Lasker∼Koshland to genetics pioneer.

The 2014 Lasker∼Koshland Special Achievement Award will be presented to Mary-Claire King, a pioneer and visionary who revolutionized the use of genetics to identify disease genes, provide insights into human evolution, and champion human rights causes.

A genome-scale yeast library with inducible expression of individual genes.

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

Pervasive genetic hitchhiking and clonal interference in forty evolving yeast populations.

The dynamics of adaptation determine which mutations fix in a population, and hence how reproducible evolution will be. This is central to understanding the spectra of mutations recovered in the evolution of antibiotic resistance, the response of pathogens to immune selection, and the dynamics of cancer progression. In laboratory evolution experiments, demonstrably beneficial mutations are found repeatedly, but are often accompanied by other mutations with no obvious benefit. Here we use whole-genome whole-population sequencing to examine the dynamics of genome sequence evolution at high temporal resolution in 40 replicate Saccharomyces cerevisiae populations growing in rich medium for 1,000 generations. We find pervasive genetic hitchhiking: multiple mutations arise and move synchronously through the population as mutational 'cohorts'. Multiple clonal cohorts are often present simultaneously, competing with each other in the same population. Our results show that patterns of sequence evolution are driven by a balance between these chance effects of hitchhiking and interference, which increase stochastic variation in evolutionary outcomes, and the deterministic action of selection on individual mutations, which favours parallel evolutionary solutions in replicate populations.

Extrachromosomal circular DNA is common in yeast.

Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥ 15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.

Characterizing the in vivo role of trehalose in Saccharomyces cerevisiae using the AGT1 transporter.

Trehalose is a highly stable, nonreducing disaccharide of glucose. A large body of research exists implicating trehalose in a variety of cellular phenomena, notably response to stresses of various kinds. However, in very few cases has the role of trehalose been examined directly in vivo. Here, we describe the development and characterization of a system in Saccharomyces cerevisiae that allows us to manipulate intracellular trehalose concentrations independently of the biosynthetic enzymes and independently of any applied stress. We found that many physiological roles heretofore ascribed to intracellular trehalose, including heat resistance, are not due to the presence of trehalose per se. We also found that many of the metabolic and growth defects associated with mutations in the trehalose biosynthesis pathway are not abolished by providing abundant intracellular trehalose. Instead, we made the observation that intracellular accumulation of trehalose or maltose (another disaccharide of glucose) is growth-inhibitory in a carbon source-specific manner. We conclude that the physiological role of the trehalose pathway is fundamentally metabolic: i.e., more complex than simply the consequence of increased concentrations of the sugar and its attendant physical properties (with the exception of the companion paper where Tapia et al. [Tapia H, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1506415112] demonstrate a direct role for trehalose in protecting cells against desiccation).

Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae.

A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of ß-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input-output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.

Yeast metabolic and signaling genes are required for heat-shock survival and have little overlap with the heat-induced genes.

Genome-wide gene-expression studies have shown that hundreds of yeast genes are induced or repressed transiently by changes in temperature; many are annotated to stress response on this basis. To obtain a genome-scale assessment of which genes are functionally important for innate and/or acquired thermotolerance, we combined the use of a barcoded pool of ~4,800 nonessential, prototrophic Saccharomyces cerevisiae deletion strains with Illumina-based deep-sequencing technology. As reported in other recent studies that have used deletion mutants to study stress responses, we observed that gene deletions resulting in the highest thermosensitivity generally are not the same as those transcriptionally induced in response to heat stress. Functional analysis of identified genes revealed that metabolism, cellular signaling, and chromatin regulation play roles in regulating thermotolerance and in acquired thermotolerance. However, for most of the genes identified, the molecular mechanism behind this action remains unclear. In fact, a large fraction of identified genes are annotated as having unknown functions, further underscoring our incomplete understanding of the response to heat shock. We suggest that survival after heat shock depends on a small number of genes that function in assessing the metabolic health of the cell and/or regulate its growth in a changing environment.

Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.

A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys(2)His(2) zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.

Comparing whole genomes using DNA microarrays.

The rapid accumulation of complete genomic sequences offers the opportunity to carry out an analysis of inter- and intra-individual genome variation within a species on a routine basis. Sequencing whole genomes requires resources that are currently beyond those of a single laboratory and therefore it is not a practical approach for resequencing hundreds of individual genomes. DNA microarrays present an alternative way to study differences between closely related genomes. Advances in microarray-based approaches have enabled the main forms of genomic variation (amplifications, deletions, insertions, rearrangements and base-pair changes) to be detected using techniques that are readily performed in individual laboratories using simple experimental approaches.

Metabolic cycling without cell division cycling in respiring yeast.

Despite rapid progress in characterizing the yeast metabolic cycle, its connection to the cell division cycle (CDC) has remained unclear. We discovered that a prototrophic batch culture of budding yeast, growing in a phosphate-limited ethanol medium, synchronizes spontaneously and goes through multiple metabolic cycles, whereas the fraction of cells in the G1/G0 phase of the CDC increases monotonically from 90 to 99%. This demonstrates that metabolic cycling does not require cell division cycling and that metabolic synchrony does not require carbon-source limitation. More than 3,000 genes, including most genes annotated to the CDC, were expressed periodically in our batch culture, albeit a mere 10% of the cells divided asynchronously; only a smaller subset of CDC genes correlated with cell division. These results suggest that the yeast metabolic cycle reflects a growth cycle during G1/G0 and explains our previous puzzling observation that genes annotated to the CDC increase in expression at slow growth.

Survival of starving yeast is correlated with oxidative stress response and nonrespiratory mitochondrial function.

Survival of yeast during starvation has been shown to depend on the nature of the missing nutrient(s). In general, starvation for "natural" nutrients such as sources of carbon, phosphate, nitrogen, or sulfate results in low death rates, whereas starvation for amino acids or other metabolites in auxotrophic mutants results in rapid loss of viability. Here we characterized phenotype, gene expression, and metabolite abundance during starvation for methionine. Some methionine auxotrophs (those with blocks in the biosynthetic pathway) respond to methionine starvation like yeast starving for natural nutrients such as phosphate or sulfate: they undergo a uniform cell cycle arrest, conserve glucose, and survive. In contrast, methionine auxotrophs with defects in the transcription factors Met31p and Met32p respond poorly, like other auxotrophs. We combined physiological and gene expression data from a variety of nutrient starvations (in both respiratory competent and incompetent cells) to show that successful starvation response is correlated with expression of genes encoding oxidative stress response and nonrespiratory mitochondrial functions, but not respiration per se.

Adaptation to diverse nitrogen-limited environments by deletion or extrachromosomal element formation of the GAP1 locus.

To study adaptive evolution in defined environments, we performed evolution experiments with Saccharomyces cerevisiae (yeast) in nitrogen-limited chemostat cultures. We used DNA microarrays to identify copy-number variation associated with adaptation and observed frequent amplifications and deletions at the GAP1 locus. GAP1 encodes the general amino acid permease, which transports amino acids across the plasma membrane. We identified a self-propagating extrachromosomal circular DNA molecule that results from intrachromosomal recombination between long terminal repeats (LTRs) flanking GAP1. Extrachromosomal DNA circles (GAP1(circle)) contain GAP1, the replication origin ARS1116, and a single hybrid LTR derived from recombination between the two flanking LTRs. Formation of the GAP1(circle) is associated with deletion of chromosomal GAP1 (gap1Δ) and production of a single hybrid LTR at the GAP1 chromosomal locus. The GAP1(circle) is selected following prolonged culturing in L-glutamine-limited chemostats in a manner analogous to the selection of oncogenes present on double minutes in human cancers. Clones carrying only the gap1Δ allele were selected under various non-amino acid nitrogen limitations including ammonium, urea, and allantoin limitation. Previous studies have shown that the rate of intrachromosomal recombination between tandem repeats is stimulated by transcription of the intervening sequence. The high level of GAP1 expression in nitrogen-limited chemostats suggests that the frequency of GAP1(circle) and gap1Δ generation may be increased under nitrogen-limiting conditions. We propose that this genomic architecture facilitates evolvability of S. cerevisiae populations exposed to variation in levels and sources of environmental nitrogen.

Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes.

The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (T(m)) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal T(m) by varying the length of probes. We demonstrate using such a microarray that we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.

Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate.

Oscillations in patterns of expression of a large fraction of yeast genes are associated with the "metabolic cycle," usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in chemostats. Gene-expression data from synchronized cultures were used to predict coincident appearance of mRNAs from pairs of genes in the unsynchronized cells. Quantitative analysis of the FISH results shows that individual unsynchronized cells growing slowly because of glucose limitation or phosphate limitation show the predicted oscillations. We conclude that the yeast metabolic cycle is an intrinsic property of yeast metabolism and does not depend on either synchronization or external limitation of growth by the carbon source.

Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease.

The past two decades have witnessed an explosion in the identification, largely by positional cloning, of genes associated with mendelian diseases. The roughly 1,200 genes that have been characterized have clarified our understanding of the molecular basis of human genetic disease. The principles derived from these successes should be applied now to strategies aimed at finding the considerably more elusive genes that underlie complex disease phenotypes. The distribution of types of mutation in mendelian disease genes argues for serious consideration of the early application of a genomic-scale sequence-based approach to association studies and against complete reliance on a positional cloning approach based on a map of anonymous single nucleotide polymorphism haplotypes.

Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data.

Much of a cell's activity is organized as a network of interacting modules: sets of genes coregulated to respond to different conditions. We present a probabilistic method for identifying regulatory modules from gene expression data. Our procedure identifies modules of coregulated genes, their regulators and the conditions under which regulation occurs, generating testable hypotheses in the form 'regulator X regulates module Y under conditions W'. We applied the method to a Saccharomyces cerevisiae expression data set, showing its ability to identify functionally coherent modules and their correct regulators. We present microarray experiments supporting three novel predictions, suggesting regulatory roles for previously uncharacterized proteins.

Global analysis of the yeast knockout phenome.

Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae, enabled by its knockout collection, have produced the largest, richest, and most systematic phenotypic description of any organism. However, integrative analyses of this rich data source have been virtually impossible because of the lack of a central data repository and consistent metadata annotations. Here, we describe the aggregation, harmonization, and analysis of ~14,500 yeast knockout screens, which we call Yeast Phenome. Using this unique dataset, we characterized two unknown genes (YHR045W and YGL117W) and showed that tryptophan starvation is a by-product of many chemical treatments. Furthermore, we uncovered an exponential relationship between phenotypic similarity and intergenic distance, which suggests that gene positions in both yeast and human genomes are optimized for function.

Saccharomyces Genome Database provides mutant phenotype data.

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is a scientific database for the molecular biology and genetics of the yeast Saccharomyces cerevisiae, which is commonly known as baker's or budding yeast. The information in SGD includes functional annotations, mapping and sequence information, protein domains and structure, expression data, mutant phenotypes, physical and genetic interactions and the primary literature from which these data are derived. Here we describe how published phenotypes and genetic interaction data are annotated and displayed in SGD.

The cost of gene expression underlies a fitness trade-off in yeast.

Natural selection optimizes an organism's genotype within the context of its environment. Adaptations to one environment can decrease fitness in another, revealing evolutionary trade-offs. Here, we show that the cost of gene expression underlies a trade-off between growth rate and mating efficiency in the yeast Saccharomyces cerevisiae. During asexual growth, mutations that eliminate the ability to mate provide an approximately 2% per-generation growth-rate advantage. Some strains, including most laboratory strains, carry an allele of GPA1 (an upstream component of the mating pathway) that increases mating efficiency by approximately 30% per round of mating at the cost of an approximately 1% per-generation growth-rate disadvantage. In addition to demonstrating a trade-off between growth rate and mating efficiency, our results illustrate differences in the selective pressures defining fitness in the laboratory versus the natural environment and show that selection, acting on the cost of gene expression, can optimize expression levels and promote gene loss.

Novel insights from a multiomics dissection of the Hayflick limit.

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-ß2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.