Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.

Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.

Integrated Multi-Omics Analysis of Mechanisms Underlying Yeast Ethanol Tolerance.

For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype.

Power provides protection: Genetic robustness in yeast depends on the capacity to generate energy.

The functional basis of genetic robustness, the ability of organisms to suppress the effects of mutations, remains incompletely understood. We exposed a set of 15 strains of Saccharomyces cerevisiae form diverse environments to increasing doses of the chemical mutagen EMS. The number of the resulting random mutations was similar for all tested strains. However, there were differences in immediate mortality after the mutagenic treatment and in defective growth of survivors. An analysis of gene expression revealed that immediate mortality was lowest in strains with lowest expression of transmembrane proteins, which are rich in thiol groups and thus vulnerable to EMS. A signal of genuine genetic robustness was detected for the other trait, the ability to grow well despite bearing non-lethal mutations. Increased tolerance of such mutations correlated with high expression of genes responsible for the oxidative energy metabolism, suggesting that the negative effect of mutations can be buffered if enough energy is available. We confirmed this finding in three additional tests of the ability to grow on (i) fermentable or non-fermentable sources of carbon, (ii) under chemical inhibition of the electron transport chain and (iii) during overexpression of its key component, cytochrome c. Our results add the capacity to generate energy as a general mechanism of genetic robustness.

Lighting up protein design.

Using a neural network to predict how green fluorescent proteins respond to genetic mutations illuminates properties that could help design new proteins.

Heterosis is prevalent among domesticated but not wild strains of Saccharomyces cerevisiae.

Crosses between inbred but unrelated individuals often result in an increased fitness of the progeny. This phenomenon is known as heterosis and has been reported for wild and domesticated populations of plants and animals. Analysis of heterosis is often hindered by the fact that the genetic relatedness between analyzed organisms is only approximately known. We studied a collection of Saccharomyces cerevisiae isolates from wild and human-created habitats whose genomes were sequenced and thus their relatedness was fully known. We reasoned that if these strains accumulated different deleterious mutations at an approximately constant rate, then heterosis should be most visible in F1 heterozygotes from the least related parents. We found that heterosis was substantial and positively correlated with sequence divergence, but only in domesticated strains. More than 80% of the heterozygous hybrids were more fit than expected from the mean of their homozygous parents, and approximately three-quarters of those exceeded even the fittest parent. Our results support the notion that domestication brings about relaxation of selection and accumulation of deleterious mutations. However, other factors may have contributed as well. In particular, the observed build-up of genetic load might be facilitated by a decrease, and not increase, in the rate of inbreeding.

Generation of stable, non-aggregating Saccharomyces cerevisiae wild isolates.

Cellular aggregates observed during growth of Saccharomyces cerevisiae strains derived from various natural environments makes most laboratory techniques optimized for non-aggregating laboratory strains inappropriate. We describe a method to reduce the size and percentage of the aggregates. This is achieved by replacing the native allele of the AMN1 gene with an allele found in the W303 laboratory strain. The reduction in aggregates is consistent across various environments and generations, with no change in maximum population density or strain viability, and only minor changes in maximum growth rate and colony morphology.