chromosome-wide association analysis against the five disomic strains reveals a 20-kb region that might play an essential role in the observed fitness improvement. Each red dot represents the statistical significance of each gene for the association between its deletion pattern and relative colony size. The five selected strains are ranked by their corresponding growth recovery rate. The regions deleted and reserved after SCRaMbLE are presented in gray and blue, respectively. (C) Doubling time measurement of YSy142, syn-del20K, wt-del20K, and YSy142 carrying the single-gene knockout strains under the Cyc condition. The error bar indicates standard error of the mean (SEM) in seven biological replicates. The p value was calculated using single-tailed Student’s t test. ∗∗∗p < 10E−3. YPD + Cyc (0.01 μg/mL) at 30°C; YPD + HU (100 mM) at 30°C. See also Figures S10 and S11.