Publications by authors named "Charles Boone"

257 Publications

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1.

Cell Chem Biol 2021 Sep 11. Epub 2021 Sep 11.

Departments of Chemistry and Earth Ocean & Atmospheric Sciences, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada. Electronic address:

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.
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http://dx.doi.org/10.1016/j.chembiol.2021.07.017DOI Listing
September 2021

Improving Measures of Chemical Structural Similarity Using Machine Learning on Chemical-Genetic Interactions.

J Chem Inf Model 2021 Jul 28. Epub 2021 Jul 28.

Department of Computer Science and Engineering, University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455, United States.

A common strategy for identifying molecules likely to possess a desired biological activity is to search large databases of compounds for high structural similarity to a query molecule that demonstrates this activity, under the assumption that structural similarity is predictive of similar biological activity. However, efforts to systematically benchmark the diverse array of available molecular fingerprints and similarity coefficients have been limited by a lack of large-scale datasets that reflect biological similarities of compounds. To elucidate the relative performance of these alternatives, we systematically benchmarked 11 different molecular fingerprint encodings, each combined with 13 different similarity coefficients, using a large set of chemical-genetic interaction data from the yeast as a systematic proxy for biological activity. We found that the performance of different molecular fingerprints and similarity coefficients varied substantially and that the all-shortest path fingerprints paired with the Braun-Blanquet similarity coefficient provided superior performance that was robust across several compound collections. We further proposed a machine learning pipeline based on support vector machines that offered a fivefold improvement relative to the best unsupervised approach. Our results generally suggest that using high-dimensional chemical-genetic data as a basis for refining molecular fingerprints can be a powerful approach for improving prediction of biological functions from chemical structures.
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http://dx.doi.org/10.1021/acs.jcim.0c00993DOI Listing
July 2021

Retention of duplicated genes in evolution.

Trends Genet 2021 Jul 20. Epub 2021 Jul 20.

Department of Molecular Genetics, Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON, Canada M5S 3E1; RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama, Japan, 351-0198.

Gene duplication is a prevalent phenomenon across the tree of life. The processes that lead to the retention of duplicated genes are not well understood. Functional genomics approaches in model organisms, such as yeast, provide useful tools to test the mechanisms underlying retention with functional redundancy and divergence of duplicated genes, including fates associated with neofunctionalization, subfunctionalization, back-up compensation, and dosage amplification. Duplicated genes may also be retained as a consequence of structural and functional entanglement. Advances in human gene editing have enabled the interrogation of duplicated genes in the human genome, providing new tools to evaluate the relative contributions of each of these factors to duplicate gene retention and the evolution of genome structure.
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http://dx.doi.org/10.1016/j.tig.2021.06.016DOI Listing
July 2021

Single-cell image analysis to explore cell-to-cell heterogeneity in isogenic populations.

Cell Syst 2021 Jun;12(6):608-621

The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada. Electronic address:

Single-cell image analysis provides a powerful approach for studying cell-to-cell heterogeneity, which is an important attribute of isogenic cell populations, from microbial cultures to individual cells in multicellular organisms. This phenotypic variability must be explained at a mechanistic level if biologists are to fully understand cellular function and address the genotype-to-phenotype relationship. Variability in single-cell phenotypes is obscured by bulk readouts or averaging of phenotypes from individual cells in a sample; thus, single-cell image analysis enables a higher resolution view of cellular function. Here, we consider examples of both small- and large-scale studies carried out with isogenic cell populations assessed by fluorescence microscopy, and we illustrate the advantages, challenges, and the promise of quantitative single-cell image analysis.
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http://dx.doi.org/10.1016/j.cels.2021.05.010DOI Listing
June 2021

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

Mol Syst Biol 2021 06;17(6):e10207

Calico Life Sciences LLC, South San Francisco, CA, USA.

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, Z EB42, that gives lower expression than Z EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.
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http://dx.doi.org/10.15252/msb.202110207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8182650PMC
June 2021

A method for benchmarking genetic screens reveals a predominant mitochondrial bias.

Mol Syst Biol 2021 05;17(5):e10013

Department of Computer Science and Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, USA.

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.
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http://dx.doi.org/10.15252/msb.202010013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138267PMC
May 2021

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Mol Cell 2021 06 10;81(11):2460-2476.e11. Epub 2021 May 10.

Institute of Molecular Biology (IMB), Mainz, Germany. Electronic address:

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
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http://dx.doi.org/10.1016/j.molcel.2021.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189435PMC
June 2021

Environmental robustness of the global yeast genetic interaction network.

Science 2021 05;372(6542)

The Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.

Phenotypes associated with genetic variants can be altered by interactions with other genetic variants (GxG), with the environment (GxE), or both (GxGxE). Yeast genetic interactions have been mapped on a global scale, but the environmental influence on the plasticity of genetic networks has not been examined systematically. To assess environmental rewiring of genetic networks, we examined 14 diverse conditions and scored 30,000 functionally representative yeast gene pairs for dynamic, differential interactions. Different conditions revealed novel differential interactions, which often uncovered functional connections between distantly related gene pairs. However, the majority of observed genetic interactions remained unchanged in different conditions, suggesting that the global yeast genetic interaction network is robust to environmental perturbation and captures the fundamental functional architecture of a eukaryotic cell.
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http://dx.doi.org/10.1126/science.abf8424DOI Listing
May 2021

Genome-wide Screening of Genes Associated with Momilactone B Sensitivity in the Fission Yeast.

G3 (Bethesda) 2021 May 6. Epub 2021 May 6.

Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.

Momilactone B is a natural product with dual biological activities, including antimicrobial and allelopathic properties, and play a major role in plant chemical defense against competitive plants and pathogens. The pharmacological effects of momilactone B on mammalian cells have also been reported. However, little is known about the molecular and cellular mechanisms underlying its broad bioactivity. In this study, the genetic determinants of momilactone B sensitivity in yeast were explored to gain insight into its mode of action. We screened fission yeast mutants resistant to momilactone B from a pooled culture containing genome-wide gene-overexpressing strains in a drug-hypersensitive genetic background. Overexpression of pmd1, bfr1, pap1, arp9, or SPAC9E9.06c conferred resistance to momilactone B. In addition, a drug-hypersensitive, barcoded deletion library was newly constructed and the genes that imparted altered sensitivity to momilactone B upon deletion were identified. Gene Ontology and fission yeast phenotype ontology enrichment analyses predicted the biological pathways related to the mode of action of momilactone B. The validation of predictions revealed that momilactone B induced abnormal phenotypes such as multiseptated cells and disrupted organization of the microtubule structure. This is the first investigation of the mechanism underlying the antifungal activity of momilactone B against yeast. The results and datasets obtained in this study narrow the possible targets of momilactone B and facilitate further studies regarding its mode of action.
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http://dx.doi.org/10.1093/g3journal/jkab156DOI Listing
May 2021

Reduced gene dosage of histone H4 prevents CENP-A mislocalization and chromosomal instability in Saccharomyces cerevisiae.

Genetics 2021 May;218(1)

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20894, USA.

Mislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to noncentromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A have been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in proteolysis of overexpressed Cse4 (GALCSE4) lead to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCFMet30, and SCFCdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of overexpressed cse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 leads to defects in sumoylation and reduced mislocalization of overexpressed Cse4, which contributes to suppression of CIN when Cse4 is overexpressed. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to noncentromeric regions, leading to CIN when Cse4 is overexpressed.
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http://dx.doi.org/10.1093/genetics/iyab033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128410PMC
May 2021

Trigenic Synthetic Genetic Array (τ-SGA) Technique for Complex Interaction Analysis.

Methods Mol Biol 2021 ;2212:377-400

The Donnelly Centre, Toronto, ON, Canada.

Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation based on the combination of phenotypes associated with individual mutant alleles. Trigenic Synthetic Genetic Array (τ-SGA) methodology was developed for the systematic analysis of complex interactions involving combinations of three gene perturbations. With a series of replica pinning steps of the τ-SGA procedure, haploid triple mutants are constructed through automated mating and meiotic recombination. For example, a double-mutant query strain carrying two mutant alleles of interest, such as a deletion allele of a nonessential gene and a conditional temperature-sensitive allele of an essential gene, is crossed to an input array of yeast mutants, such as the diagnostic array set of ~1200 mutants, to generate an output array of triple mutants. The colony-size measurements of the resulting triple mutants are used to estimate cellular fitness and quantify trigenic interactions by incorporating corresponding single- and double-mutant fitness estimates. Trigenic interaction networks can be further analyzed for functional modules using various clustering and enrichment analysis tools. Complex genetic interactions are rich in functional information and provide insight into the genotype-to-phenotype relationship, genome size, and speciation.
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http://dx.doi.org/10.1007/978-1-0716-0947-7_23DOI Listing
April 2021

τ-SGA: synthetic genetic array analysis for systematically screening and quantifying trigenic interactions in yeast.

Nat Protoc 2021 02 18;16(2):1219-1250. Epub 2021 Jan 18.

The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

Systematic complex genetic interaction studies have provided insight into high-order functional redundancies and genetic network wiring of the cell. Here, we describe a method for screening and quantifying trigenic interactions from ordered arrays of yeast strains grown on agar plates as individual colonies. The protocol instructs users on the trigenic synthetic genetic array analysis technique, τ-SGA, for high-throughput screens. The steps describe construction of the double-mutant query strains and the corresponding single-mutant control query strains, which are screened in parallel in two replicates. The screening experimental set-up consists of sequential replica-pinning steps that enable automated mating, meiotic recombination and successive haploid selection steps for the generation of triple mutants, which are scored for colony size as a proxy for fitness, which enables the calculation of trigenic interactions. The procedure described here was used to conduct 422 trigenic interaction screens, which generated ~460,000 yeast triple mutants for trigenic interaction analysis. Users should be familiar with robotic equipment required for high-throughput genetic interaction screens and be proficient at the command line to execute the scoring pipeline. Large-scale screen computational analysis is achieved by using MATLAB pipelines that score raw colony size data to produce τ-SGA interaction scores. Additional recommendations are included for optimizing experimental design and analysis of smaller-scale trigenic interaction screens by using a web-based analysis system, SGAtools. This protocol provides a resource for those who would like to gain a deeper, more practical understanding of trigenic interaction screening and quantification methodology.
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http://dx.doi.org/10.1038/s41596-020-00456-3DOI Listing
February 2021

Genetic profiling of protein burden and nuclear export overload.

Elife 2020 11 4;9. Epub 2020 Nov 4.

Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan.

Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.
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http://dx.doi.org/10.7554/eLife.54080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673788PMC
November 2020

Systematic analysis of bypass suppression of essential genes.

Mol Syst Biol 2020 09;16(9):e9828

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.

Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project.
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http://dx.doi.org/10.15252/msb.20209828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507402PMC
September 2020

TheCellVision.org: A Database for Visualizing and Mining High-Content Cell Imaging Projects.

G3 (Bethesda) 2020 11 5;10(11):3969-3976. Epub 2020 Nov 5.

The Donnelly Centre, University of Toronto, Ontario, M5S 3E1, Canada

Advances in genome engineering and high throughput imaging technologies have enabled genome-scale screens of single cells for a variety of phenotypes, including subcellular morphology and protein localization. We constructed TheCellVision.org, a freely available and web-accessible image visualization and data browsing tool that serves as a central repository for fluorescence microscopy images and associated quantitative data produced by high-content screening experiments. Currently, TheCellVision.org hosts ∼575,590 images and associated analysis results from two published high-content screening (HCS) projects focused on the budding yeast TheCellVision.org allows users to access, visualize and explore fluorescence microscopy images, and to search, compare, and extract data related to subcellular compartment morphology, protein abundance, and localization. Each dataset can be queried independently or as part of a search across multiple datasets using the advanced search option. The website also hosts computational tools associated with the available datasets, which can be applied to other projects and cell systems, a feature we demonstrate using published images of mammalian cells. Providing access to HCS data through websites such as TheCelllVision.org enables new discovery and independent re-analyses of imaging data.
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http://dx.doi.org/10.1534/g3.120.401570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642925PMC
November 2020

Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.

Nat Metab 2020 06 1;2(6):499-513. Epub 2020 Jun 1.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.
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http://dx.doi.org/10.1038/s42255-020-0211-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566881PMC
June 2020

Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

Science 2020 06;368(6498)

Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors.
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http://dx.doi.org/10.1126/science.aaz5667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539174PMC
June 2020

Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture.

mBio 2020 05 5;11(3). Epub 2020 May 5.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.
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http://dx.doi.org/10.1128/mBio.00449-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403777PMC
May 2020

Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in .

G3 (Bethesda) 2020 06 1;10(6):2057-2068. Epub 2020 Jun 1.

Genetics Branch, Center for Cancer Research, National Cancer Institute,

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 () by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and strains display synthetic dosage lethality (SDL) with We previously performed a genome-wide screen and identified five alleles of and that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with We determined that strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4 Mutation of ( ) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a strain. We determined that does not rescue the SDL and defects in proteolysis of in a strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a strain were similar to that of and strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1 Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.
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http://dx.doi.org/10.1534/g3.120.401131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263675PMC
June 2020

Publisher Correction: Integrating yeast chemical genomics and mammalian cell pathway analysis.

Acta Pharmacol Sin 2020 05;41(5):729

The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, 201203, China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41401-019-0355-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471429PMC
May 2020

Systematic genetics and single-cell imaging reveal widespread morphological pleiotropy and cell-to-cell variability.

Mol Syst Biol 2020 02;16(2):e9243

The Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Our ability to understand the genotype-to-phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single-cell level. To systematically assess cell-to-cell phenotypic variability, we combined automated yeast genetics, high-content screening and neural network-based image analysis of single cells, focussing on genes that influence the architecture of four subcellular compartments of the endocytic pathway as a model system. Our unbiased assessment of the morphology of these compartments-endocytic patch, actin patch, late endosome and vacuole-identified 17 distinct mutant phenotypes associated with ~1,600 genes (~30% of all yeast genes). Approximately half of these mutants exhibited multiple phenotypes, highlighting the extent of morphological pleiotropy. Quantitative analysis also revealed that incomplete penetrance was prevalent, with the majority of mutants exhibiting substantial variability in phenotype at the single-cell level. Our single-cell analysis enabled exploration of factors that contribute to incomplete penetrance and cellular heterogeneity, including replicative age, organelle inheritance and response to stress.
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http://dx.doi.org/10.15252/msb.20199243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025093PMC
February 2020

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast.

PLoS Genet 2020 02 7;16(2):e1008597. Epub 2020 Feb 7.

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.
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http://dx.doi.org/10.1371/journal.pgen.1008597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032732PMC
February 2020

Discovering genetic interactions bridging pathways in genome-wide association studies.

Nat Commun 2019 09 19;10(1):4274. Epub 2019 Sep 19.

Department of Computer Science and Engineering, University of Minnesota, Minneapolis, MN, 55455, USA.

Genetic interactions have been reported to underlie phenotypes in a variety of systems, but the extent to which they contribute to complex disease in humans remains unclear. In principle, genome-wide association studies (GWAS) provide a platform for detecting genetic interactions, but existing methods for identifying them from GWAS data tend to focus on testing individual locus pairs, which undermines statistical power. Importantly, a global genetic network mapped for a model eukaryotic organism revealed that genetic interactions often connect genes between compensatory functional modules in a highly coherent manner. Taking advantage of this expected structure, we developed a computational approach called BridGE that identifies pathways connected by genetic interactions from GWAS data. Applying BridGE broadly, we discover significant interactions in Parkinson's disease, schizophrenia, hypertension, prostate cancer, breast cancer, and type 2 diabetes. Our novel approach provides a general framework for mapping complex genetic networks underlying human disease from genome-wide genotype data.
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http://dx.doi.org/10.1038/s41467-019-12131-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753138PMC
September 2019

Decyl Gallate as a Possible Inhibitor of N-Glycosylation Process in .

Antimicrob Agents Chemother 2019 11 22;63(11). Epub 2019 Oct 22.

Department of Clinical Analysis, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil

The available antifungal therapeutic arsenal is limited. The search for alternative drugs with fewer side effects and new targets remains a major challenge. Decyl gallate (G14) is a derivative of gallic acid with a range of biological activities and broad-spectrum antifungal activity. Previously, our group demonstrated the promising anti- activity of G14. In this work, to evaluate the antifungal characteristics of G14 for , a chemical-genetic interaction analysis was conducted on a model. N-glycosylation and/or the unfolded protein response pathway was identified as a high-confidence process for drug target prediction. The overactivation of unfolded protein response (UPR) signaling was confirmed using this model with IRE1/ATF6/PERK genes tagged with green fluorescent protein (GFP). In , this prediction was confirmed by the low activity of glycosylated enzymes [α-(1,3)-glucanase, -acetyl-β-d-glucosaminidase (NAGase), and α-(1,4)-amylase], by hyperexpression of genes involved with the UPR and glycosylated enzymes, and by the reduction in the amounts of glycosylated proteins and chitin. All of these components are involved in fungal cell wall integrity and are dependent on the N-glycosylation process. This loss of integrity was confirmed by the reduction in mitochondrial activity, impaired budding, enhancement of wall permeability, and a decrease in viability. These events led to a reduction of the ability of fungi to adhere on human lung epithelial cells (A549) Therefore, G14 may have an important role in balancing the inflammatory reaction caused by fungal infection, without interfering with the microbicidal activity of nitric oxide. This work provides new information on the activity of G14, a potential anti- compound.
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http://dx.doi.org/10.1128/AAC.01909-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811398PMC
November 2019

Functional link between mitochondria and Rnr3, the minor catalytic subunit of yeast ribonucleotide reductase.

Microb Cell 2019 May 20;6(6):286-294. Epub 2019 May 20.

School of Medical Sciences, North West Cancer Research Institute, Bangor University, Deniol Road, Bangor, LL57 2UW, United Kingdom.

Ribonucleotide reductase (RNR) is an essential holoenzyme required for synthesis of dNTPs. The genome encodes for two catalytic subunits, Rnr1 and Rnr3. While Rnr1 is required for DNA replication and DNA damage repair, the function(s) of Rnr3 is unknown. Here, we show that carbon source, an essential nutrient, impacts Rnr1 and Rnr3 abundance: Non-fermentable carbon sources or limiting concentrations of glucose down regulate Rnr1 and induce Rnr3 expression. Oppositely, abundant glucose induces Rnr1 expression and down regulates Rnr3. The carbon source dependent regulation of Rnr3 is mediated by Mec1, the budding yeast ATM/ATR checkpoint response kinase. Unexpectedly, this regulation is independent of all currently known components of the Mec1 DNA damage response network, including Rad53, Dun1, and Tel1, implicating a novel Mec1 signalling axis. Δ leads to growth defects under respiratory conditions and rescues temperature sensitivity conferred by the absence of Tom6, a component of the mitochondrial TOM (translocase of outer membrane) complex responsible for mitochondrial protein import. Together, these results unveil involvement of Rnr3 in mitochondrial functions and Mec1 in mediating the carbon source dependent regulation of Rnr3.
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http://dx.doi.org/10.15698/mic2019.06.680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545439PMC
May 2019

Integrating yeast chemical genomics and mammalian cell pathway analysis.

Acta Pharmacol Sin 2019 Sep 28;40(9):1245-1255. Epub 2019 May 28.

The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, 201203, China.

Chemical genomics has been applied extensively to evaluate small molecules that modulate biological processes in Saccharomyces cerevisiae. Here, we use yeast as a surrogate system for studying compounds that are active against metazoan targets. Large-scale chemical-genetic profiling of thousands of synthetic and natural compounds from the Chinese National Compound Library identified those with high-confidence bioprocess target predictions. To discover compounds that have the potential to function like therapeutic agents with known targets, we also analyzed a reference library of approved drugs. Previously uncharacterized compounds with chemical-genetic profiles resembling existing drugs that modulate autophagy and Wnt/β-catenin signal transduction were further examined in mammalian cells, and new modulators with specific modes of action were validated. This analysis exploits yeast as a general platform for predicting compound bioactivity in mammalian cells.
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http://dx.doi.org/10.1038/s41401-019-0231-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786357PMC
September 2019

Genetic interaction networks in cancer cells.

Curr Opin Genet Dev 2019 02 8;54:64-72. Epub 2019 Apr 8.

Donnelly Centre, University of Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, ON, Canada. Electronic address:

The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer.
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http://dx.doi.org/10.1016/j.gde.2019.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820710PMC
February 2019

Global Genetic Networks and the Genotype-to-Phenotype Relationship.

Cell 2019 03;177(1):85-100

The Donnelly Centre, University of Toronto, 160 College Street, Toronto ON, Canada; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto ON, Canada. Electronic address:

Genetic interactions identify combinations of genetic variants that impinge on phenotype. With whole-genome sequence information available for thousands of individuals within a species, a major outstanding issue concerns the interpretation of allelic combinations of genes underlying inherited traits. In this Review, we discuss how large-scale analyses in model systems have illuminated the general principles and phenotypic impact of genetic interactions. We focus on studies in budding yeast, including the mapping of a global genetic network. We emphasize how information gained from work in yeast translates to other systems, and how a global genetic network not only annotates gene function but also provides new insights into the genotype-to-phenotype relationship.
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http://dx.doi.org/10.1016/j.cell.2019.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817365PMC
March 2019

Complex modifier landscape underlying genetic background effects.

Proc Natl Acad Sci U S A 2019 03 25;116(11):5045-5054. Epub 2019 Feb 25.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada;

The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual strains, S288c and Σ1278b, ∼1% of yeast genes were previously identified as "conditional essential." Here, in addition to confirming that some conditional essential genes are modified by a nonchromosomal element, we show that most cases involve a complex set of genomic modifiers. From tetrad analysis of S288C/Σ1278b hybrid strains and whole-genome sequencing of viable hybrid spore progeny, we identified complex sets of multiple genomic regions underlying conditional essentiality. For a smaller subset of genes, including and , each of which encodes components of the cysteine biosynthesis pathway, we observed a segregation pattern consistent with a single modifier associated with conditional essentiality. In natural yeast isolates, we found that the / conditional essentiality can be caused by variation in two independent modifiers, and , each with roles associated with cellular cysteine physiology. Interestingly, the allelic variation appears to have arisen independently from separate lineages, with rare allele frequencies below 0.5%. Thus, while conditional gene essentiality is usually driven by genetic interactions associated with complex modifier architectures, our analysis also highlights the role of functionally related, genetically independent, and rare variants.
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http://dx.doi.org/10.1073/pnas.1820915116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421401PMC
March 2019

Yeast Two-Hybrid Analysis for Ubiquitin Variant Inhibitors of Human Deubiquitinases.

J Mol Biol 2019 03 11;431(6):1160-1171. Epub 2019 Feb 11.

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada; Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S3E1, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.
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http://dx.doi.org/10.1016/j.jmb.2019.02.007DOI Listing
March 2019
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