Sasha F. Levy

Network hubs buffer environmental variation in Saccharomyces cerevisiae.

Regulatory and developmental systems produce phenotypes that are robust to environmental and genetic variation. A gene product that normally contributes to this robustness is termed a phenotypic capacitor. When a phenotypic capacitor fails, for example when challenged by a harsh environment or mutation, the system becomes less robust and thus produces greater phenotypic variation. A functional phenotypic capacitor provides a mechanism by which hidden polymorphism can accumulate, whereas its failure provides a mechanism by which evolutionary change might be promoted. The primary example to date of a phenotypic capacitor is Hsp90, a molecular chaperone that targets a large set of signal transduction proteins. In both Drosophila and Arabidopsis, compromised Hsp90 function results in pleiotropic phenotypic effects dependent on the underlying genotype. For some traits, Hsp90 also appears to buffer stochastic variation, yet the relationship between environmental and genetic buffering remains an important unresolved question. We previously used simulations of knockout mutations in transcriptional networks to predict that many gene products would act as phenotypic capacitors. To test this prediction, we use high-throughput morphological phenotyping of individual yeast cells from single-gene deletion strains to identify gene products that buffer environmental variation in Saccharomyces cerevisiae. We find more than 300 gene products that, when absent, increase morphological variation. Overrepresented among these capacitors are gene products that control chromosome organization and DNA integrity, RNA elongation, protein modification, cell cycle, and response to stimuli such as stress. Capacitors have a high number of synthetic-lethal interactions but knockouts of these genes do not tend to cause severe decreases in growth rate. Each capacitor can be classified based on whether or not it is encoded by a gene with a paralog in the genome. Capacitors with a duplicate are highly connected in the protein–protein interaction network and show considerable divergence in expression from their paralogs. In contrast, capacitors encoded by singleton genes are part of highly interconnected protein clusters whose other members also tend to affect phenotypic variability or fitness. These results suggest that buffering and release of variation is a widespread phenomenon that is caused by incomplete functional redundancy at multiple levels in the genetic architecture.

Bet Hedging in Yeast by Heterogeneous, Age-Correlated Expression of a Stress Protectant

A new experimental approach reveals a bet-hedging strategy in unstressed, clonal yeast cells, whereby they adopt a range of growth states that correlate with expression of a trehalose-synthesis regulator and predict resistance to future stress.

Quantitative evolutionary dynamics using high-resolution lineage tracking

Evolution of large asexual cell populations underlies ∼30% of deaths worldwide, including those caused by bacteria, fungi, parasites, and cancer. However, the dynamics underlying these evolutionary processes remain poorly understood because they involve many competing beneficial lineages, most of which never rise above extremely low frequencies in the population. To observe these normally hidden evolutionary dynamics, we constructed a sequencing-based ultra high-resolution lineage tracking system in Saccharomyces cerevisiae that allowed us to monitor the relative frequencies of ∼500,000 lineages simultaneously. In contrast to some expectations, we found that the spectrum of fitness effects of beneficial mutations is neither exponential nor monotonic. Early adaptation is a predictable consequence of this spectrum and is strikingly reproducible, but the initial small-effect mutations are soon outcompeted by rarer large-effect mutations that result in variability between replicates. These results suggest that early evolutionary dynamics may be deterministic for a period of time before stochastic effects become important.

Three- and Four-repeat Tau Regulate the Dynamic Instability of Two Distinct Microtubule Subpopulations in Qualitatively Different Manners IMPLICATIONS FOR NEURODEGENERATION

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wild-type ∼50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a “microtubule misregulation” model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.

Development of a Comprehensive Genotype-to-Fitness Map of Adaptation-Driving Mutations in Yeast

Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene.

Histone variant HTZ1 shows extensive epistasis with, but does not increase robustness to, new mutations.

Biological systems produce phenotypes that appear to be robust to perturbation by mutations and environmental variation. Prior studies identified genes that, when impaired, reveal previously cryptic genetic variation. This result is typically interpreted as evidence that the disrupted gene normally increases robustness to mutations, as such robustness would allow cryptic variants to accumulate. However, revelation of cryptic genetic variation is not necessarily evidence that a mutationally robust state has been made less robust. Demonstrating a difference in robustness requires comparing the ability of each state (with the gene perturbed or intact) to suppress the effects of new mutations. Previous studies used strains in which the existing genetic variation had been filtered by selection. Here, we use mutation accumulation (MA) lines that have experienced minimal selection, to test the ability of histone H2A.Z (HTZ1) to increase robustness to mutations in the yeast Saccharomyces cerevisiae. HTZ1, a regulator of chromatin structure and gene expression, represents a class of genes implicated in mutational robustness. It had previously been shown to increase robustness of yeast cell morphology to fluctuations in the external or internal microenvironment. We measured morphological variation within and among 79 MA lines with and without HTZ1. Analysis of within-line variation confirms that HTZ1 increases microenvironmental robustness. Analysis of between-line variation shows the morphological effects of eliminating HTZ1 to be highly dependent on the line, which implies that HTZ1 interacts with mutations that have accumulated in the lines. However, lines without HTZ1 are, as a group, not more phenotypically diverse than lines with HTZ1 present. The presence of HTZ1, therefore, does not confer greater robustness to mutations than its absence. Our results provide experimental evidence that revelation of cryptic genetic variation cannot be assumed to be caused by loss of robustness, and therefore force reevaluation of prior claims based on that assumption.

FTDP-17 mutations in Tau alter the regulation of microtubule dynamics: an "alternative core" model for normal and pathological Tau action.

Mutations affecting either the structure or regulation of the microtubule-associated protein Tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among the most characterized activities of Tau is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here we have tested the hypothesis that Tau mutations causing neurodegeneration also alter the ability of Tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions, and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or interrepeat regions of Tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of Tau regulatory action based on an “alternative core” of microtubule binding and regulatory activities composed of two repeats and the interrepeat between them. In this model, the interrepeat serves as the primary regulator of microtubule dynamics, whereas the flanking repeats serve as tethers to properly position the interrepeat on the microtubule. Importantly, since there are multiple interrepeats on each Tau molecule, there are also multiple cores on each Tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for Tau-mediated neuronal cell death and provide a novel mechanistic model for normal and pathological Tau action.

The robustness continuum.

Organisms are subject to random changes in their external environments, as well as in their internal components. A central goal of evolutionary systems biology is to understand how living systems cope with-and in some cases exploit-this variation. Many cellular and developmental processes operate with high fidelity to produce stereotyped, irreversible outcomes despite environmental and genetic perturbation. These processes are said to be robust or insensitive to variation. Robustness can lead to single, invariant phenotypes, or it can take the form of phenotypic plasticity, in which different environmental conditions reproducibly induce distinct phenotypes. Some organisms cope with environmental variation not with robust responses but with stochastic, reversible fate decisions. In those organisms, lower robustness yields heterogeneity among individuals, which in turn serves as a bet-hedging mechanism for the population. Considering high-fidelity and bet-hedging processes together-as a robustness continuum-provides a unifying framework for analyzing and conceptualizing variation in complex evolving systems. This framework can be applied to understanding the architectures and dynamics of the regulatory networks that underlie fate decisions in microbes, plants, animals, and cancer cells.

Beyond genome sequencing: Lineage tracking with barcodes to study the dynamics of evolution, infection, and cancer

Evolving cellular communities, such as the gut microbiome, pathogenic infections, and cancer, consist of large populations of ~10(7)-10(14) cells. Because of their large population sizes, adaptation within these populations can be driven by many beneficial mutations that never rise above extremely low frequencies. Genome sequencing methods such as clonal, single cell, or whole population sequencing are poorly suited to detect these rare beneficial lineages, and, more generally, to characterize which mutations are most important to the population dynamics. Here, we introduce an alternative approach: high-resolution lineage tracking with DNA barcodes. In contrast to whole genome sequencing, lineage tracking can detect a beneficial mutation at an extremely low frequency within the population, and estimate its time of occurrence and fitness effect. Many lineage trajectories can be observed in parallel, allowing one to observe the population dynamics in exquisite detail. We describe some of the technical and analytical challenges to lineage tracking with DNA barcodes and discuss its applications to studies of evolution, infectious disease and cancer.

A method for high‐throughput production of sequence‐verified DNA libraries and strain collections

The low costs of array‐synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost‐effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site‐specific recombination to index library DNA, and next‐generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost‐effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.