Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

Abstract

Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1’s proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102–104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18–21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.

Aneuploidy in yeast: Segregation error or adaptation mechanism?

“…In cancer cells, for instance, aneuploidy is known to result in deregulation of the transcriptome and proteome (Dürrbaum & Storchová, ; Ried et al, ). Aneuploidy, as with any other alteration in gene copy number (Lauer et al, ), can result in differential gene expression (Sheltzer, Torres, Dunham, & Amon, ). Under certain conditions, such changes in gene dosage may provide fitness benefits to the cell that outweigh the costs of maintaining the aneuploidy.…”

“…The consistency with which aneuploid cells are recovered following the exposure of yeasts to stress in the laboratory suggests that it can serve as an adaptation mechanism to harsh environments (see Box 1 and Table 1 for more detailed information). In S. cerevisiae, adaptation though aneuploidy is likely due to the amplified or modified expression of genes on aneuploid chromosomes, which can lead to increased tolerance to environmental stress (Chen et al, 2012;Lauer et al, 2018;Linder, Greco, Seidl, Matsui, & Ehrenreich, 2017;Millet, Ausiannikava, Le Bihan, Granneman, & Makovets, 2015;Selmecki et al, 2015;Yona et al, 2012). This includes low nutrient availability (Gresham et al, 2008;Hong & Gresham, 2014;Selmecki et al, 2015;Sunshine et al, 2015), high ethanol concentrations (Voordeckers et al, 2015), high temperature ( Figure 2 adapted from Yona et al, 2012), and telomerase insufficiency (Millet et al, 2015).…”

Transcription-induced formation of extrachromosomal DNA during yeast ageing

“…Re-integration of eccDNA provides an efficient pathway for gene amplification, with various chromosomal adaptations in lower eukaryotes being known or predicted to have emerged through eccDNA intermediates [14][15][16][17][18]. In tumour cells, aggregation of smaller eccDNA to large (1-5MB), microscopically visible double minutes, and re-integration in homogeneously staining regions to yield high-copy chromosomal repeats has also been reported [7,19,20].…”

A natural variant of the essential host geneMMS21restricts the parasitic 2-micron plasmid inSaccharomyces cerevisiae

“…These dual markers ensured the reporter plasmid could be initially introduced and be retained in plasmid-lacking strains. In addition the GFP allows strains to be easily quantified for plasmid presence, absence, and copy number that scales with GFP intensity in yeast [63][64][65][66] . Our analyses revealed that GFP intensity for this 2µ reporter plasmid is roughly normally distributed across single cells (Supplementary Figure S1) indicating that GFP signal did not saturate the detector at high copy number.…”