Biology subjects

Gasch, A. P.

Publications and source records attributed to Gasch, A. P..

18 recordsLinked to original sources

bioRxiv2026

Domains of RNA-binding protein Ssd1 required for tolerating chromosome amplification in yeast

Many RNA-binding proteins play pleiotropic roles in the cell, often through disparate domains that can be decoupled. Here we undertook a mutational analysis of yeast RNA-binding protein Ssd1 to better understand domains and processes required for tolerating extra chromosomes, a special class of aneuploidy. Ssd1 is important for cellular fitness when cells carry extra chromosomes, for reasons that are not clear. Ssd1 has multiple distinct domains, many of which are poorly characterized, including multiple RNA binding folds, nuclear import and export signals, an intrinsically disordered domain, an RNA polymerase II-interacting region, and many phosphorylation sites. We measured multiple phenotypes, including the ability to tolerate an extra copy of yeast chromosome XII as a representative aneuploid. Mutating RNA binding domains disrupts fitness in aneuploid, but not euploid, cells and ablates binding of a representative Ssd1 target. However, mutations affecting phase separation or interaction with the kinase Cbk1, which regulates Ssd1 granule formation during the cell cycle, did not affect aneuploidy tolerance. Evaluating correlations across these and other growth phenotypes informed on functional relationships among domains. We discuss implications for Ssd1 function and its role in aneuploidy tolerance in yeast.

molecular biology↗

bioRxiv2026

Investigating the role of phosphodiesterase Pde2 in coordinating the yeast Environmental Stress Response

Yeast responding to acute stress reallocate cellular resources, in part via the Environmental Stress Response (ESR) that induces stress-defense genes while repressing ribosome-biogenesis and growth genes. The purpose and regulation of coordinated induction and repression is incompletely understood, but both responses are influenced by ESR transcription factors Msn2 and Msn4 (Msn2/4). Here we used single-cell microscopy and transcriptomic analysis to investigate the role of upstream regulator Pde2 in ESR regulation and post-stress fitness. Loss of PDE2 weakened and shortened Msn2 activation following salt stress and produced muted induction of Msn2/4 targets, similar to a msn2{triangleup}msn4{triangleup} strain. In contrast, Pde2 had at most a minor impact on ESR repressor Dot6, yet was important for repression of its targets beyond Msn2/4 influence. Consistent with our recent resource-reallocation model, pde2{triangleup} cells had normal or faster post-stress growth rates, despite weaker activation of the ESR. We discuss implications for ESR regulation and function.

genetics↗

bioRxiv2025

Inferring fungal cis-regulatory networks from genome sequences

Gene expression patterns are determined to a large extent by transcription factor binding to non-coding regulatory regions in the genome. However, gene expression cannot yet be systematically predicted from genome sequences, in part because non-functional matches to the sequence patterns (motifs) recognized by transcription factors (TFs) occur frequently throughout the genome. Large-scale functional genomics data for many TFs has enabled characterization of regulatory networks in experimentally accessible cells such as budding yeast. Beyond yeast, fungi are important industrial organisms and pathogens, but large-scale functional data is only sporadically available. Uncharacterized regulatory networks control key pathways and gene expression programs associated with fungal phenotypes. Here we explore a sequence-only approach to inferring regulatory networks by leveraging the 100s of genomes now available for many clades of fungi. We use gene orthology as the learning signal to infer interpretable, TF motif-based representations of non-coding regulatory regions. Using these representations to identify conserved signals for motifs, comparative genomics can be scaled to evolutionary comparisons where sequence similarity cannot be detected. We show that similarity of these conserved motif signals predicts gene expression and regulation better than using experimental data, and that we can infer known and novel regulatory connections in diverse fungi. Our new predictions include a pathway for recombination in C. albicans and pathways for mating and an RNAi immune response in Neurospora. Taken together, our results indicate that specific hypotheses about transcriptional regulation in fungi can be obtained for many genes from genome sequence analysis alone.

systems biology↗

bioRxiv2024

Regulated resource reallocation is transcriptionally hard wired into the yeast stress response

Many organisms maintain generalized stress responses activated by adverse conditions. Although details vary, a common theme is the redirection of transcriptional and translational capacity away from growth-promoting genes and toward defense genes. Yet the precise roles of these coupled programs are difficult to dissect. Here we investigated Saccharomyces cerevisiae responding to salt as a model stressor. We used molecular, genomic, and single-cell microfluidic methods to examine the interplay between transcription factors Msn2 and Msn4 that induce stress-defense genes and Dot6 and Tod6 that transiently repress growth-promoting genes during stress. Surprisingly, loss of Dot6/Tod6 led to slower acclimation to salt, whereas loss of Msn2/4 produced faster growth during stress. This supports a model where transient repression of growth-promoting genes accelerates the Msn2/4 response, which is essential for acquisition of subsequent peroxide tolerance. Remarkably, we find that Msn2/4 regulate DOT6 mRNA production, influence Dot6 activation dynamics, and are required for full repression of growth-promoting genes. Thus, Msn2/4 directly regulate resource reallocation needed to mount their own response. We discuss broader implications for common stress responses across organisms. SYNOPSISThis study investigates how genes induced and repressed in the yeast Environmental Stress Response contribute to stress tolerance, growth rate, and resource allocation. The work uses molecular, genomic, and systems biology approaches to present new insights into eukaryotic responses to acute stress. HIGHLIGHTSO_LICells lacking stress-activated transcription factors have a faster post-stress growth rate C_LIO_LICells lacking repressors of growth-promoting genes have a slower post-stress growth rate C_LIO_LIStress-defense factors control the induction of growth-promoting gene repressors, thereby coordinating the resource re-allocation needed for the response C_LI

systems biology↗

bioRxiv2024

On the rate of aneuploidy reversion in a wild yeast model

Aneuploidy, arising from gain or loss of chromosomes due to nondisjunction, is a special class of mutation. It can create significant phenotypic changes by altering abundance of hundreds of genes in a single event, providing material for adaptive evolution. But it can also incur large fitness costs relative to other types of mutations. Understanding mutational dynamics of aneuploidy is important for modeling its impact in nature, but aneuploidy rates are difficult to measure accurately. One challenge is that aneuploid karyotypes may revert back to euploidy, biasing forward mutation rate estimates - yet the rate of aneuploidy reversion is largely uncharacterized. Furthermore, current rate estimates are confounded because fitness differences between euploids and aneuploids are typically not accounted for in rate calculations. We developed a unique fluctuation assay in a wild-yeast model to measure the rate of extra-chromosome loss across three aneuploid chromosomes, while accounting for fitness differences between aneuploid and euploid cells. We show that incorporating fitness effects is essential to obtain accurate estimates of aneuploidy rates. Furthermore, the rate of extra-chromosome loss, separate from karyotype fitness differences, varies across chromosomes. We also measured rates in a strain lacking RNA-binding protein Ssd1, important for aneuploidy tolerance and implicated in chromosome segregation. We found no role for Ssd1 in the loss of native aneuploid chromosomes, although it did impact an engineered chromosome XV with a perturbed centromeric sequence. We discuss the impacts and challenges of modeling aneuploidy dynamics in real world situations. ARTICLE SUMMARYErrors in chromosome segregation can produce aneuploid cells with an abnormal number of chromosomes. Aneuploidy is not uncommon in wild populations of fungi and can underlie emergence of drug-resistant pathogens. But modeling the impact of aneuploidy on evolution has been challenging, because rates of aneuploidy emergence and reversion have been difficult to measure. This work used a novel fluctuation assay that incorporates euploid-aneuploid fitness differences to calculate rates of extra-chromosome loss in aneuploid Saccharomyces cerevisiae, across several chromosomes. The results present for the first time estimates of aneuploidy reversion ("back mutation") rates and implications for previously measured rates of aneuploidy.

genetics↗

bioRxiv2024

Investigating the role of RNA-binding protein Ssd1 in aneuploidy tolerance through network analysis

RNA-binding proteins (RBPs) play critical cellular roles by mediating various stages of RNA life cycles. Ssd1, an RBP with pleiotropic effects, has been implicated in aneuploidy tolerance in Saccharomyces cerevisiae but its mechanistic role remains unclear. Here we used a network-based approach to inform on Ssd1s role in aneuploidy tolerance, by identifying and experimentally perturbing a network of RBPs that share mRNA targets with Ssd1. We identified RBPs whose bound mRNA targets significantly overlap with Ssd1 targets. For 14 identified RBPs, we then used a genetic approach to generate all combinations of genotypes for euploid and aneuploid yeast with an extra copy of chromosome XII, with and without SSD1 and/or the RBP of interest. Deletion of 10 RBPs either exacerbated or alleviated the sensitivity of wild-type and/or ssd1{Delta} cells to chromosome XII duplication, in several cases indicating genetic interactions with SSD1 in the context of aneuploidy. We integrated these findings with results from a global over-expression screen that identified genes whose duplication complements ssd1{Delta} aneuploid sensitivity. The resulting network points to a sub-group of proteins with shared roles in translational repression and p-body formation, implicating these functions in aneuploidy tolerance. Our results reveal a role for new RBPs in aneuploidy tolerance and support a model in which Ssd1 mitigates translation-related stresses in aneuploid cells.

genetics↗

bioRxiv2024

Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.

molecular biology↗

bioRxiv2022

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phosphor-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1{Delta} strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1{Delta} parental strain. Genetic mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes underscored a role for lipid homeostasis, which was further supported by evolved changes in lipid profiles and gene expression. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization. Author SummaryAll organisms utilize an energy source to generate the cellular resources needed to grow and divide. These individual processes have been well study, but the coordination and crosstalk between the process is not well understood. To study growth and metabolism coupling, we used a yeast strain that was genetically engineered to ferment the sugar xylose but lacked growth on the sugar. The decoupled growth and metabolism was caused by a single gene deletion in a highly conserved signaling pathway found in all eukaryotes. While our work is focused on xylose metabolism, we address the fundamental question of how cells coordinate growth with metabolism under non-ideal conditions. We identified vast changes in gene expression that implicated altered regulatory mechanisms involved in lipid metabolism correlating with decouple growth and metabolism. Our work highlights the complexity of engineering new cellular functions and that global regulatory modifications, rather than altering individual pathways, may be required for broad cellular changes.

genetics↗

bioRxiv2021

Integrating multiple single-cell phenotypes links stress acclimation to prior life history in yeast

Stress defense and cell growth are inversely related in bulk culture analyses; however, these studies miss substantial cell-to-cell heterogeneity, thus obscuring true phenotypic relationships. Here, we devised a microfluidics system to characterize multiple phenotypes in single yeast cells over time before, during, and after salt stress. The system measured cell and colony size, growth rate, and cell-cycle phase along with nuclear trans-localization of two transcription factors: stress-activated Msn2 that regulates defense genes and Dot6 that represses ribosome biogenesis genes during an active stress response. By tracking cells dynamically, we discovered unexpected discordance between Msn2 and Dot6 behavior that revealed subpopulations of cells with distinct growth properties. Surprisingly, post-stress growth recovery was positively corelated with activation of the Dot6 repressor. In contrast, cells lacking Dot6 displayed slower growth acclimation, even though they grow normally in the absence of stress. We show that wild-type cells with a larger Dot6 response display faster production of Msn2-regulated Ctt1 protein, separable from the contribution of Msn2. These results are consistent with the model that transcriptional repression during acute stress in yeast provides a protective response, likely by redirecting translational capacity to induced transcripts.

molecular biology↗

bioRxiv2021

Natural variation in the consequences of gene overexpression and its implications for evolutionary trajectories

Copy number variation (CNV) through gene or chromosome amplification provides a route for rapid phenotypic variation and supports long-term evolution of gene functions. Although the evolutionary importance of CNV is known, little is understood about how genetic background influences CNV tolerance. Here, we measured fitness costs of over 4,000 over-expressed genes in 15 Saccharomyces cerevisiae strains representing different lineages, to explore natural variation in tolerating gene overexpression (OE). Strain-specific effects dominated the fitness costs of gene OE. We report global differences in the consequences of gene OE, independent of the amplified gene, as well as gene-specific effects that were dependent on the genetic background. Natural variation in the response to gene OE could be explained by several models, including strain-specific physiological differences, resource limitations, and regulatory sensitivities. This work provides new insight on how genetic background influences tolerance to gene amplification and the evolutionary trajectories accessible to different backgrounds.

genomics↗

bioRxiv2020

CRISpy-pop: a web tool for designing CRISPR/Cas9-driven genetic modifications in diverse populations

CRISPR/Cas9 is a powerful tool for editing genomes, but design decisions are generally made with respect to a single reference genome. With population genomic data becoming available for an increasing number of model organisms, researchers are interested in manipulating multiple strains and lines. CRISpy-pop is a web application that generates and filters guide RNA sequences for CRISPR/Cas9 genome editing for diverse yeast and bacterial strains. The current implementation designs and predicts the activity of guide RNAs against more than 1000 Saccharomyces cerevisiae genomes, including 167 strains frequently used in bioenergy research. Zymomonas mobilis, an increasingly popular bacterial bioenergy research model, is also supported. CRISpy-pop is available as a web application (https://CRISpy-pop.glbrc.org/) with an intuitive graphical user interface. CRISpy-pop also cross-references the human genome to allow users to avoid the selection of sgRNAs with potential biosafety concerns. Additionally, CRISpy-pop predicts the strain coverage of each guide RNA within the supported strain sets, which aids in functional population genetic studies. Finally, we validate how CRISpy-pop can accurately predict the activity of guide RNAs across strains using population genomic data.

genetics↗

bioRxiv2019

PKA and HOG signaling contribute separable roles to anaerobic xylose fermentation in yeast engineered for biofuel production

Lignocellulosic biomass offers a sustainable source for biofuel production that does not compete with food-based cropping systems. Importantly, two critical bottlenecks prevent economic adoption: many industrially relevant microorganisms cannot ferment pentose sugars prevalent in lignocellulosic medium, leaving a significant amount of carbon unutilized. Furthermore, chemical biomass pretreatment required to release fermentable sugars generates a variety of toxins, which inhibit microbial growth and metabolism, specifically limiting pentose utilization in engineered strains. Here we dissected genetic determinants of anaerobic xylose fermentation and stress tolerance in chemically pretreated corn stover biomass, called hydrolysate. We previously revealed that loss-of-function mutations in the stress-responsive MAP kinase HOG1 and negative regulator of the RAS/Protein Kinase A (PKA) pathway, IRA2, enhances anaerobic xylose fermentation. However, these mutations likely reduce cells ability to tolerate the toxins present in lignocellulosic hydrolysate, making the strain especially vulnerable to it. We tested the contributions of Hog1 and PKA signaling via IRA2 or PKA negative regulatory subunit BCY1 to metabolism, growth, and stress tolerance in corn stover hydrolysate and laboratory medium with mixed sugars. We found mutations causing upregulated PKA activity increase growth rate and glucose consumption in various media but do not have a specific impact on xylose fermentation. In contrast, mutation of HOG1 specifically increased xylose usage. We hypothesized improving stress tolerance would enhance the rate of xylose consumption in hydrolysate. Surprisingly, increasing stress tolerance did not augment xylose fermentation in lignocellulosic medium in this strain background, suggesting other mechanisms besides cellular stress limit this strains ability for anaerobic xylose fermentation in hydrolysate.

genetics↗

bioRxiv2018

Independent mechanisms for acquired salt tolerance versus growth resumption induced by mild ethanol pretreatment in Saccharomyces cerevisiae

All living organisms must recognize and respond to various environmental stresses throughout their lifetime. In natural environments, cells frequently encounter fluctuating concentrations of different stressors that can occur in combination or sequentially. Thus, the ability to anticipate an impending stress is likely ecologically relevant. One possible mechanism for anticipating future stress is acquired stress resistance, where cells pre-exposed to a mild sub-lethal dose of stress gain the ability to survive an otherwise lethal dose of stress. We have been leveraging wild strains of Saccharomyces cerevisiae to investigate natural variation in the yeast ethanol stress response and its role in acquired stress resistance. Here, we report that a wild vineyard isolate possesses ethanol-induced cross-protection against severe concentrations of salt. Because this phenotype correlates with ethanol-dependent induction of the ENA genes, which encode sodium efflux pumps already associated with salt resistance, we hypothesized that variation in ENA expression was responsible for differences in acquired salt tolerance across strains. Surprisingly, we found that the ENA genes were completely dispensable for ethanol-induced survival of high salt concentrations in the wild vineyard strain. Instead, the ENA genes were necessary for the ability to resume growth on high concentrations of salt following a mild ethanol pretreatment. Surprisingly, this growth acclimation phenotype was also shared by the lab yeast strain despite lack of ENA induction under this condition. This study underscores that cross protection can affect both viability and growth through distinct mechanisms, both of which likely confer fitness effects that are ecologically relevant.\n\nIMPORTANCEMicrobes in nature frequently experience \"boom or bust\" cycles of environmental stress. Thus, microbes that can anticipate the onset of stress would have an advantage. One way microbes anticipate future stress is through acquired stress resistance, where cells exposed to a mild dose of one stress gain the ability survive an otherwise lethal dose of a subsequent stress. In the budding yeast Saccharomyces cerevisiae, certain stressors can cross protect against high salt concentrations, though the mechanisms governing this acquisition of higher stress resistance are not well understood. In this study, we took advantage of wild yeast strains to understand the mechanism underlying ethanol-induced cross protection against high salt concentrations. We found that mild ethanol stress allows cells to resume growth on high salt, which involves a novel role for a well-studied salt transporter. Overall, this discovery highlights how leveraging natural variation can provide new insights into well-studied stress defense mechanisms.

microbiology↗

bioRxiv2018

Genotype-by-environment-by-environment interactions in the Saccharomyces cerevisiae transcriptomic response to alcohols and anaerobiosis

Next generation biofuels including longer-chain alcohols such as butanol are attractive as renewable, high-energy fuels. A barrier to microbial production of butanols is the increased toxicity compared to ethanol; however, the cellular targets and microbial defense mechanisms remain poorly understood, especially under anaerobic conditions used frequently in industry. Here we took a comparative approach to understand the response of Saccharomyces cerevisiae to 1-butanol, isobutanol, or ethanol, across three genetic backgrounds of varying tolerance in aerobic and anaerobic conditions. We find that strains have different growth properties and alcohol tolerances with and without oxygen availability, as well as unique and common responses to each of the three alcohols. Our results provide evidence for strain-by-alcohol-by-oxygen interactions that moderate how cells respond to alcohol stress.\n\nARTICLE SUMMARYEfforts to promote sustainable bioenergy focus on microbial production of biofuels including butanols, which can be blended into gasoline engines and condensed for higher energy fuels. The stress of these end products can limit microbial production; yet it remains unclear how higher-energy butanols impact cell physiology compared to the well studied ethanol. This study compares the transcriptomic response to 1-butaonol, isobutanol, and ethanol in three strains of Saccharomyces cerevisiae from diverse natural populations, with and without oxygen. Results show that oxygen availability and strain background significantly impact the response to each alcohol and point to shared responses to alcohol stress.

genomics↗

bioRxiv2017

Genome-wide association across Saccharomyces cerevisiae strains reveals substantial variation in underlying gene requirements for toxin tolerance.

Cellulosic plant biomass is a promising sustainable resource for generating alternative biofuels and biochemicals with microbial factories. But a remaining bottleneck is engineering microbes that are tolerant of toxins generated during biomass processing, because mechanisms of toxin defense are only beginning to emerge. Here, we exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no significant effect in another strain. In fact, whether or not the gene was involved in tolerance in each strain background had a bigger contribution to strain-specific variation than allelic differences. Our results suggest a major difference in the underlying network of causal genes in different strains, suggesting that mechanisms of hydrolysate tolerance are very dependent on the genetic background. These results could have significant implications for interpreting GWA results and raise important considerations for engineering strategies for industrial strain improvement.\n\nAuthor summaryUnderstanding the genetic architecture of complex traits is important for elucidating the genotype-phenotype relationship. Many studies have sought genetic variants that underlie phenotypic variation across individuals, both to implicate causal variants and to inform on architecture. Here we used genome-wide association analysis to identify genes and processes involved in tolerance of toxins found in plant-biomass hydrolysate, an important substrate for sustainable biofuel production. We found substantial variation in whether or not individual genes were important for tolerance across genetic backgrounds. Whether or not a gene was important in a given strain background explained more variation than the alleleic differences in the gene. These results suggest substantial variation in gene contributions, and perhaps underlying mechanisms, of toxin tolerance.

genomics↗

bioRxiv2017

Single-cell RNA-seq reveals intrinsic and extrinsic regulatory heterogeneity in yeast responding to stress

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single S. cerevisiae cells treated with and without salt stress, to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells decouple expression of ribosomal protein genes from the environmental stress response, in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results reveal that cells maintain a cellular filter against decoupled bursts of transcription-factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.

systems biology↗

bioRxiv2017

Network inference reveals novel connections in pathways regulating growth and defense in the yeast salt response

Cells respond to stressful conditions by coordinating a complex, multi-faceted response that spans many levels of physiology. Much of the response is coordinated by changes in protein phosphorylation. Although the regulators of transcriptome changes during stress are well characterized in Saccharomyces cerevisiae, the upstream regulatory network controlling protein phosphorylation is less well dissected. Here, we developed a computational approach to infer the signaling network that regulates phosphorylation changes in response to salt stress. The method uses integer linear programming (ILP) to integrate stress-responsive phospho-proteome responses in wild-type and mutant strains, predicted phosphorylation motifs on groups of coregulated peptides, and published protein interaction data. A key advance is that by grouping peptides into submodules before inference, the method can overcome missing protein interactions in published datasets to predict novel, stress-dependent protein interactions and phosphorylation events. The network we inferred predicted new regulatory connections between stress-activated and growth-regulating pathways and suggested mechanisms coordinating metabolism, cell-cycle progression, and growth during stress. We confirmed several network predictions with co-immunoprecipitations coupled with mass-spectrometry protein identification and mutant phospho-proteomic analysis. Results show that the cAMP-phosphodiesterase Pde2 physically interacts with many stress-regulated transcription factors targeted by PKA, and that reduced phosphorylation of those factors during stress requires the Rck2 kinase that we show physically interacts with Pde2. Together, our work shows how a high-quality computational network model can facilitate discovery of new pathway interactions during osmotic stress.

systems biology↗

bioRxiv2016

SCnorm: A quantile-regression based approach for robust normalization of single-cell RNA-seq data

Normalization of RNA-sequencing data is essential for accurate downstream inference, but the assumptions upon which most methods are based do not hold in the single-cell setting. Consequently, applying existing normalization methods to single-cell RNA-seq data introduces artifacts that bias downstream analyses. To address this, we introduce SCnorm for accurate and efficient normalization of scRNA-seq data.

genomics↗