Yeast cell responses and survival during periodic osmotic stress are controlled by glucose availability.

Natural environments of living organisms are often dynamic and multifactorial, with multiple parameters fluctuating over time. To better understand how cells respond to dynamically interacting factors, we quantified the effects of dual fluctuations of osmotic stress and glucose deprivation on yeast cells using microfluidics and time-lapse microscopy. Strikingly, we observed that cell proliferation, survival, and signaling depend on the phasing of the two periodic stresses. Cells divided faster, survived longer, and showed decreased transcriptional response when fluctuations of hyperosmotic stress and glucose deprivation occurred in phase than when the two stresses occurred alternatively. Therefore, glucose availability regulates yeast responses to dynamic osmotic stress, showcasing the key role of metabolic fluctuations in cellular responses to dynamic stress. We also found that mutants with impaired osmotic stress response were better adapted to alternating stresses than wild-type cells, showing that genetic mechanisms of adaptation to a persistent stress factor can be detrimental under dynamically interacting conditions.

Plasticity and environment-specific relationships between gene expression and fitness in Saccharomyces cerevisiae.

Phenotypic evolution is shaped by interactions between organisms and their environments. The environment influences how an organism's genotype determines its phenotype and how this phenotype affects its fitness. To better understand this dual role of the environment in the production and selection of phenotypic variation, we empirically determined and compared the genotype-phenotype-fitness relationship for mutant strains of the budding yeast Saccharomyces cerevisiae in four environments. Specifically, we measured how mutations in the promoter of the metabolic gene TDH3 modified its expression level and affected its growth on media with four different carbon sources. In each environment, we observed a clear relationship between TDH3 expression level and fitness, but this relationship differed among environments. Genetic variants with similar effects on TDH3 expression in different environments often had different effects on fitness and vice versa. Such environment-specific relationships between phenotype and fitness can shape the evolution of phenotypic plasticity. The set of mutants we examined also allowed us to compare the effects of mutations disrupting binding sites for key transcriptional regulators and the TATA box, which is part of the core promoter sequence. Mutations disrupting the binding sites for the transcription factors had more variable effects on expression among environments than mutations disrupting the TATA box, yet mutations with the most environmentally variable effects on fitness were located in the TATA box. This observation suggests that mutations affecting different molecular mechanisms are likely to contribute unequally to regulatory sequence evolution in changing environments. Significance Statement: Environments can affect the phenotypic traits an organism produces as well as the adaptive value of these traits (i.e. whether those traits will allow the organism to better survive and pass their genes on to the next generation). This study shows how the environment impacts both the production and selection of traits using the expression of a metabolic gene in the baker's yeast as a model system. This study further shows that some types of genetic changes make gene expression traits more responsive to environmental changes than others, suggesting that genetic changes affecting different molecular mechanisms of gene regulation may contribute differently to genetic evolution.

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae.

Heritable variation in a gene's expression arises from mutations impacting cis- and trans-acting components of its regulatory network. Here, we investigate how trans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans-regulatory effects on expression of the same focal gene in Saccharomyces cerevisiae. Relative to 1766 mutations without effects on expression of this focal gene, we found that these trans-regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of the trans-regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism, and signal transduction. These data show how genetic changes in diverse types of genes can impact a gene's expression in trans, revealing properties of trans-regulatory mutations that provide the raw material for trans-regulatory variation segregating within natural populations.

Empirical measures of mutational effects define neutral models of regulatory evolution in Saccharomyces cerevisiae.

Understanding how phenotypes evolve requires disentangling the effects of mutation generating new variation from the effects of selection filtering it. Tests for selection frequently assume that mutation introduces phenotypic variation symmetrically around the population mean, yet few studies have tested this assumption by deeply sampling the distributions of mutational effects for particular traits. Here, we examine distributions of mutational effects for gene expression in the budding yeast Saccharomyces cerevisiae by measuring the effects of thousands of point mutations introduced randomly throughout the genome. We find that the distributions of mutational effects differ for the 10 genes surveyed and are inconsistent with normality. For example, all 10 distributions of mutational effects included more mutations with large effects than expected for normally distributed phenotypes. In addition, some genes also showed asymmetries in their distribution of mutational effects, with new mutations more likely to increase than decrease the gene's expression or vice versa. Neutral models of regulatory evolution that take these empirically determined distributions into account suggest that neutral processes may explain more expression variation within natural populations than currently appreciated.

Fitness effects of altering gene expression noise in Saccharomyces cerevisiae.

Gene expression noise is an evolvable property of biological systems that describes differences in expression among genetically identical cells in the same environment. Prior work has shown that expression noise is heritable and can be shaped by selection, but the impact of variation in expression noise on organismal fitness has proven difficult to measure. Here, we quantify the fitness effects of altering expression noise for the TDH3 gene in Saccharomyces cerevisiae. We show that increases in expression noise can be deleterious or beneficial depending on the difference between the average expression level of a genotype and the expression level maximizing fitness. We also show that a simple model relating single-cell expression levels to population growth produces patterns consistent with our empirical data. We use this model to explore a broad range of average expression levels and expression noise, providing additional insight into the fitness effects of variation in expression noise.

Effects of mutation and selection on plasticity of a promoter activity in Saccharomyces cerevisiae.

Phenotypic plasticity is an evolvable property of biological systems that can arise from environment-specific regulation of gene expression. To better understand the evolutionary and molecular mechanisms that give rise to plasticity in gene expression, we quantified the effects of 235 single-nucleotide mutations in the Saccharomyces cerevisiae TDH3 promoter (PTDH3 ) on the activity of this promoter in media containing glucose, galactose, or glycerol as a carbon source. We found that the distributions of mutational effects differed among environments because many mutations altered the plastic response exhibited by the wild-type allele. Comparing the effects of these mutations with the effects of 30 PTDH3 polymorphisms on expression plasticity in the same environments provided evidence of natural selection acting to prevent the plastic response in PTDH3 activity between glucose and galactose from becoming larger. The largest changes in expression plasticity were observed between fermentable (glucose or galactose) and nonfermentable (glycerol) carbon sources and were caused by mutations located in the RAP1 and GCR1 transcription factor binding sites. Mutations altered expression plasticity most frequently between the two fermentable environments, with mutations causing significant changes in plasticity between glucose and galactose distributed throughout the promoter, suggesting they might affect chromatin structure. Taken together, these results provide insight into the molecular mechanisms underlying gene-by-environment interactions affecting gene expression as well as the evolutionary dynamics affecting natural variation in plasticity of gene expression.

Fitness Effects of Cis-Regulatory Variants in the Saccharomyces cerevisiae TDH3 Promoter.

Variation in gene expression is widespread within and between species, but fitness consequences of this variation are generally unknown. Here, we use mutations in the Saccharomyces cerevisiae TDH3 promoter to assess how changes in TDH3 expression affect cell growth. From these data, we predict the fitness consequences of de novo mutations and natural polymorphisms in the TDH3 promoter. Nearly all mutations and polymorphisms in the TDH3 promoter were found to have no significant effect on fitness in the environment assayed, suggesting that the wild-type allele of this promoter is robust to the effects of most new cis-regulatory mutations.

Contrasting Frequencies and Effects of cis- and trans-Regulatory Mutations Affecting Gene Expression.

Heritable differences in gene expression are caused by mutations in DNA sequences encoding cis-regulatory elements and trans-regulatory factors. These two classes of regulatory change differ in their relative contributions to expression differences in natural populations because of the combined effects of mutation and natural selection. Here, we investigate how new mutations create the regulatory variation upon which natural selection acts by quantifying the frequencies and effects of hundreds of new cis- and trans-acting mutations altering activity of the TDH3 promoter in the yeast Saccharomyces cerevisiae in the absence of natural selection. We find that cis-regulatory mutations have larger effects on expression than trans-regulatory mutations and that while trans-regulatory mutations are more common overall, cis- and trans-regulatory changes in expression are equally abundant when only the largest changes in expression are considered. In addition, we find that cis-regulatory mutations are skewed toward decreased expression while trans-regulatory mutations are skewed toward increased expression. We also measure the effects of cis- and trans-regulatory mutations on the variability in gene expression among genetically identical cells, a property of gene expression known as expression noise, finding that trans-regulatory mutations are much more likely to decrease expression noise than cis-regulatory mutations. Because new mutations are the raw material upon which natural selection acts, these differences in the frequencies and effects of cis- and trans-regulatory mutations should be considered in models of regulatory evolution.

Selection on noise constrains variation in a eukaryotic promoter.

Genetic variation segregating within a species reflects the combined activities of mutation, selection, and genetic drift. In the absence of selection, polymorphisms are expected to be a random subset of new mutations; thus, comparing the effects of polymorphisms and new mutations provides a test for selection. When evidence of selection exists, such comparisons can identify properties of mutations that are most likely to persist in natural populations. Here we investigate how mutation and selection have shaped variation in a cis-regulatory sequence controlling gene expression by empirically determining the effects of polymorphisms segregating in the TDH3 promoter among 85 strains of Saccharomyces cerevisiae and comparing their effects to a distribution of mutational effects defined by 236 point mutations in the same promoter. Surprisingly, we find that selection on expression noise (that is, variability in expression among genetically identical cells) appears to have had a greater impact on sequence variation in the TDH3 promoter than selection on mean expression level. This is not necessarily because variation in expression noise impacts fitness more than variation in mean expression level, but rather because of differences in the distributions of mutational effects for these two phenotypes. This study shows how systematically examining the effects of new mutations can enrich our understanding of evolutionary mechanisms. It also provides rare empirical evidence of selection acting on expression noise.

Mapping small effect mutations in Saccharomyces cerevisiae: impacts of experimental design and mutational properties.

Genetic variants identified by mapping are biased toward large phenotypic effects because of methodologic challenges for detecting genetic variants with small phenotypic effects. Recently, bulk segregant analysis combined with next-generation sequencing (BSA-seq) was shown to be a powerful and cost-effective way to map small effect variants in natural populations. Here, we examine the power of BSA-seq for efficiently mapping small effect mutations isolated from a mutagenesis screen. Specifically, we determined the impact of segregant population size, intensity of phenotypic selection to collect segregants, number of mitotic generations between meiosis and sequencing, and average sequencing depth on power for mapping mutations with a range of effects on the phenotypic mean and standard deviation as well as relative fitness. We then used BSA-seq to map the mutations responsible for three ethyl methanesulfonate-induced mutant phenotypes in Saccharomyces cerevisiae. These mutants display small quantitative variation in the mean expression of a fluorescent reporter gene (-3%, +7%, and +10%). Using a genetic background with increased meiosis rate, a reliable mating type marker, and fluorescence-activated cell sorting to efficiently score large segregating populations and isolate cells with extreme phenotypes, we successfully mapped and functionally confirmed a single point mutation responsible for the mutant phenotype in all three cases. Our simulations and experimental data show that the effects of a causative site not only on the mean phenotype, but also on its standard deviation and relative fitness should be considered when mapping genetic variants in microorganisms such as yeast that require population growth steps for BSA-seq.

Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae.

BACKGROUND: The nematode Caenorhabditis elegans is a major model organism in laboratory biology. Very little is known, however, about its ecology, including where it proliferates. In the past, C. elegans was mainly isolated from human-made compost heaps, where it was overwhelmingly found in the non-feeding dauer diapause stage. RESULTS: C. elegans and C. briggsae were found in large, proliferating populations in rotting plant material (fruits and stems) in several locations in mainland France. Both species were found to co-occur in samples isolated from a given plant species. Population counts spanned a range from one to more than 10,000 Caenorhabditis individuals on a single fruit or stem. Some populations with an intermediate census size (10 to 1,000) contained no dauer larvae at all, whereas larger populations always included some larvae in the pre-dauer or dauer stages. We report on associated micro-organisms, including pathogens. We systematically sampled a spatio-temporally structured set of rotting apples in an apple orchard in Orsay over four years. C. elegans and C. briggsae were abundantly found every year, but their temporal distributions did not coincide. C. briggsae was found alone in summer, whereas both species co-occurred in early fall and C. elegans was found alone in late fall. Competition experiments in the laboratory at different temperatures show that C. briggsae out-competes C. elegans at high temperatures, whereas C. elegans out-competes C. briggsae at lower temperatures. CONCLUSIONS: C. elegans and C. briggsae proliferate in the same rotting vegetal substrates. In contrast to previous surveys of populations in compost heaps, we found fully proliferating populations with no dauer larvae. The temporal sharing of the habitat by the two species coincides with their temperature preference in the laboratory, with C. briggsae populations growing faster than C. elegans at higher temperatures, and vice at lower temperatures.

Role of pleiotropy in the evolution of a cryptic developmental variation in Caenorhabditis elegans.

Robust biological systems are expected to accumulate cryptic genetic variation that does not affect the system output in standard conditions yet may play an evolutionary role once phenotypically expressed under a strong perturbation. Genetic variation that is cryptic relative to a robust trait may accumulate neutrally as it does not change the phenotype, yet it could also evolve under selection if it affects traits related to fitness in addition to its cryptic effect. Cryptic variation affecting the vulval intercellular signaling network was previously uncovered among wild isolates of Caenorhabditis elegans. Using a quantitative genetic approach, we identify a non-synonymous polymorphism of the previously uncharacterized nath-10 gene that affects the vulval phenotype when the system is sensitized with different mutations, but not in wild-type strains. nath-10 is an essential protein acetyltransferase gene and the homolog of human NAT10. The nath-10 polymorphism also presents non-cryptic effects on life history traits. The nath-10 allele carried by the N2 reference strain leads to a subtle increase in the egg laying rate and in the total number of sperm, a trait affecting the trade-off between fertility and minimal generation time in hermaphrodite individuals. We show that this allele appeared during early laboratory culture of N2, which allowed us to test whether it may have evolved under selection in this novel environment. The derived allele indeed strongly outcompetes the ancestral allele in laboratory conditions. In conclusion, we identified the molecular nature of a cryptic genetic variation and characterized its evolutionary history. These results show that cryptic genetic variation does not necessarily accumulate neutrally at the whole-organism level, but may evolve through selection for pleiotropic effects that alter fitness. In addition, cultivation in the laboratory has led to adaptive evolution of the reference strain N2 to the laboratory environment, which may modify other phenotypes of interest.

Intraspecific evolution of the intercellular signaling network underlying a robust developmental system.

Many biological systems produce an invariant output when faced with stochastic or environmental variation. This robustness of system output to variation affecting the underlying process may allow for "cryptic" genetic evolution within the system without change in output. We studied variation of cell fate patterning of Caenorhabditis elegans vulva precursors, a developmental system that relies on a simple intercellular signaling network and yields an invariant output of cell fates and lineages among C. elegans wild isolates. We first investigated the system's genetic variation in C. elegans by means of genetic tools and cell ablation to break down its buffering mechanisms. We uncovered distinct architectures of quantitative variation along the Ras signaling cascade, including compensatory variation, and differences in cell sensitivity to induction along the anteroposterior axis. In the unperturbed system, we further found variation between isolates in spatio-temporal dynamics of Ras pathway activity, which can explain the phenotypic differences revealed upon perturbation. Finally, the variation mostly affects the signaling pathways in a tissue-specific manner. We thus demonstrate and characterize microevolution of a developmental signaling network. In addition, our results suggest that the vulva genetic screens would have yielded a different mutation spectrum, especially for Wnt pathway mutations, had they been performed in another C. elegans genetic background.

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13.

Wnt target gene transcription is mediated by nuclear translocation of stabilized beta-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of beta-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.