Biology subjects

Nislow, C.

Publications and source records attributed to Nislow, C..

12 recordsLinked to original sources

bioRxiv2026

Defining the spectrum of cellular protection against cadmium stress by Dsup expressed in yeast

The intrinsically disordered tardigrade Dsup protein has garnered attention for its ability to protect cells and organisms from radiation and oxidative stress. Our lab has previously described Dsups protective effect in yeast, finding that, while Dsup expression comes with a fitness cost, it improves survival under many stressors, including the heavy metal cadmium. While Dsups protection against oxidative stress and radiation is well-established, the mechanism by which it protects against other stressors is underinvestigated. To address this knowledge gap, we screened a collection of 4000 yeast deletion mutants expressing Dsup in the presence of three doses of cadmium to identify synergistic/synthetically lethal genes and processes. We find that Dsup broadly buffers many processes/genes at lower levels of cadmium stress but at higher doses, cells shift to buffering specific genes/processes. These results identify host genetic processes that influence Dsups ability to confer cadmium resistance to organisms.

genomics↗

bioRxiv2025

Friend and Foe: Genome-Wide Analysis of the Tardigrade Dsup Protein Expressed in Yeast Reveals Trade-offs of DNA Protection

Tardigrades are among the most resilient eukaryotes known, capable of surviving a wide range of extreme conditions. While their genomes contain thousands of tardigrade-specific genes, one key contributor to their stress resistance is the Damage suppressor (Dsup) protein. Previous work showed that expression of Dsup in cancer cells reduced DNA damage caused by X-rays. This observation has sparked growing interest in Dsup across diverse model organisms. Most studies to date have focused on phenotypic outcomes, while the mechanisms and consequences of Dsup remain poorly understood. Deciphering its mechanism is crucial, especially because Dsup expression is deleterious to some cells and organisms. In this work, we show that Dsup protects yeast against a variety of DNA-damaging agents. However, this protection is not universal, and it has a fitness cost. Analysis of nucleosome occupancy of cells expressing Dsup shows that effects on chromatin are modest, without global remodeling, and transcriptome analysis reveals a robust induction of oxidative stress pathways, which might be a form of preconditioning for oxidative stress. Genome-wide screens of [~]4300 loss-of-function mutants expressing Dsup shows that deletions incompatible with Dsup are highly enriched for DNA repair processes and that under DNA damage stress, Dsup can protect some mutants in a pathway-specific manner. Our findings demonstrate that while Dsup broadly enhances resistance to genotoxic stress, it also imposes physiological costs and generates new dependencies on DNA repair and genome maintenance pathways.

genomics↗

bioRxiv2025

A platform for the systematic interrogation of genetic interactions in human cells: 26 gene by genome-wide double knockout screens as a proof of concept

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term "gene by genome-wide" knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca).

genetics↗

bioRxiv2024

Mining Yeast Diversity Unveils Novel Targets for Improved Heterologous Laccase Production in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a widely utilized host cell for recombinant protein production due to its well studied and annotated genome, its ability to secrete large and post-translationally modified proteins, fast growth and cost-effective culturing. However, recombinant protein yields from S. cerevisiae often fall behind that of other host systems. To address this, we developed a high throughput screen of wild, industrial and laboratory S. cerevisiae isolates to identify strains with a natural propensity for greater recombinant protein production, specifically focussing on laccase multicopper oxidases from the fungi Trametes trogii and Myceliophthora thermophila. Using this method, we identified 20 non-laboratory strains with higher capacity to produce active laccase. Interestingly, lower levels of laccase mRNA were measured in most cases, indicating that the drivers of elevated protein production capacity lie beyond the regulation of recombinant gene expression. We characterized the identified strains using complementary genomic and proteomic approaches to reveal several potential pathways driving the improved expression phenotype. Gene ontology analysis suggests broad changes in cellular metabolism, specifically in genes/proteins involved in carbohydrate catabolism, thiamine biosynthesis, transmembrane transport and vacuolar degradation. Targeted deletions of the hexose transporter HXT11 and the Coat protein complex II interacting paralogs PRM8 and 9, involved in ER to Golgi transport, resulted in significantly improved laccase production from the S288C laboratory strain. Whereas the deletion of the Hsp110 SSE1 gene, guided by our proteomic analysis, also led to higher laccase activity, we did not observe major changes of the protein homeostasis network within the strains with higher laccase activity. This study opens new avenues to leverage the vast diversity of Saccharomyces cerevisiae for recombinant protein production, as well as offers new strategies and insights to enhance recombinant protein yields of current strains.

molecular biology↗

bioRxiv2023

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase, along with PMT1 or PMT2, two ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of Scj1p had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. ImportanceOur study showcases a newly developed high throughput screening technique to identify yeast mutant strains that exhibit an enhanced capacity for recombinant protein production. Using a genome-wide approach, we show that vesicle trafficking plays a crucial role in protein production, as the genes associated with this process are notably enriched in our screen. Furthermore, we demonstrate that a specific set of gene deletions, which were not previously recognized for their impact on recombinant laccase production, can be effectively manipulated in combination to increase the production of heterologous proteins. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

microbiology↗

bioRxiv2022

Persistent acetylation of newly synthesized histones inhibits DNA replication origin activity

In Saccharomyces cerevisiae, newly synthesized histone H3 are acetylated on lysine 56 (H3 K56ac) by the Rtt109 acetyltransferase prior to their deposition on nascent DNA behind replication forks. Two deacetylases of the sirtuin family, Hst3 and Hst4, remove H3 K56ac from chromatin following S phase. hst3{Delta} hst4{Delta} cells present constitutive H3 K56ac, which sensitizes cells to replicative stress via mechanisms that remain unclear. We performed a screen to identify genes that influence cell fitness upon nicotinamide (NAM)-induced inhibition of sirtuins. The screen revealed that DBF4 heterozygosity causes NAM sensitivity. DBF4 and CDC7 encode subunits of the Dbf4-dependent kinase, which activates origins of DNA replication. We show that i) cells harboring the dbf4-1 or cdc7-4 hypomorphic alleles are sensitive to NAM, ii) Rif1, an inhibitor of Cdc7-dependent activation of origins, causes DNA damage and replication defects in NAM-treated cells and hst3{Delta} hst4{Delta} mutants, and iii) cdc7-4 hst3{Delta} hst4{Delta} cells display synthetic temperature sensitivity associated with delayed initiation of DNA replication. Such replication defects are not due to activation of the intra-S phase checkpoint but require Rtt109-dependent H3 K56ac. Overall, these results suggest that persistent H3 K56ac sensitizes cells to replicative stress in part by negatively influencing replication origin activity.

molecular biology↗

bioRxiv2021

Combining chemogenomic and gene-dose assays to investigate drug synergy

From the earliest days of using natural remedies to modern applications of clinically tested medications, combining therapies for disease treatment has been standard practice. Combination treatments can exhibit synergistic effects, broadly defined as a greater-than-additive effect of two or more therapeutic agents. Indeed, clinicians often use their experience and expertise to tailor such combinations in the hopes of maximizing the therapeutic effect. Alongside these efforts, computational studies into understanding and predicting the biophysical underpinnings of how synergy is achieved have benefitted from high-throughput screening and computational biology. One challenge is how to best design and analyze the results of synergy studies performed at scale, especially because the number of possible combinations to test quickly becomes unmanageable, and the tools to analyze the resulting data are quite new. Nevertheless, the benefits of such studies are clear -- by combining multiple drugs in the treatment of infectious disease and cancer, for instance, one can lessen host toxicity and simultaneously reduce the likelihood of resistance to treatment. In this study, we extend the widely validated chemogenomic HIPHOP assay to drug combinations. We identify a class of "combination-specific sensitive strains" that suggest mechanisms for the synergies we observe and further suggest focused follow-up studies.

genomics↗

bioRxiv2020

Beta-cell specific insulin resistance promotes glucose-stimulated insulin hypersecretion

Abstract Insulin receptor (Insr) protein can be found at higher levels in pancreatic {beta}-cells than in most other tissues, but the consequences of {beta}-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in {beta}-cells of both female and male mice. Experimental mice were compared to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined {beta}-cells revealed transcriptomic consequences of Insr loss, which differed between female and male mice. Action potential and calcium oscillation frequencies were increased in Insr knockout {beta}- cells from female, but not male mice, whereas only male {beta}InsrKO mice had reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female {beta}InsrKO and {beta}InsrHET mice exhibited elevated insulin release in perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr did not alter {beta}-cell area up to 9 months of age, nor did it impair hyperglycemia-induced proliferation. Based on our data, we adapted a mathematical model to include {beta}-cell insulin resistance, which predicted that {beta}-cell Insr knockout would improve glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance was significantly improved in female {beta}InsrKO and {beta}InsrHET mice when compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We did not observe improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of {beta}-cell specific insulin resistance. The propensity for hyperinsulinemia was associated with mildly reduced fasting glucose and increased body weight. We further validated our main in vivo findings using the Ins1-CreERT transgenic line and found that male mice had improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that loss of {beta}-cell Insr contributes to glucose-induced hyperinsulinemia, thereby improving glucose homeostasis in otherwise insulin sensitive dietary and age contexts.

physiology↗

bioRxiv2019

Broad-Spectrum Antifungal Activities and Mechanism of Drimane Sesquiterpenoids

Eight drimane sesquiterpenoids including (-)-drimenol and (+)-albicanol were synthesized from (+)-sclareolide and evaluated for their antifungal activities. Three compounds, (-)-drimenol, (+)-albicanol, and (1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyl-decahydronaphthalene-1-carbaldehyde (4) showed strong activity against C. albicans. (-)-Drimenol, the strongest inhibitor of the three, (at concentrations of 8 - 64 g/ml, causing 100% death of fungi), acts not only against C. albicans as a fungicidal manner, but also inhibits other fungi such as Aspergillus, Cryptococcus, Pneumocystis, Blastomyces, Fusarium, Rhizopus, Saksenaea and FLU resistant strains of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. auris. These observations suggest drimenol is a broad-spectrum antifungal agent. At high concentration (100 g/ml), drimenol caused a rupture of the fungal cell wall/membrane. In a nematode model of C. albicans infection, drimenol rescued the worms from C. albicans-mediated death, indicating drimenol is tolerable and bioactive in a metazoan. Genome-wide fitness profiling assays of both S. cerevisiae (nonessential homozygous and essential heterozygous) and C. albicans (Tn-insertion mutants) collections revealed putative genes and pathways affected by drimenol. Using a C. albicans mutants spot assay, the Crk1 kinase associated gene products, Ret2, Cdc37, and novel putative targets orf19.759, orf19.1672, and orf19.4382 were revealed to be the potential targets of drimenol. Further, computational modeling results suggest possible modification of the structure of drimenol including the A ring for improving antifungal activity.

microbiology↗

bioRxiv2019

A competence-regulated toxin-antitoxin system in Haemophilus influenzae

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ( toxTA), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy-and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.\n\nABBREVIATED SUMMARYThe competence regulon of Haemophilus influenzae includes an unprecedented toxin/antitoxin gene pair. When not opposed by antitoxin, the toxin completely prevents DNA uptake but causes only very minor decreases in cell growth and competence gene expression. The TA gene pair was acquired by horizontal gene transfer, and the toxin gene has undergone repeated deletions in other strains.

microbiology↗

bioRxiv2019

The Paf1 complex broadly impacts the transcriptome of Saccharomyces cerevisiae

The Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate the Saccharomyces cerevisiae transcriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here we took advantage of a genetic background that enriches for unstable transcripts and demonstrate that deletion of PAF1 affects all classes of Pol II transcripts including multiple classes of noncoding RNAs. By conducting a de novo differential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected by PAF1 deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 tri-methylation. Finally, we showed that one iron regulon gene, FET4, is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.

genomics↗

bioRxiv2018

A competence-regulated toxin-antitoxin system in Haemophilus influenzae

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenae, the DNA uptake machinery is induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that HI0659--which is absolutely required for DNA uptake-- encodes the antitoxin of a competence-regulated toxin-antitoxin operon ( toxTA), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the toxin restores uptake to the antitoxin mutant. In addition to the expected Sxy-and CRP-dependent-competence promoter, transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We present evidence that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture, and we show that the toxin gene has been inactivated by deletion in many H. influenzae strains. We suggest that this competence-regulated toxin-antitoxin system may facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions, or alternatively be a simple genetic parasite.

microbiology↗