RESUMO
The many functions of microbial communities emerge from a complex web of interactions between organisms and their environment. This poses a significant obstacle to engineering microbial consortia, hindering our ability to harness the potential of microorganisms for biotechnological applications. In this study, we demonstrate that the collective effect of ecological interactions between microbes in a community can be captured by simple statistical models that predict how adding a new species to a community will affect its function. These predictive models mirror the patterns of global epistasis reported in genetics, and they can be quantitatively interpreted in terms of pairwise interactions between community members. Our results illuminate an unexplored path to quantitatively predicting the function of microbial consortia from their composition, paving the way to optimizing desirable community properties and bringing the tasks of predicting biological function at the genetic, organismal, and ecological scales under the same quantitative formalism.
Assuntos
Microbiologia Ambiental , Epistasia Genética , Consórcios Microbianos , Biologia Sintética , Interações Microbianas , BioengenhariaRESUMO
Many sequence variants have additive effects on blood lipid levels and, through that, on the risk of coronary artery disease (CAD). We show that variants also have non-additive effects and interact to affect lipid levels as well as affecting variance and correlations. Variance and correlation effects are often signatures of epistasis or gene-environmental interactions. These complex effects can translate into CAD risk. For example, Trp154Ter in FUT2 protects against CAD among subjects with the A1 blood group, whereas it associates with greater risk of CAD in others. His48Arg in ADH1B interacts with alcohol consumption to affect lipid levels and CAD. The effect of variants in TM6SF2 on blood lipids is greatest among those who never eat oily fish but absent from those who often do. This work demonstrates that variants that affect variance of quantitative traits can allow for the discovery of epistasis and interactions of variants with the environment.
Assuntos
Doença da Artéria Coronariana , Animais , Humanos , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/genética , Epistasia Genética , Fenótipo , Lipídeos/sangue , Sistema ABO de Grupos SanguíneosRESUMO
Structural variants (SVs) underlie important crop improvement and domestication traits. However, resolving the extent, diversity, and quantitative impact of SVs has been challenging. We used long-read nanopore sequencing to capture 238,490 SVs in 100 diverse tomato lines. This panSV genome, along with 14 new reference assemblies, revealed large-scale intermixing of diverse genotypes, as well as thousands of SVs intersecting genes and cis-regulatory regions. Hundreds of SV-gene pairs exhibit subtle and significant expression changes, which could broadly influence quantitative trait variation. By combining quantitative genetics with genome editing, we show how multiple SVs that changed gene dosage and expression levels modified fruit flavor, size, and production. In the last example, higher order epistasis among four SVs affecting three related transcription factors allowed introduction of an important harvesting trait in modern tomato. Our findings highlight the underexplored role of SVs in genotype-to-phenotype relationships and their widespread importance and utility in crop improvement.
Assuntos
Produtos Agrícolas/genética , Regulação da Expressão Gênica de Plantas , Variação Estrutural do Genoma , Solanum lycopersicum/genética , Alelos , Sistema Enzimático do Citocromo P-450/genética , Ecótipo , Epistasia Genética , Frutas/genética , Duplicação Gênica , Genoma de Planta , Genótipo , Endogamia , Anotação de Sequência Molecular , Fenótipo , Melhoramento Vegetal , Locos de Características Quantitativas/genéticaRESUMO
Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modeling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighboring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations.
Assuntos
Processamento Alternativo/genética , Splicing de RNA/genética , Receptor fas/genética , Animais , Éxons/genética , Técnicas Genéticas , Genética , Genótipo , Humanos , Íntrons/genética , Camundongos , Modelos Teóricos , Mutação/genética , Fenótipo , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismoRESUMO
Recent studies of the tumor genome seek to identify cancer pathways as groups of genes in which mutations are epistatic with one another or, specifically, "mutually exclusive." Here, we show that most mutations are mutually exclusive not due to pathway structure but to interactions with disease subtype and tumor mutation load. In particular, many cancer driver genes are mutated preferentially in tumors with few mutations overall, causing mutations in these cancer genes to appear mutually exclusive with numerous others. Researchers should view current epistasis maps with caution until we better understand the multiple cause-and-effect relationships among factors such as tumor subtype, positive selection for mutations, and gross tumor characteristics including mutational signatures and load.
Assuntos
Epistasia Genética/genética , Genes Neoplásicos/genética , Neoplasias/genética , Algoritmos , Biologia Computacional/métodos , Epistasia Genética/fisiologia , Genes Neoplásicos/fisiologia , Humanos , Modelos Genéticos , Mutação/genética , Oncogenes/genéticaRESUMO
Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.
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Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.
Assuntos
Biomarcadores/metabolismo , Colesterol/metabolismo , Epistasia Genética , Redes Reguladoras de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Jurkat , Células K562 , Mapeamento de Interação de ProteínasRESUMO
Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.
Assuntos
Marcação de Genes/métodos , Animais , Linhagem Celular , Células-Tronco Embrionárias , Camundongos , Camundongos TransgênicosRESUMO
Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing "jointless" fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms. VIDEO ABSTRACT.
Assuntos
Epistasia Genética , Proteínas de Domínio MADS/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Sequência de Aminoácidos , Domesticação , Inflorescência/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/fisiologia , Proteínas de Domínio MADS/química , Proteínas de Domínio MADS/metabolismo , Meristema/metabolismo , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.
Assuntos
RNA Helicases DEAD-box/metabolismo , Epistasia Genética , Subunidade alfa de Receptor de Interleucina-7/genética , Splicing de RNA , RNA Helicases DEAD-box/genética , Éxons , Células HeLa , Humanos , Esclerose Múltipla/genética , Biossíntese de Proteínas , RNA Interferente Pequeno/metabolismo , Linfócitos T/imunologiaRESUMO
The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.
Assuntos
Evolução Biológica , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Especificidade de Hospedeiro , África Ocidental/epidemiologia , Animais , Quirópteros/virologia , Surtos de Doenças , Ebolavirus/classificação , Ebolavirus/genética , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/transmissão , Humanos , Mutação , Filogenia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , ZoonosesRESUMO
Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.
Assuntos
Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Ciclo Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Retroalimentação , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/metabolismoRESUMO
Recent work from Nguyen et al. unveils massively parallel measurements of epistatic interactions between two enzymes, dihydrofolate reductase and thymidylate synthase, in their natural cellular context. Almost 3000 mutations of DHFR in three TYMS backgrounds reveal a complex interaction network. The authors capture much of this complexity using a simple model.
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Uncovering the genes, variants, and interactions underlying crop diversity is a frontier in plant genetics. Phenotypic variation often does not reflect the cumulative effect of individual gene mutations. This deviation is due to epistasis, in which interactions between alleles are often unpredictable and quantitative in effect. Recent advances in genomics and genome-editing technologies are elevating the study of epistasis in crops. Using the traits and developmental pathways that were major targets in domestication and breeding, we highlight how epistasis is central in guiding the behavior of the genetic variation that shapes quantitative trait variation. We outline new strategies that illuminate how quantitative epistasis from modified gene dosage defines background dependencies. Advancing our understanding of epistasis in crops can reveal new principles and approaches to engineering targeted improvements in agriculture.
Assuntos
Produtos Agrícolas/genética , Epistasia Genética/genética , Variação Genética/genética , Locos de Características Quantitativas/genética , Animais , Domesticação , Edição de Genes/métodos , Genoma de Planta/genética , Genômica/métodos , Humanos , Melhoramento Vegetal/métodosRESUMO
We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.
Assuntos
Epistasia Genética , Infecções por HIV/genética , Fator Regulador 7 de Interferon/genética , Fatores de Transcrição/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Interferons/genética , Mutação , Transdução de Sinais/genéticaRESUMO
Dominance is usually considered a constant value that describes the relative difference in fitness or phenotype between heterozygotes and the average of homozygotes at a focal polymorphic locus. However, the observed dominance can vary with the genetic background of the focal locus. Here, alleles at other loci modify the observed phenotype through position effects or dominance modifiers that are sometimes associated with pathogen resistance, lineage, sex, or mating type. Theoretical models have illustrated how variable dominance appears in the context of multi-locus interaction (epistasis). Here, we review empirical evidence for variable dominance and how the observed patterns may be captured by proposed epistatic models. We highlight how integrating epistasis and dominance is crucial for comprehensively understanding adaptation and speciation.
Assuntos
Epistasia Genética , Modelos Genéticos , Heterozigoto , Fenótipo , Homozigoto , AlelosRESUMO
Alternative to the conventional search for single-target, single-compound treatments, combination therapies can open entirely new opportunities to fight antibiotic resistance. However, combinatorial complexity prohibits experimental testing of drug combinations on a large scale, and methods to rationally design combination therapies are lagging behind. Here, we developed a combined experimental-computational approach to predict drug-drug interactions using high-throughput metabolomics. The approach was tested on 1,279 pharmacologically diverse drugs applied to the gram-negative bacterium Escherichia coli. Combining our metabolic profiling of drug response with previously generated metabolic and chemogenomic profiles of 3,807 single-gene deletion strains revealed an unexpectedly large space of inhibited gene functions and enabled rational design of drug combinations. This approach is applicable to other therapeutic areas and can unveil unprecedented insights into drug tolerance, side effects, and repurposing. The compendium of drug-associated metabolome profiles is available at https://zampierigroup.shinyapps.io/EcoPrestMet, providing a valuable resource for the microbiological and pharmacological communities.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Redes e Vias Metabólicas/efeitos dos fármacos , Medicamentos sob Prescrição/farmacologia , Antibacterianos/química , Quimioinformática/métodos , Combinação de Medicamentos , Interações Medicamentosas , Reposicionamento de Medicamentos/métodos , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Deleção de Genes , Internet , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Medicamentos sob Prescrição/químicaRESUMO
Natural selection makes evolutionary adaptation possible even if the overwhelming majority of new mutations are deleterious. However, in rapidly evolving populations where numerous linked mutations occur and segregate simultaneously, clonal interference and genetic hitchhiking can limit the efficiency of selection, allowing deleterious mutations to accumulate over time. This can in principle overwhelm the fitness increases provided by beneficial mutations, leading to an overall fitness decline. Here, we analyze the conditions under which evolution will tend to drive populations to higher versus lower fitness. Our analysis focuses on quantifying the boundary between these two regimes, as a function of parameters such as population size, mutation rates, and selection pressures. This boundary represents a state in which adaptation is precisely balanced by Muller's ratchet, and we show that it can be characterized by rapid molecular evolution without any net fitness change. Finally, we consider the implications of global fitness-mediated epistasis and find that under some circumstances, this can drive populations toward the boundary state, which can thus represent a long-term evolutionary attractor.
Assuntos
Taxa de Mutação , Seleção Genética , Mutação , Evolução Molecular , Densidade Demográfica , Modelos GenéticosRESUMO
Protein engineering often targets binding pockets or active sites which are enriched in epistasis-nonadditive interactions between amino acid substitutions-and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable ß-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences-a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling.
Assuntos
Domínio Catalítico , Epistasia Genética , Triptofano Sintase , Domínio Catalítico/genética , Triptofano Sintase/genética , Triptofano Sintase/metabolismo , Triptofano Sintase/química , Engenharia de Proteínas/métodos , Substituição de Aminoácidos , Modelos MolecularesRESUMO
How emerging adaptive variants interact is an important factor in the evolution of wild populations, but the opportunity to empirically study this interaction is rare. We recently documented the emergence of an adaptive phenotype "curly-wing" in Hawaiian populations of field crickets (Teleogryllus oceanicus). Curly-wing inhibits males' ability to sing, protecting them from eavesdropping parasitoid flies (Ormia ochracea). Surprisingly, curly-wing co-occurs with similarly protective silent "flatwing" phenotypes in multiple populations, in which neither phenotype has spread to fixation. These two phenotypes are frequently coexpressed, but since either sufficiently reduces song amplitude to evade the fly, their coexpression confers no additional fitness benefit. Numerous "off-target" phenotypic changes are known to accompany flatwing, and we find that curly-wing, too, negatively impacts male courtship ability and affects mass and survival of females under lab conditions. We show through crosses and genomic and mRNA sequencing that curly-wing expression is associated with variation on a single autosome. In parallel analyses of flatwing, our results reinforce previous findings of X-linked single-locus inheritance. By combining insights into the genetic architecture of these alternative phenotypes with simulations and field observations, we show that the co-occurrence of these two adaptations impedes either from fixing, despite extreme fitness benefits, due to fitness epistasis. This co-occurrence of similar adaptive forms in the same populations might be more common than is generally considered and could be an important force inhibiting adaptive evolution in wild populations of sexually reproducing organisms.