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Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis., Author summary Genomes are in flux, as genes are constantly added and lost throughout evolution. New genes were once thought to arise almost exclusively via the modification or duplication of existing genes. Recently, however, interest has grown in alternative modes of new gene origination, such as de novo evolution from genetic material that previously did not encode proteins. Many de novo genes are expressed in male reproductive tissues, but their significance for fertility is not well understood. We screened likely de novo genes expressed in the Drosophila testis for reproductive roles and found one gene, atlas, essential for male fertility. We leveraged genetic and cell biological experiments to investigate roles for Atlas protein in reproduction and found that it is required during sperm development for proper packaging of DNA in the sperm nucleus. Evolutionary analyses of this gene revealed a complicated history, including loss in some lineages, movement between chromosomes, and fusion with a non-protein-coding gene. Studying both the functions and evolutionary histories of new proteins illustrates how they might evolve critical roles in biological processes despite their relative novelty. Furthermore, the study of atlas identifies an essential genetic player in the fly testis, an important model system for understanding how gametes are produced.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation., Author summary Vps13 family proteins are conserved lipid transfer proteins that function at organelle contact sites and have been implicated in a number of different neurological diseases. In the yeast Saccharomyces cerevisiae, Vps13 is encoded by a single gene and is localized to various contact sites by interaction with different adaptor proteins and/or lipids, however its regulation is yet to be clarified. We have previously shown that during the developmental process of sporulation, Vps13 is recruited to de novo membrane structures called prospore membranes (PSMs) by a specific adaptor complex, and Vps13 and its adaptors are required for PSM extension. Here we reveal that loss of an adaptor can be overcome by lowering phosphatidylinositol-4-phosphate (PI4P) levels, either by inhibiting PI 4-kinase on the PSM or recruiting PI 4-phospatase to the PSM and that PI4P levels in the PSM affect Vps13 function. Further, we show that Vps13 forms endoplasmic reticulum (ER)-PSM contact sites, that ER-plasma membrane tethering proteins are recruited to ER-PSM contacts, and these proteins may function in conjunction with Vps13. Thus, our work shines light on both the mechanisms of intracellular remodeling and the function of this important class of lipid transfer proteins.

Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. We conducted a genome-wide genetic screen to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington’s disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases., Author summary Polyglutamine (polyQ) diseases are neurodegenerative diseases caused by the expansion of glutamine repeats in specific proteins. After >100 years of effort trying to decipher the pathogenesis of polyQ diseases, the mechanisms by which these mutant proteins induced neuronal death are still unknown. Here, we performed a genome-wide genetic screen in Drosophila for mutations that alleviate polyQ cytotoxicity. We found that a mutation in the gene rack1, which encodes a scaffold protein, suppressed polyQ-induced neurodegeneration when polyQ tracts were expresses, and in models of HD and MJD. Importantly, this effect was seen without clearance of the polyQ proteins. Further, we found that RACK1 binds both the E3 ubiquitin ligase POE and ERK, thereby promoting ERK degradation. These data suggest that the RACK1/POE/ERK pathway is involved in pathogenesis of polyQ diseases, and identify RACK1/POE/ERK as potential therapeutic targets for treating polyQ diseases.

The metabolic reprogramming of cancer cells creates metabolic vulnerabilities that can be therapeutically targeted. However, our understanding of metabolic dependencies and the pathway crosstalk that creates these vulnerabilities in cancer cells remains incomplete. Here, by integrating gene expression data with genetic loss-of-function and pharmacological screening data from hundreds of cancer cell lines, we identified metabolic vulnerabilities at the level of pathways rather than individual genes. This approach revealed that metabolic pathway dependencies are highly context-specific such that cancer cells are vulnerable to inhibition of one metabolic pathway only when activity of another metabolic pathway is altered. Notably, we also found that the no single metabolic pathway was universally essential, suggesting that cancer cells are not invariably dependent on any metabolic pathway. In addition, we confirmed that cell culture medium is a major confounding factor for the analysis of metabolic pathway vulnerabilities. Nevertheless, we found robust associations between metabolic pathway activity and sensitivity to clinically approved drugs that were independent of cell culture medium. Lastly, we used parallel integration of pharmacological and genetic dependency data to confidently identify metabolic pathway vulnerabilities. Taken together, this study serves as a comprehensive characterization of the landscape of metabolic pathway vulnerabilities in cancer cell lines., Author summary Cancer cells rewire their metabolism, which creates targetable metabolic vulnerabilities. Previous analyses of metabolic vulnerabilities in cancer cells have been limited to the analysis of individual genes or metabolites. However, metabolic pathways exhibit significant cross talk and compensation for one another. We developed a computational method to answer the question: when a metabolic pathway’s activity is high, which other metabolic pathways become more essential or less essential? By integrating genetic screen data with drug response data from FDA approved drugs, we identified cancer cell line dependence on metabolic pathways as opposed to individual genes. For example, we found that identifying key regulators of metabolic pathways, such as the Pentose Phosphate Pathway, may serve as a biomarker to identify which patients may benefit from antifolate chemotherapies (e.g. methotrexate, 5-fluorouracil). The efforts outlined here serve as the first characterization of the landscape of metabolic pathway vulnerabilities in cancer cell lines. Our results demonstrate the benefit of analyzing dependencies on metabolic pathways as opposed to metabolic genes.

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest., Author summary Nerve connections that are severed in spinal cord injury do not heal, which can lead to permanent paralysis. Lack of repair may in part be due to prolonged inflammation of the injury site. In contrast, zebrafish show excellent repair of nerve connections after spinal injury and this is associated with controlling inflammation. Due to recent advances in genetic technology (CRISPR/Cas9) we can now determine the function of genes that influence regeneration in the living zebrafish in a matter of days. Here we devise a very rapid screening method for the function of inflammation-related genes in zebrafish larvae after spinal cord injury. We find a number of genes that are necessary for repair of nerve connections and control of the inflammation after injury. This provides important leads to improve our understanding of the role of inflammation in spinal cord injury. Moreover, our fast and robust screening method can be adopted by other researchers to screen for gene functions in a whole animal, which was previously not easily possible.

Background Downstream targets for a large number of RNA-binding proteins remain to be identified. The Drosophila master sex-switch protein Sex-lethal (SXL) is an RNA-binding protein that controls splicing, polyadenylation, or translation of certain mRNAs to mediate female-specific sexual differentiation. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens. Methodology/Principal findings Here, we have used an alternative molecular approach of GEnomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) using both the genomic DNA and cDNA pools from several Drosophila developmental stages to identify new potential targets of SXL. Our systematic analysis provides a comprehensive view of the Drosophila transcriptome for potential SXL-binding sites. Conclusion/Significance We have successfully identified new SXL-binding sites in the Drosophila transcriptome. We discuss the significance of our analysis and that the newly identified binding sites and sequences could serve as a useful resource for the research community. This approach should also be applicable to other RNA-binding proteins for which downstream targets are unknown.

The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in our forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function.

The eyeless C. elegans exhibits robust phototaxis behavior in response to short-wavelength light, particularly UV light. C. elegans senses light through LITE-1, a unique photoreceptor protein that belongs to the invertebrate taste receptor family. However, it remains unclear how LITE-1 is regulated. Here, we performed a forward genetic screen for genes that when mutated suppress LITE-1 function. One group of lite-1 suppressors are the genes required for producing the two primary antioxidants thioredoxin and glutathione, suggesting that oxidization of LITE-1 inhibits its function. Indeed, the oxidant hydrogen peroxide (H2O2) suppresses phototaxis behavior and inhibits the photoresponse in photoreceptor neurons, whereas other sensory behaviors are relatively less vulnerable to H2O2. Conversely, antioxidants can rescue the phenotype of lite-1 suppressor mutants and promote the photoresponse. As UV light illumination generates H2O2, we propose that upon light activation of LITE-1, light-produced H2O2 then deactivates LITE-1 to terminate the photoresponse, while antioxidants may promote LITE-1’s recovery from its inactive state. Our studies provide a potential mechanism by which H2O2 and antioxidants act synergistically to regulate photosensation in C. elegans., Author summary The nematode C. elegans possesses a unique photoreceptor protein, LITE-1, which mediates a light-avoidance behavior upon light exposure. C. elegans avoids short-wavelength light, particularly UV light, providing a potential mechanism by which worms escape from the dangerous UV rays in the sunlight. However, it is not clear how LITE-1 is regulated. Here, we performed a genetic screen to identify genes regulating LITE-1. We uncovered six genes that when mutated suppress LITE-1 function. All these genes are involved in regenerating cellular antioxidants that function to clear reactive oxygen species, particularly hydrogen peroxide (H2O2), suggesting that the function of LITE-1 is vulnerable to H2O2. Indeed, we show that H2O2 exposure inhibits LITE-1 function, while antioxidants promote it. Notably, other sensory functions are relatively less sensitive to H2O2. As UV light illumination is known to generate H2O2 within the cell, this provides a potential mechanism to turn off LITE-1. Our results uncover a potential mechanism of LITE-1 regulation, where antioxidants and oxidants act to promote and suppress LITE-1 function, respectively.

Predicting the phenotypes resulting from molecular perturbations is one of the key challenges in genetics. Both forward and reverse genetic screen are employed to identify the molecular mechanisms underlying phenotypes and disease, and these resulted in a large number of genotype–phenotype association being available for humans and model organisms. Combined with recent advances in machine learning, it may now be possible to predict human phenotypes resulting from particular molecular aberrations. We developed DeepPheno, a neural network based hierarchical multi-class multi-label classification method for predicting the phenotypes resulting from loss-of-function in single genes. DeepPheno uses the functional annotations with gene products to predict the phenotypes resulting from a loss-of-function; additionally, we employ a two-step procedure in which we predict these functions first and then predict phenotypes. Prediction of phenotypes is ontology-based and we propose a novel ontology-based classifier suitable for very large hierarchical classification tasks. These methods allow us to predict phenotypes associated with any known protein-coding gene. We evaluate our approach using evaluation metrics established by the CAFA challenge and compare with top performing CAFA2 methods as well as several state of the art phenotype prediction approaches, demonstrating the improvement of DeepPheno over established methods. Furthermore, we show that predictions generated by DeepPheno are applicable to predicting gene–disease associations based on comparing phenotypes, and that a large number of new predictions made by DeepPheno have recently been added as phenotype databases., Author summary Gene–phenotype associations can help to understand the underlying mechanisms of many genetic diseases. However, experimental identification, often involving animal models, is time consuming and expensive. Computational methods that predict gene–phenotype associations can be used instead. We developed DeepPheno, a novel approach for predicting the phenotypes resulting from a loss of function of a single gene. We use gene functions and gene expression as information to prediction phenotypes. Our method uses a neural network classifier that is able to account for hierarchical dependencies between phenotypes. We extensively evaluate our method and compare it with related approaches, and we show that DeepPheno results in better performance in several evaluations. Furthermore, we found that many of the new predictions made by our method have been added to phenotype association databases released one year later. Overall, DeepPheno simulates some aspects of human physiology and how molecular and physiological alterations lead to abnormal phenotypes.

Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2., Author summary Diphthamide is a unique post-translationally modified histidine residue (His699 in yeast, His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). Mice that are deficient in diphthamide biosynthesis are embryonic lethal, attesting to the importance of diphthamide in normal physiology. It has taken four decades to identify the seven non-redundant genes in diphthamide biosynthesis, but whether additional factors are required and how the pathway is regulated remained elusive. To address these issues, we performed two saturating, independent, and unbiased genome-wide CRISPR knockout screens. The screens concluded independently that Dph1-Dph7 and additionally transcription factor Miz1 are the key factors required for diphthamide biosynthesis. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. While diphthamide biosynthesis machinery (Dph1-Dph7) exists across eukaryotes, Miz1 orthologues do not exist in lower species such as yeast, C. elegans, and Drosophila, indicating that the regulation of diphthamide modification by Miz1 emerged much later in evolution. The work opens a new avenue for understanding the role that diphthamide modification plays in normal physiology and bacterial toxin pathogenesis.

The conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, expression of O-GlcNAcylation-deficient forms of human or worm OGT rescue the hypertonic stress response phenotype. However, expression of an OGT protein lacking the tetracopeptide repeat (TPR) domain does not rescue. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins., Author summary The ability to sense and adapt to changes in the environment is an essential feature of cellular life. Changes in environmental salt and water concentrations can rapidly cause cell volume swelling or shrinkage and, if left unchecked, will lead to cell and organismal death. All organisms have developed similar physiological strategies for maintaining cell volume. However, the molecular mechanisms that control these physiological outputs are not well understood in animals. Using unbiased genetic screening in C. elegans, we discovered that a highly conserved enzyme called O-GlcNAc transferase (OGT) is essential for regulating physiological responses to increased environmental solute levels. A human form of OGT can functionally substitute for worm OGT, showing that this role is conserved across evolution. Surprisingly, the only known enzymatic activity of OGT was not required for this role, suggesting this enzyme has important undescribed molecular functions. Our studies reveal a new animal-specific role for OGT in the response to osmotic stress and show that C. elegans is an important model for defining the conserved molecular mechanisms that respond to alterations in cell volume.

Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, we identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). We showed that NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. We showed that NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling., Author summary Nt-acetylation is often regarded as a constitutive, irreversible, and static modification that is not suited to serve regulatory functions. Our observation that Nt-acetylation by NatB coordinately regulates some of the highly conserved positive and negative components of the EGFR/MAPK pathway suggest that Nt-acetylation and N-end rule pathways can play important roles regulating important signaling pathways. As these positive and negative pathway components are conserved and shared by multiple growth factor signaling pathways and as Acetyl-CoA level, which is influenced by cell metabolism, can be rate limiting for Nt-acetylation, our results also suggest a potentially new mechanism by which cellular metabolic status can regulate growth factor signaling.

Enhancers are powerful and versatile agents of cell-type specific gene regulation, which are thought to play key roles in human disease. Enhancers are short DNA elements that function primarily as clusters of transcription factor binding sites that are spatially coordinated to regulate expression of one or more specific target genes. These regulatory connections between enhancers and target genes can therefore be characterized as enhancer-gene links that can affect development, disease, and homeostatic cellular processes. Despite their implication in disease and the establishment of cell identity during development, most enhancer-gene links remain unknown. Here we introduce a new, publicly accessible database of predicted enhancer-gene links, PEREGRINE. The PEREGRINE human enhancer-gene links interactive web interface incorporates publicly available experimental data from ChIA-PET, eQTL, and Hi-C assays across 78 cell and tissue types to link 449,627 enhancers to 17,643 protein-coding genes. These enhancer-gene links are made available through the new Enhancer module of the PANTHER database and website where the user may easily access the evidence for each enhancer-gene link, as well as query by target gene and enhancer location.

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington’s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis., Author summary Circadian clocks evolved to anticipate 24 h environmental rhythms driven by the earth’s daily rotation and regulate nearly all aspects of behavior, physiology and the genome. Disruptions of the circadian clock have been associated with a wide range of human diseases, including neurodegenerative diseases such as Huntington’s disease (HD). Using an HD animal model in which a mutant Huntingtin (mHtt) protein is expressed, we identify a role for the RNA binding protein and neurodegenerative disease gene Ataxin-2 (Atx2) in mediating mHtt effects on circadian behavioral rhythms. Using transcriptomics, we identify the transcription factor CrebA as a potential target of both Atx2 and the circadian clock. Finally, we demonstrate a role for CrebA in mediating mHtt effects on circadian behavior, defining a novel Atx2-CrebA pathway in a neurodegenerative disease model. These studies define the molecular mechanisms by which mHtt can disrupt circadian rhythms identifying potential novel therapeutic targets for this uniformly fatal disease.

Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections., Author summary Life threatening infections are an increasing reality. Multi-drug resistant bacteria e.g. MRSA are present in nearly all hospitals, and community acquired TB is often recalcitrant to treatment. Less well known, but causing four to five times more deaths in the UK than MRSA, are fungi in the genus Candida. These are commonly associated with mucosal infections such as “thrush”, but are responsible for > 400,000 life-threatening infections worldwide every year, especially in the immunosuppressed patient population. One of the principle pathogens is Candida glabrata. This fungus grows as a single celled yeast, and alarmingly is highly resistant to both host innate defences and many clinically used antifungal drugs. Even with the best possible medical care, infections with this yeast cause ~50% mortality and alarmingly the incidence of C. glabrata infections is steadily climbing. This demands that novel antifungal therapies are developed that can target drug resistant fungal infections such as C. glabrata. We have identified genes that prevent the growth of fungal cells that express gain-of-function mutations in Pdr1, a key mediator of antifungal resistance in C. glabrata. We also provide proof of concept that targeting a protein essential for Pdr1 function significantly inhibits the emergence of antifungal drug resistance. Thus such an approach could provide a powerful tool in developing treatments for drug resistant infections.

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously., Author summary The protozoan parasite Leishmania is auxotroph for folate and unconjugated pterins and salvages both from the mammalian host. Two enzymes of the folate metabolism pathway, namely the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the pteridine reductase 1 (PTR1), are being evaluated for drug discovery and repurposing of existing anti-metabolites. Despite their apparent potential, development of DHFR-TS and PTR1 targeted chemotherapy against Leishmania is still awaiting. Here we revisited folate metabolism at the genomic level and report on the identification of known resistance genes alongside some new ones. Through gene disruption studies we found that L. infantum DHFR-TS null mutants are thymidine auxotroph and that these can be rescued by the bacterial flavin dependent thymidylate synthase ThyX. We also found that PTR1 is essential in the absence of a functional DHFR-TS even in the presence of ThyX or thymidine supplementation, indicating the essential role of reduced pterins or folate beyond thymidine synthesis. This study indicates that simultaneous targeting of DHFR-TS and PTR1 will be required for the development of anti-folate chemotherapy against Leishmania.

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo., Author summary MicroRNAs play an essential contribution among the many mechanisms used by cells to maintain a proper gene expression program. These endogenous small RNAs regulate gene expression by binding to target mRNAs and blocking their translation into functional proteins. To achieve this, microRNAs rely on Argonaute and other proteins that form a complex called the miRISC. While research on microRNAs has brought a lot of interest over the last years, it is still unclear what modulators affect the activity of the miRISC. Our study identified proteins implicated in intracellular trafficking as important factors for miRISC function. We showed that the intracellular location of the Argonaute protein needs to be tightly regulated to assure proper microRNA-mediated mRNA repression in animals.

The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, we sought to uncover the genetic modifiers of flight using a measure of flies’ reaction and response to an abrupt drop in a vertical flight column. We conducted a genome wide association study (GWAS) using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and identified a combination of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. We functionally validated 13 of the additive candidate genes’ (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo), and introduce a novel approach to whole gene significance screens: PEGASUS_flies. Additionally, we identified ppk23, an Acid Sensing Ion Channel (ASIC) homolog, as an important hub for epistatic interactions. We propose a model that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects., Author summary Insect flight is a widely recognizable phenotype of many winged insects, hence the name: flies. While fruit flies, or Drosophila melanogaster, are a genetically tractable model, flight performance is a highly integrative phenotype, and therefore challenging to identify comprehensively which genetic modifiers contribute to its genetic architecture. Accordingly, we screened 197 Drosophila Genetic Reference Panel lines for their ability to react and respond to an abrupt drop. Using several computational approaches, we identified additive, marginal, and epistatic variants, as well as whole genes and altered sub-networks of gene-gene and protein-protein interaction networks that contribute to variation in flight performance. More generally, we demonstrate the benefits of employing multiple methodologies to elucidate the genetic architecture of complex traits. Many variants and genes mapped to regions of the genome that affect neurodevelopment, wing and muscle development, and regulation of gene expression. We also introduce PEGASUS_flies, a Drosophila-adapted version of the PEGASUS platform first used in human studies, to infer gene-level significance of association based on the gene’s distribution of individual variant P-values. Our results contribute to the debate over the relative importance of individual, additive factors and epistatic, or higher order, interactions, in the mapping of genotype to phenotype.

The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer., Author summary RB1 is a tumor suppressor gene that is inactivated in a significant proportion of all cancer cases. A therapeutic approach that specifically targets defects associated with this tumor suppressor is currently not available. A synthetic lethal (SL) interaction occurs between two genes when the inactivation of either gene alone is viable but the inactivation of both genes simultaneously results in the loss of viability. Screening for SL interactions in model organisms can identify candidate genes/pathways that can be targeted to specifically kill tumor cells with a specific mutation. Here we used a cross-species approach to identify evolutionarily conserved SL targets for RB1-deficient cells. We demonstrated that drugs targeting the identified targets, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol, can selectively kill human RB1-deficient cancer cells. Because cancer cells are characterized by the accumulation of mutations in multiple oncogenic pathways, we further confirmed that these interactions are preserved in the presence of additional strong oncogenic alterations. Moreover, low activity of the identified SL genes in human tumors, when combined with low levels of RB1, was associated with improved patient survival. Altogether, we identified several novel targets of RB1-deficient cells that may be further adapted for the treatment of human tumors.

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR., Author summary The level of cholesterol circulating in the blood in low-density lipoproteins (LDL) is an important determinant of overall risk for cardiovascular diseases, including heart attack and stroke. This level is regulated by the removal of LDL from circulation into liver cells. While many molecules involved in LDL uptake have been characterized, we hypothesized that other currently unrecognized genetic interactions are also involved in this process. We therefore applied CRISPR-mediated genome editing to systematically test the contribution of every gene in the human genome to the uptake of LDL by a liver-derived cell line. We synthesized a secondary CRISPR library targeting the top candidate genes from this initial genome-wide screen to confirm their role in LDL uptake and to test their influence on other cellular functions. Our findings confirm the role of genes previously known to participate in LDL uptake and also provide novel insight into the overall regulation of this process.

The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance., Author summary Cancer cells rely on the ATR replication stress response pathway to ensure DNA replication and continued cellular proliferation. As such, inhibitors of the ATR kinase activity represent promising new anti-cancer drugs. However, the tumors’ susceptibility to these drugs can be markedly impacted by their genomic profile. To address this, we employed dual CRISPR knockout and activation genome-wide genetic screens to catalog the genetic determinants of the cellular resistance to multiple ATR inhibitors. We identified several mechanisms which control this resistance, including regulation of apoptosis and stabilization of replication fork stability. Our work lays the foundation for personalized deployment of ATR inhibitors in cancer therapy.

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators., Author summary Cells sense and respond to changes in their surrounding environment through G protein-coupled receptors (GPCRs) and their associated cascades. The proper function of these pathways is essential to human physiology, and GPCRs have become a prime target for drug development for a range of human diseases. Therefore, it is of utmost importance to be able to map how these pathways operate to enable cells to fine-tune their responsiveness. Here, we describe a screening approach that we have devised to systematically identify regulators of GPCR function. We have developed a sortable reporter system and coupled that with silencing of genes across the entire human genome in order to uncover a range of novel mediators of GPCR activity. We characterize a few of these new regulators and show that they function at different steps of the cascade. Therefore, this study serves as proof of principle for the new screening platform. We envision that the approach can be used to dissect additional dimensions of GPCR function, including regulators of drug-specific responses, functional characterization of receptor features, and identification of novel drugs, and thus advance a genome-scale understanding of these critical pathways.

CRISPR screens are a powerful technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By targeting non-coding sequences for perturbation, CRISPR screens have the potential to systematically discover novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from CRISPR screens. RELICS specifically addresses many of the challenges of non-coding CRISPR screens such as the unknown locations of functional sequences, overdispersion in the observed single guide RNA counts, and the need to combine information across multiple pools in an experiment. RELICS outperforms existing methods with higher precision, higher recall, and finer-resolution predictions on simulated datasets. We apply RELICS to published CRISPR interference and CRISPR activation screens to predict and experimentally validate novel regulatory sequences that are missed by other analysis methods. In summary, RELICS is a powerful new analysis method for CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy., Author summary Non-coding genome sequences contain a disproportionate number of genetic variants associated with human traits and diseases, however, interpretation of non-coding genetic variants is difficult because the molecular function of most non-coding sequences is unknown. By perturbing the genome using CRISPR, the function of non-coding sequences can be tested. Here we develop a new computational tool, RELICS, for the analysis of high-throughput CRISPR screens in which thousands of genome sequences are perturbed in a single experiment. Using simulated data, we find that RELICS has higher accuracy and resolution than other analysis methods. We apply RELICS to existing datasets to discover novel functional sequences and verify these predictions with experiments. In summary, RELICS is a powerful new analysis method for the discovery of functional sequences from CRISPR screens.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid—the apicoplast—which originated from a secondary (eukaryote–eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(−) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases., The apicoplast of the malaria parasite Plasmodium falciparum provides critical metabolites during host cell infection. An unbiased forward genetic screen identifies previously unknown genes required for biogenesis of this organelle, potentially yielding novel antimalarial drug targets., Author summary Plasmodium parasites, which cause malaria, and related apicomplexan parasites evolved from photosynthetic algae that acquired their chloroplast through two successive endosymbioses. Although no longer photosynthetic, the apicomplexan plastid—or apicoplast—was retained in these pathogens and provides critical metabolites during host cell infection. The apicoplast is of major interest for its unique biology and potential to yield new antimalarial drug targets. Here, we focused on the critical genes required to grow, divide, and inherit new apicoplasts during parasite replication. Given the apicoplast’s divergent evolution, most of these cannot be recognized by their homology to genes with known functions. Instead, we overcame significant technical challenges in the Plasmodium experimental system to perform an unbiased screen to search for these critical genes. Our screen has uncovered new genes with intriguing evolution and function that open up opportunities to understand and ultimately exploit apicoplast biology. Finally, assigning new, essential gene functions in Plasmodium parasites remains a daunting task. The successful identification of essential gene functions using an unbiased approach in this study provides a viable route for expansion of this screen or developing screens for other novel Plasmodium pathways in the future.

The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

There is a need to investigate and better understand the inherited risk of cancer to ensure that clinical applications provide more accurate assessments and management strategies. Developing research-based next-generation sequencing gene panels that not only target (present-day) clinically actionable susceptibility genes but also genes that currently lack sufficient evidence for risk as well as candidate genes, such as those in DNA repair pathways, can help aid this effort. Therefore, gene panel B.O.P. (Breast, Ovarian, and Prostate) was developed to evaluate the genetic risk of breast, ovarian and/or prostate cancer, and this manuscript serves as an introduction to B.O.P. and highlights its initial analytical validity assessment. B.O.P targets 87 genes that have been suggested, predicted, or clinically proven to be associated with breast, ovarian, and/or prostate cancer risk using Agilent Technologies Haloplex probes. The probes were designed for 100 base pair reads on an Illumina platform and target both coding and non-coding exons as well as 10 intronic base pairs flanking the intron-exon boundaries. The initial B.O.P screening involved 43 individuals from the Alabama Hereditary Cancer Cohort, and an average sequencing depth of 809X was obtained. Upon variant filtering and validation, true positives had an average sequencing depth of 659X and allele balance of 0.51. The average false positive sequencing depth was 34X and allele balance was 0.33. Although low sequencing depth was not always indicative of a false positive, high sequencing depths (>100X) signified a true positive. Furthermore, sensitivity and specificity of BRCA1/2 were calculated to be 100% and 92.3%, respectively. Overall, this screening enabled the establishment of criteria to alleviate future validation efforts and strongly supports the use of B.O.P. to further elucidate hereditary cancer susceptibility. Ultimately, continued B.O.P. screening will provide insights toward the genetic risk of and overlap between breast, ovarian, and/or prostate cancer.

Immune genes are under intense, pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of “Intracellular Pathogen Response” or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans., Author summary Infection by microbial pathogens imposes selective pressure on animal and plant hosts. For this reason, host immune genes tend to vary in DNA sequence over evolutionary time and become ‘species-specific’. In this work we describe a pair of species-specific genes called pals-22/pals-25 that promote resistance against natural pathogens of the small roundworm Caenorhabditis elegans. This gene pair acts as an on/off switch for genes that are activated after infection and rewires the physiology of worms to either be in an immune state or a growth state. In particular, the pals-22 gene promotes development and increases lifespan, while the pals-25 gene promotes resistance against heat shock and natural pathogens including virus and fungal-related microbes. While the DNA sequence of pals-22 and pals-25 appears specific to worms, the way that these two genes affect physiology may provide insight into how organisms balance growth and immunity.

Real-time fluorescent quantitative PCR (qRT-PCR) is often chosen as an effective experimental method for analyzing gene expression. However, an appropriate reference gene as a standard is needed to obtain accurate gene expression data. To date, no internal reference genes have been reported for research on cranberries. Expanding the selection of internal reference genes for cranberry will enable reliable gene expression analysis, and, at the same time, can also lay a solid foundation for revealing the biological characteristics of cranberry. Here, we selected ten candidate reference gene families and used three statistical software tools—geNorm, NormFinder and BestKeeper—to evaluate their expression stability under the influence of different experimental factors. The results showed that protein phosphatase 2A regulatory subunit (PP2A) or RNA helicase-like 8 (RH 8) was the best choice for an internal reference gene when analyzing different cranberry cultivars. In two sample sets comprising different cranberry organs and three abiotic stress treatments, sand family protein (SAND) was the best choice as a reference gene. In this study, we screened genes that are stably expressed under the influence of various experimental factors by qRT-PCR. Our results will guide future studies involving gene expression analysis of cranberry.

Hearing loss (HL) is a common sensory disorder. More than half of HL cases can be attributed to genetic causes. There is no effective therapy for genetic HL at present, early diagnosis to reduce the incidence of genetic HL is important for clinical intervention in genetic HL. Previous studies have identified 111 nonsyndromic hearing loss genes. The most frequently mutated genes identified in NSHL patients in China include GJB2, SLC26A4, and the mitochondrial gene MT-RNR1. It is important to develop HL gene panels in Chinese population, which allow for etiologic diagnosis of both SHL and NSHL. In this study, a total of 220 unrelated Han Chinese patients with bilateral progressive SNHL and 50 unrelated healthy controls were performed Single nucleotide polymorphism (SNP) genotyping using an improved multiplex ligation detection reaction (iMLDR) technique, is to simultaneously detect a total of 32 mutations in ten HL genes, covering all currently characterized mutations involved in the etiology of nonsyndromic or syndromic hearing loss in the Chinese population. The 49 positive samples with known mutations were successfully detected using the iMLDR Technique. For 171 SNHL patients, gene variants were found in 57 cases (33.33%), among which, 30 patients carried mutations in GJB2, 14 patients carried mutations in SLC26A4, seven patients carried mutations in GJB3, and six patients carried mutations in MT-RNR1. The molecular etiology of deafness was confirmed in 12.9% (22/171) of patients carried homozygous variants. These results were verified by Sanger sequencing, indicating that the sensitivity and specificity of the iMLDR technique was 100%. We believe that the implementation of this population-specific technology at an efficient clinical level would have great value in HL diagnosis and treatment.

The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates., Author summary The earliest stages of animal development are regulated by factors in the egg that are made during oogenesis and are required for the embryo to develop prior to genome activation of the embryo itself. Because eggs are large, the cells of the early embryo are large and so unique mechanisms act during these stages of development. To study these molecular-genetic processes in a vertebrate, we have chemically induced mutations in the zebrafish germline and screened for mutant mothers (maternal-effect mutants) with defects in these processes. We identified three distinct classes of mutations affecting cell division in the early embryo. One of these mutations also exhibits a male-sterile phenotype. We identify the mutated genes in three of these mutants. We expect the remaining mutant lines will serve as important tools for elucidating molecular mechanisms involved in cell organization and/or positioning during the cleavage stage, as well as mechanisms critical for proper patterning of the early embryo.

Our ability to identify genes that participate in cell growth and division is limited because their loss often leads to lethality. A solution to this is to isolate conditional mutants where the phenotype is visible under restrictive conditions. Here, we capitalize on the haploid growth-phase of the moss Physcomitrella patens to identify conditional loss-of-growth (CLoG) mutants with impaired growth at high temperature. We used whole-genome sequencing of pooled segregants to pinpoint the lesion of one of these mutants (clog1) and validated the identified mutation by rescuing the conditional phenotype by homologous recombination. We found that CLoG1 is a novel and ancient gene conserved in plants. At the restrictive temperature, clog1 plants have smaller cells but can complete cell division, indicating an important role of CLoG1 in cell growth, but not an essential role in cell division. Fluorescent protein fusions of CLoG1 indicate it is localized to microtubules with a bias towards depolymerizing microtubule ends. Silencing CLoG1 decreases microtubule dynamics, suggesting that CLoG1 plays a critical role in regulating microtubule dynamics. By discovering a novel gene critical for plant growth, our work demonstrates that P. patens is an excellent genetic system to study genes with a fundamental role in plant cell growth., Author summary Genes important for cell growth are difficult to identify because their disruption often results in the death of the organism. A solution to this problem is to isolate temperature-sensitive mutants where growth is blocked only at high temperatures. Here, we used the moss Physcomitrella patens, a simple model plant, to isolate temperature-sensitive mutants with reduced growth. We used whole-genome sequencing to identify the gene disrupted in one of these mutants (clog1). We found that CLoG1 is a previously uncharacterized gene present in algae and plants. Localization studies of CLoG1 protein in living cells showed CLoG1 concentrates on microtubules and tracks depolymerizing ends. Loss-of-function analysis suggests a possible role in controlling microtubule dynamics. Our approach establishes the moss P. patens as a valuable model-organism to investigate genes important for cell growth in plants.

Thrombin and other inflammatory mediators may induce vascular permeability through the disruption of adherens junctions between adjacent endothelial cells. If uncontrolled, hyperpermeability leads to an impaired barrier, fluid leakage and edema, which can contribute to multi-organ failure and death. RhoGTPases control cytoskeletal dynamics, adhesion and migration and are known regulators of endothelial integrity. Knowledge of the precise role of each RhoGTPase, and their associated regulatory and effector genes, in endothelial integrity is incomplete. Using a combination of a RNAi screen with electrical impedance measurements, we quantified the effect of individually silencing 270 Rho-associated genes on the barrier function of thrombin-activated, primary endothelial cells. Known and novel RhoGTPase-associated regulators that modulate the response to thrombin were identified (RTKN, TIAM2, MLC1, ARPC1B, SEPT2, SLC9A3R1, RACGAP1, RAPGEF2, RHOD, PREX1, ARHGEF7, PLXNB2, ARHGAP45, SRGAP2, ARHGEF5). In conclusion, with this siRNA screen, we confirmed the roles of known regulators of endothelial integrity but also identified new, potential key players in thrombin-induced endothelial signaling.

Pooled CRISPR libraries are widely used in high-throughput screening to study various biological processes. Various pooled CRISPR libraries have been shared for CRISPR screens and useful tools have been developed to construct researcher’s own libraries, however, many researchers are struggling to create their own pooled CRISPR libraries: it is a time-consuming, labor-intensive, and expensive process. In this study, we develop a simple method to customize conventional pooled CRISPR libraries using the CRISPR/Cas9 system. We show that conventional pooled CRISPR libraries can be modified by eliminating gRNAs that target positive genes, enabling the identification of unknown target genes in CRISPR screening. CRISPR/Cas9 system can be applied as a precise tool for customizing conventional pooled CRISPR libraries and will broaden the scope of high-throughput screening technology.

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria,Rickettsiaare highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improvedmariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group speciesRickettsia parkeri. These mutants targeted genes implicated in a variety of pathways, including bacterial replication and metabolism, hypothetical proteins, the type IV secretion system, as well as factors with previously established roles in host cell interactions and pathogenesis. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

Nemaline Myopathy (NM) is a rare genetic disorder that encompasses a large spectrum of myopathies characterized by hypotonia and generalized muscle weakness. To date, mutations in thirteen different genes have been associated with NM. The most frequently responsible genes are NEB (50% of cases) and ACTA1 (15-25% of cases). In this report all known NM related genes were screened by Next Generation Sequencing in five Spanish patients in order to genetically confirm the clinical and histological diagnosis of NM. Four mutations in NEB (c.17779_17780delTA, c.11086A>C, c.21076C>T and c.2310+5G>A) and one mutation in ACTA1 (c.871A>T) were found in four patients. Three of the four mutations in NEB were novel. A cDNA sequencing assay of the nove/variants c.17779_17780delTA, c.11086A>C and c.2310+5G>A revealed that the intronic variant c.2310+5G>A affected the splicing process. Mutations reported here could help clinicians and geneticists in NM diagnosis.

Morphotype switch is a cellular response to external and internal cues. The Cryptococcus neoformans species complex can undergo morphological transitions between the yeast and the hypha form, and such morphological changes profoundly affect cryptococcal interaction with various hosts. Filamentation in Cryptococcus was historically considered a mating response towards pheromone. Recent studies indicate the existence of pheromone-independent signaling pathways but their identity or the effectors remain unknown. Here, we demonstrated that glucosamine stimulated the C. neoformans species complex to undergo self-filamentation. Glucosamine-stimulated filamentation was independent of the key components of the pheromone pathway, which is distinct from pheromone-elicited filamentation. Glucosamine stimulated self-filamentation in H99, a highly virulent serotype A clinical isolate and a widely used reference strain. Through a genetic screen of the deletion sets made in the H99 background, we found that Crz1, a transcription factor downstream of calcineurin, was essential for glucosamine-stimulated filamentation despite its dispensability for pheromone-mediated filamentation. Glucosamine promoted Crz1 translocation from the cytoplasm to the nucleus. Interestingly, multiple components of the high osmolality glycerol response (HOG) pathway, consisting of the phosphorelay system and some of the Hog1 MAPK module, acted as repressors of glucosamine-elicited filamentation through their calcineurin-opposing effect on Crz1’s nuclear translocation. Surprisingly, glucosamine-stimulated filamentation did not require Hog1 itself and was distinct from the conventional general stress response. The results demonstrate that Cryptococcus can resort to multiple genetic pathways for morphological transition in response to different stimuli. Given that the filamentous form attenuates cryptococcal virulence and is immune-stimulatory in mammalian models, the findings suggest that morphogenesis is a fertile ground for future investigation into novel means to compromise cryptococcal pathogenesis., Author summary Cryptococcal meningitis claims half a million lives each year. There is no clinically available vaccine and the current antifungal therapies have serious limitations. Thus identifying cryptococcal specific programs that can be targeted for antifungal or vaccine development is of great value. We have shown previously that switching from the yeast to the hypha form drastically attenuates/abolishes cryptococcal virulence. Cryptococcal cells in the filamentous form also trigger host immune responses that can protect the host from a subsequent lethal challenge. However, self-filamentation is rarely observed in serotype A isolates that are responsible for the vast majority of cryptococcosis cases. In this study, we found that glucosamine stimulated self-filamentation in genetically distinct strains of the Cryptococcus species complex, including the most commonly used serotype A reference strain H99. We demonstrated that filamentation elicited by glucosamine did not depend on the pheromone pathway, but it requires the calcineurin transcription factor Crz1. Glucosamine promotes nuclear translocation of Crz1, which is positively controlled by the phosphatase calcineurin and is suppressed by the HOG pathway. These findings raise the possibility of manipulating genetic pathways controlling fungal morphogenesis against diseases caused by the Cryptococcus species complex.

In almost every field in genomics, large-scale biomedical datasets are used to report associations. Extracting associations that recur across multiple studies while controlling the false discovery rate is a fundamental challenge. Here, we propose a new method to allow joint analysis of multiple studies. Given a set of p-values obtained from each study, the goal is to identify associations that recur in at least k > 1 studies while controlling the false discovery rate. We propose several new algorithms that differ in how the study dependencies are modeled, and compare them and extant methods under various simulated scenarios. The top algorithm, SCREEN (Scalable Cluster-based REplicability ENhancement), is our new algorithm that works in three stages: (1) clustering an estimated correlation network of the studies, (2) learning replicability (e.g., of genes) within clusters, and (3) merging the results across the clusters. When we applied SCREEN to two real datasets it greatly outperformed the results obtained via standard meta-analysis. First, on a collection of 29 case-control gene expression cancer studies, we detected a large set of consistently up-regulated genes related to proliferation and cell cycle regulation. These genes are both consistently up-regulated across many cancer studies, and are well connected in known gene networks. Second, on a recent pan-cancer study that examined the expression profiles of patients with and without mutations in the HLA complex, we detected a large active module of up-regulated genes that are both related to immune responses and are well connected in known gene networks. This module covers thrice more genes as compared to the original study at a similar false discovery rate, demonstrating the high power of SCREEN. An implementation of SCREEN is available in the supplement., Author summary When analyzing results from multiple studies, extracting replicated associations is the first step towards making new discoveries. The standard approach for this task is to use meta-analysis methods, which usually make an underlying null hypothesis that a gene has no effect in all studies. On the other hand, in replicability analysis we explicitly require that the gene will manifest a recurring pattern of effects. In this study we develop new algorithms for replicability analysis that are both scalable (i.e., can handle many studies) and allow controlling the false discovery rate. We show that our main algorithm called SCREEN (Scalable Cluster-based REplicability ENhancement) outperforms the other methods in simulated scenarios. Moreover, when applied to real datasets, SCREEN greatly extended the results of the meta-analysis, and can even facilitate detection of new biological results.

A novel bunyavirus was recently found to cause severe febrile illness with high mortality in agricultural regions of China, Japan, and South Korea. This virus, named severe fever with thrombocytopenia syndrome virus (SFTSV), represents a new group within the Phlebovirus genus of the Bunyaviridae. Little is known about the viral entry requirements beyond showing dependence on dynamin and endosomal acidification. A haploid forward genetic screen was performed to identify host cell requirements for SFTSV entry. The screen identified dependence on glucosylceramide synthase (ugcg), the enzyme responsible for initiating de novo glycosphingolipid biosynthesis. Genetic and pharmacological approaches confirmed that UGCG expression and enzymatic activity were required for efficient SFTSV entry. Furthermore, inhibition of UGCG affected a post-internalization stage of SFTSV entry, leading to the accumulation of virus particles in enlarged cytoplasmic structures, suggesting impaired trafficking and/or fusion of viral and host membranes. These findings specify a role for glucosylceramide in SFTSV entry and provide a novel target for antiviral therapies., Author summary As obligate, intracellular parasites, viruses depend upon numerous host-derived factors to replicate. For many bunyaviruses, these pro-viral host factors are largely uncharacterized. In the present study, we used an unbiased genetic screening strategy to identify human genes that facilitate the entry process of a newly discovered pathogenic bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV). Our screen and subsequent validation confirmed a role for the glucose-modified lipid, glucosylceramide (GlcCer), in the entry of SFTSV. We found that depletion of GlcCer in the cell leads to the accumulation of virus particles in membranous compartments, suggesting that the virions cannot properly access the cytoplasm to initiate replication. Clinically available compounds that target GlcCer formation may provide a novel antiviral therapeutic strategy for this newly emerged virus.

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., Author summary Genetic material on each chromosome must be faithfully transmitted to the daughter cell during cell division and chromosomal instability (CIN) results in aneuploidy, a hallmark of cancers. The kinetochore (centromeric DNA and associated proteins) regulates faithful chromosome segregation. Restricting the localization of CENP-A (Cse4 in yeast) to kinetochores is essential for chromosomal stability. Mislocalization of CENP-A contributes to CIN in yeast, fly and human cells and is observed in cancers where it correlates with increased invasiveness and poor prognosis. Hence, identification of pathways that regulate CENP-A levels will help us understand the correlation between CENP-A mislocalization and aneuploidy in cancers. We used a genetic screen to identify essential genes for Cse4 homeostasis and identified a major ubiquitin-dependent pathway where both nuclear F-box proteins, Met30 and Cdc4 of the SCF complex, cooperatively regulate proteolysis of Cse4 to prevent its mislocalization and CIN under physiological conditions. Our studies define a role for SCF-mediated proteolysis of Cse4 as a critical mechanism to ensure faithful chromosome segregation. These studies are significant because mutations in human homologs of Met30 (β-TrCP) and Cdc4 (Fbxw7) have been implicated in cancers, and future studies will determine if SCF-mediated proteolysis of CENP-A prevents its mislocalization for chromosomal stability in human cells.

The aim of this study was to construct the spleen differential genes library of broilers fed with probiotic Bacillus cereus PAS38 by suppression subtractive hybridization (SSH) and screen the immune-related genes. Sixty seven-day-old broilers were randomly divided into two groups. The control group was fed with basal diet, and the treated group was fed with basal diet containing Bacillus cereus PAS38 1×106 CFU/g. Spleen tissues were taken and extracted its total RNA at 42 days old, then SSH was used to construct differential gene library and screen immune-related genes. A total of 119 differentially expressed sequence tags (ESTs) were isolated by SSH and 9 immune-related genes were screened out by Gene ontology analysis. Nine differentially expressed genes were identified by qRT-PCR. JCHAIN, FTH1, P2RX7, TLR7, IGF1R, SMAD7, and SLC7A6 were found to be significantly up-regulated in the treated group. Which was consistent with the results of SSH. These findings imply that probiotic Bacillus cereus PAS38-induced differentially expressed genes in spleen might play an important role in the improvement of immunity for broilers, which provided useful information for further understanding of the molecular mechanism of probiotics responsible to affect the poultry immunity.

What makes a gene essential for cellular survival? In model organisms, such as budding yeast, systematic gene deletion studies have revealed that paralog genes are less likely to be essential than singleton genes and that this can partially be attributed to the ability of paralogs to buffer each other's loss. However, the essentiality of a gene is not a fixed property and can vary significantly across different genetic backgrounds. It is unclear to what extent paralogs contribute to this variation, as most studies have analyzed genes identified as essential in a single genetic background. Here, using gene essentiality profiles of 558 genetically heterogeneous tumor cell lines, we analyze the contribution of paralogy to variable essentiality. We find that, compared to singleton genes, paralogs are less frequently essential and that this is more evident when considering genes with multiple paralogs or with highly sequence-similar paralogs. In addition, we find that paralogs derived from whole genome duplication exhibit more variable essentiality than those derived from small-scale duplications. We provide evidence that in 13–17% of cases the variable essentiality of paralogs can be attributed to buffering relationships between paralog pairs, as evidenced by synthetic lethality. Paralog pairs derived from whole genome duplication and pairs that function in protein complexes are significantly more likely to display such synthetic lethal relationships. Overall we find that many of the observations made using a single strain of budding yeast can be extended to understand patterns of essentiality in genetically heterogeneous cancer cell lines., Author summary Somewhat surprisingly, the majority of human genes can be mutated or deleted in individual cell lines without killing the cells. This observation raises a number of questions—which genes can be lost and why? Here we address these questions by analyzing data on which genes are essential for the growth of over 500 cancer cell lines. In general we find that paralog genes are essential in fewer cell lines than genes that are not paralogs. Paralogs are genes that have been duplicated at some point in evolutionary history, resulting in our genome having two copies of the same gene—a paralog pair. These paralog pairs are a potential source of redundancy, similar to a car having a spare tire. If this is the case, one might expect that losing one gene from a paralog pair could be tolerated by cells, due to the existence of a 'backup gene', but losing both members would cause cells to die. By analyzing the cancer cell lines we estimate this to be the case for 13–17% of paralog pairs, and that this provides an explanation for why some genes are essential in some cell lines but not others.

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction., Author summary Borrelia burgdorferi, the causative agent of Lyme disease, must adjust to environmental changes as it moves between its tick and vertebrate hosts. We performed a screen of a B. burgdorferi transposon library using massively parallel sequencing (Tn-seq) to identify fitness defects involved in survival in its tick host. This screen accurately identified genes known to cause decreased fitness for tick survival and identified new genes involved in B. burgdorferi survival in ticks. All of the genes tested individually confirmed the Tn-seq results. One of the genes identified encodes a protein whose function was previously unknown that appears to be involved in regulating expression of proteins known to be involved in environmental adaptation. Tn-seq is a powerful tool for understanding vector-pathogen interactions and may reveal new opportunities for interrupting the infectious cycle of vector-borne diseases.

The recently developed CRISPR screen technology, based on the CRISPR/Cas9 genome editing system, enables genome-wide interrogation of gene functions in an efficient and cost-effective manner. Although many computational algorithms and web servers have been developed to design single-guide RNAs (sgRNAs) with high specificity and efficiency, algorithms specifically designed for conducting CRISPR screens are still lacking. Here we present CRISPR-FOCUS, a web-based platform to search and prioritize sgRNAs for CRISPR screen experiments. With official gene symbols or RefSeq IDs as the only mandatory input, CRISPR-FOCUS filters and prioritizes sgRNAs based on multiple criteria, including efficiency, specificity, sequence conservation, isoform structure, as well as genomic variations including Single Nucleotide Polymorphisms and cancer somatic mutations. CRISPR-FOCUS also provides pre-defined positive and negative control sgRNAs, as well as other necessary sequences in the construct (e.g., U6 promoters to drive sgRNA transcription and RNA scaffolds of the CRISPR/Cas9). These features allow users to synthesize oligonucleotides directly based on the output of CRISPR-FOCUS. Overall, CRISPR-FOCUS provides a rational and high-throughput approach for sgRNA library design that enables users to efficiently conduct a focused screen experiment targeting up to thousands of genes. (CRISPR-FOCUS is freely available at http://cistrome.org/crispr-focus/).

International audience; Expression quantitative trait (eQTL) studies are a powerful tool for identifying genetic variants that affect levels of messenger RNA. Since gene expression is controlled by a complex network of gene-regulating factors, one way to identify these factors is to search for interaction effects between genetic variants and mRNA levels of transcription factors (TFs) and their respective target genes. However, identification of interaction effects in gene expression data pose a variety of methodological challenges, and it has become clear that such analyses should be conducted and interpreted with caution. Investigating the validity and interpretability of several interaction tests when screening for eQTL SNPs whose effect on the target gene expression is modified by the expression level of a transcription factor, we characterized two important methodological issues. First, we stress the scale-dependency of interaction effects and highlight that commonly applied transformation of gene expression data can induce or remove interactions, making interpretation of results more challenging. We then demonstrate that, in the setting of moderate to strong interaction effects on the order of what may be reasonably expected for eQTL studies, standard interaction screening can be biased due to heteroscedasticity induced by true interactions. Using simulation and real data analysis, we outline a set of reasonable minimum conditions and sample size requirements for reliable detection of variant-by-environment and variant-by-TF interactions using the heteroscedasticity consistent covariance-based approach.

Background Congenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required. Methods We developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)–associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates. Results The test was validated through direct sequencing in a blinded study with 100% concordance. Conclusions The results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.