Celia Payen

The Awesome Power of Yeast Evolutionary Genetics: New Genome Sequences and Strain Resources for the Saccharomyces sensu stricto Genus

High-quality, well-annotated genome sequences and standardized laboratory strains fuel experimental and evolutionary research. We present improved genome sequences of three species of Saccharomyces sensu stricto yeasts: S. bayanus var. uvarum (CBS 7001), S. kudriavzevii (IFO 1802T and ZP 591), and S. mikatae (IFO 1815T), and describe their comparison to the genomes of S. cerevisiae and S. paradoxus. The new sequences, derived by assembling millions of short DNA sequence reads together with previously published Sanger shotgun reads, have vastly greater long-range continuity and far fewer gaps than the previously available genome sequences. New gene predictions defined a set of 5261 protein-coding orthologs across the five most commonly studied Saccharomyces yeasts, enabling a re-examination of the tempo and mode of yeast gene evolution and improved inferences of species-specific gains and losses. To facilitate experimental investigations, we generated genetically marked, stable haploid strains for all three of these Saccharomyces species. These nearly complete genome sequences and the collection of genetically marked strains provide a valuable toolset for comparative studies of gene function, metabolism, and evolution, and render Saccharomyces sensu stricto the most experimentally tractable model genus. These resources are freely available and accessible through www.SaccharomycesSensuStricto.org.

Whole-genome sequencing of a laboratory-evolved yeast strain

BackgroundExperimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined.ResultsWe sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement.ConclusionsThis study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

The fitness consequences of aneuploidy are driven by condition-dependent gene effects.

Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell’s proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp’s fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.

The dynamics of diverse segmental amplifications in populations of Saccharomyces cerevisiae adapting to strong selection.

Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.

Origin-dependent inverted-repeat amplification: a replication-based model for generating palindromic amplicons.

(dsDNA) break and implicate DNA fusions (either homologous or non-homologous), Break-Induced Replication (BIR), Microhomology/Microsatellite-Induced Replication (MMIR), and/or inverted or directly repeated sequences that adopt unusual secondary structures for their repair [4]. From the molecular analysis [7] of a yeast strain that contains amplified copies of the gene for the high affinity sulfur transporter, SUL1 [3], we derived a new general model that explains the generation of interstitial tandem inverted repeat arrays of chromosome segments in yeast and in human cancers, and of de novo congenital inverted triplications and other chromosomal rearrangements. We propose that cells commit a singular error in replication: the ligation of the nascent leading strand to the nascent lagging strand at the replication fork. This model can potentially explain the origin of many palindromic rearrangements and their structural, enzymatic, and genetic requirements.

Coupling Unbiased Mutagenesis to High-throughput DNA Sequencing Uncovers Functional Domains in the Ndc80 Kinetochore Protein of Saccharomyces cerevisiae

During mitosis, kinetochores physically link chromosomes to the dynamic ends of spindle microtubules. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain. Ndc80 is also required for recruiting other components to the kinetochore and responding to mitotic regulatory signals. While the calponin homology domain has been the focus of biochemical and structural characterization, the function of the remainder of Ndc80 is poorly understood. Here, we utilized a new approach that couples high-throughput sequencing to a saturating linker-scanning mutagenesis screen in Saccharomyces cerevisiae. We identified domains in previously uncharacterized regions of Ndc80 that are essential for its function in vivo. We show that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex. Furthermore, a mutation in this hairpin abolishes the ability of the Dam1 complex to strengthen microtubule attachments made by the Ndc80 complex. Finally, we defined a C-terminal segment of Ndc80 required for tetramerization of the Ndc80 complex in vivo. This unbiased mutagenesis approach can be generally applied to genes in S. cerevisiae to identify functional properties and domains.

Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene’s promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1. Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology—saturation mutagenesis, chemostat selection, and DNA sequencing to track variants—should be a broadly applicable approach.

High-Throughput Identification of Adaptive Mutations in Experimentally Evolved Yeast Populations

High-throughput sequencing has enabled genetic screens that can rapidly identify mutations that occur during experimental evolution. The presence of a mutation in an evolved lineage does not, however, constitute proof that the mutation is adaptive, given the well-known and widespread phenomenon of genetic hitchhiking, in which a non-adaptive or even detrimental mutation can co-occur in a genome with a beneficial mutation and the combined genotype is carried to high frequency by selection. We approximated the spectrum of possible beneficial mutations in Saccharomyces cerevisiae using sets of single-gene deletions and amplifications of almost all the genes in the S. cerevisiae genome. We determined the fitness effects of each mutation in three different nutrient-limited conditions using pooled competitions followed by barcode sequencing. Although most of the mutations were neutral or deleterious, ~500 of them increased fitness. We then compared those results to the mutations that actually occurred during experimental evolution in the same three nutrient-limited conditions. On average, ~35% of the mutations that occurred during experimental evolution were predicted by the systematic screen to be beneficial. We found that the distribution of fitness effects depended on the selective conditions. In the phosphate-limited and glucose-limited conditions, a large number of beneficial mutations of nearly equivalent, small effects drove the fitness increases. In the sulfate-limited condition, one type of mutation, the amplification of the high-affinity sulfate transporter, dominated. In the absence of that mutation, evolution in the sulfate-limited condition involved mutations in other genes that were not observed previously—but were predicted by the systematic screen. Thus, gross functional screens have the potential to predict and identify adaptive mutations that occur during experimental evolution.

Empirical determinants of adaptive mutations in yeast experimental evolution

High-throughput sequencing technologies have enabled expansion of the scope of genetic screens to identify mutations that underlie quantitative phenotypes, such as fitness improvements that occur during the course of experimental evolution. This new capability has allowed us to describe the relationship between fitness and genotype at a level never possible before, and ask deeper questions, such as how genome structure, available mutation spectrum, and other factors drive evolution. Here we combined functional genomics and experimental evolution to first map on a genome scale the distribution of potential beneficial mutations available as a first step to an evolving population and then compare these to the mutations actually observed in order to define the constraints acting upon evolution. We first constructed a single-step fitness landscape for the yeast genome by using barcoded gene deletion and overexpression collections, competitive growth in continuous culture, and barcode sequencing. By quantifying the relative fitness effects of thousands of single-gene amplifications or deletions simultaneously we revealed the presence of hundreds of accessible evolutionary paths. To determine the actual mutation spectrum used in evolution, we built a catalog of >1000 mutations selected during experimental evolution. By combining both datasets, we were able to ask how and why evolution is constrained. We identified adaptive mutations in laboratory evolved populations, derived mutational signatures in a variety of conditions and ploidy states, and determined that half of the mutations accumulated positively affect cellular fitness. We also uncovered hundreds of potential beneficial mutations never observed in the mutational spectrum derived from the experimental evolution catalog and found that those adaptive mutations become accessible in the absence of the dominant adaptive solution. This comprehensive functional screen explored the set of potential adaptive mutations on one genetic background, and allows us for the first time at this scale to compare the mutational path with the actual, spontaneously derived spectrum of mutations. AUTHOR SUMMARY Whole genome sequencing of thousands of cancer genomes has been conducted to characterize variants including point mutations and structural changes, providing a large catalogue of critical polymorphisms associated with tumorigenesis. Despite the high prevalence of mutations in cancer and technological advances in their genotyping, cancer genetics still presents many open questions regarding the prediction of selection and the functional impact of mutations on cellular fitness. Long term experimental evolution using model organisms has allowed the selection for strains bearing recurrent and rare mutations, mimicking the genetic aberrations acquired by tumor cells. Here, we evaluate the functional impact of thousands of single gene losses and amplifications on the cellular fitness of yeast. Our results show that hundreds of beneficial mutations are possible during adaptation but not all of them have been selected in evolution experiments so far performed. Together, our results provide evidence that 50% of the mutations found in experimentally evolved populations are advantageous, and that alternative mutations with improved fitness could be selected in the absence of the main adaptive mutations with higher fitness. BLURB A combined view of potential adaptive mutations, generated by a systematic screening approach, coupled with the mutational spectrum derived from experimentally evolved yeast reveals the usage of accessible evolutionary solutions.

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.