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The zinc finger-associated domain (ZAD) is present in over 90 C2H2 zinc finger (ZNF) proteins. Despite their abundance, only a few ZAD-ZNF genes have been characterized to date. Here, we systematically analyze the function of 68 ZAD-ZNF genes in Drosophila female germ cells by performing an in vivo RNA-interference screen. We identified eight ZAD-ZNF genes required for oogenesis, and based on further characterization of the knockdown phenotypes, we uncovered defects broadly consistent with functions in germ cell specification and/or survival, early differentiation, and egg chamber maturation. These results provide a candidate pool for future studies aimed at functionalization of this large but poorly characterized gene family.

The human genome encodes five RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) that participate in various processes underpinning genomic stability. Of these enzymes, the disease-associated RECQL4 is comparatively understudied due to a variety of technical challenges. However,Saccharomyces cerevisiaeencodes a functional homolog of RECQL4 called Hrq1, which is more amenable to experimentation and has recently been shown to be involved in DNA inter-strand crosslink (ICL) repair and telomere maintenance. To expand our understanding of Hrq1 and the RecQ4 subfamily of helicases in general, we took a multi-omics approach to define the Hrq1 interactome in yeast. Using synthetic genetic array analysis, we found that mutations of genes involved in processes such as DNA repair, chromosome segregation, and transcription synthetically interact with deletion ofHRQ1and the catalytically inactivehrq1-K318Aallele. Pull-down of tagged Hrq1 and mass spectrometry identification of interacting partners similarly underscored links to these processes and others. Focusing on transcription, we found thathrq1mutant cells are sensitive to caffeine and that mutation ofHRQ1alters the expression levels of hundreds of genes. In the case ofhrq1-K318A, several of the most highly upregulated genes encode proteins of unknown function whose expression levels are also increased by DNA ICL damage. Together, our results suggest a heretofore unrecognized role for Hrq1 in transcription, as well as novel members of the Hrq1 ICL repair pathway. These data expand our understanding of RecQ4 subfamily helicase biology and help to explain why mutations in human RECQL4 cause diseases of genomic instability.

The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process we performed genetic screens using a collection of 480 P{EP} transposon insertion strains. We identified genes in gain of function and loss of function screens that significantly altered the percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 36 strains resulted in altered Rh3% in the gain of function screen where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 53 strains resulted in altered Rh3% in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the P{EP} insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, hedgehog signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.

Biological tube formation underlies organ development and, when disrupted, can cause severe birth defects. To investigate the genetic basis of tubulogenesis, we study the formation of Drosophila melanogaster eggshell structures, called dorsal appendages, which are produced by epithelial tubes. Previously we found that precise levels of Drosophila Chitinase-Like Proteins (CLPs), encoded by the Imaginal disc growth factor (Idgf) gene family, are needed to regulate dorsal-appendage tube closure and tube migration. To identify factors that act in the Idgf pathway, we developed a genetic modifier screen based on the finding that overexpressing Idgf3 causes dorsal appendage defects with ∼50% frequency. Using a library of partially overlapping heterozygous deficiencies, we scanned chromosome 3L and found regions that enhanced or suppressed the Idgf3-overexpression phenotype. Using smaller deletions, RNAi, and mutant alleles, we further mapped five regions and refined the interactions to 58 candidate genes. Importantly, mutant alleles identified combover (cmb), a substrate of Rho-kinase (Rok) and a component of the Planar Cell Polarity (PCP) pathway, as an Idgf3-interacting gene: loss of function enhanced while gain of function suppressed the dorsal appendage defects. Since PCP drives cell intercalation in other systems, we asked if cmb/+ affected cell intercalation in our model, but we found no evidence of its involvement in this step. Instead, we found that loss of cmb dominantly enhanced tube defects associated with Idgf3 overexpression by expanding the apical area of dorsal appendage cells. Apical surface area determines tube volume and shape; in this way, Idgf3 and cmb regulate tube morphology.

Genetic screens in Saccharomyces cerevisiae have allowed for the identification of many genes as sensors or effectors of DNA damage, typically by comparing the fitness of genetic mutants in the presence or absence of DNA-damaging treatments. However, these static screens overlook the dynamic nature of DNA damage response pathways, missing time-dependent or transient effects. Here, we examine gene dependencies in the dynamic response to ultraviolet radiation-induced DNA damage by integrating ultra-high-density arrays of 6144 diploid gene deletion mutants with high-frequency time-lapse imaging. We identify 494 ultraviolet radiation response genes which, in addition to recovering molecular pathways and protein complexes previously annotated to DNA damage repair, include components of the CCR4-NOT complex, tRNA wobble modification, autophagy, and, most unexpectedly, 153 nuclear-encoded mitochondrial genes. Notably, mitochondria-deficient strains present time-dependent insensitivity to ultraviolet radiation, posing impaired mitochondrial function as a protective factor in the ultraviolet radiation response.

The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumor suppressor mutants, including brain tumor and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2l(3)tb. Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5′UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumorigenesis. Our findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use.

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens inSaccharomyces cerevisiaefor genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionary conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

LRRK2 mutations cause Parkinson’s, but the molecular link from increased kinase activity to pathological neurodegeneration remains undetermined. Previous in vitro assays indicate that LRRK2 substrates include at least 8 Rab GTPases. We have now examined this hypothesis in vivo in a functional, electroretinogram screen, expressing each Rab with/without LRRK2-G2019S in selected Drosophila dopaminergic neurons. Our screen discriminated Rab10 from Rab3. The strongest Rab/LRRK2-G2019S interaction is with Rab10; the weakest with Rab3. Rab10 is expressed in a different set of dopaminergic neurons from Rab3. Thus, anatomical and physiological patterns of Rab10 are related. We conclude that Rab10 is a valid substrate of LRRK2 in dopaminergic neurons in vivo. We propose that variations in Rab expression contribute to differences in the rate of neurodegeneration recorded in different dopaminergic nuclei in Parkinson’s.

Protein quality control in the cytosol (CytoQC) is an important cellular pathway consisting of a network of components which monitor the folding of cytosolic proteins and ensure the efficient removal of aberrant ones. Our understanding of CytoQC mechanisms is limited in part by the ability of current approaches to identify new genes in the pathway. In this study, we developed a CytoQC reporter substrate, Ste6*C-HA-Ura3, for a new genetic selection of spontaneous CytoQC mutations in the yeast Saccharomyces cerevisiae. In addition to UBR1, which encodes for a known CytoQC E3 ligase, we identified six new CytoQC candidates. In the preliminary characterization of two mutants, we found that Doa4 is involved in the degradation of misfolded substrates while Pup2 functions in the selectivity of CytoQC and ERAD substrates. Overall, the strategy demonstrates the potential to identify novel genes and advance our understanding of CytoQC., G3: Genes, Genomes, Genetics, 10 (6), ISSN:2160-1836

FUS is a nucleic acid binding protein that, when mutated, cause a subset of familial amyotrophic lateral sclerosis (ALS). Expression of FUS in yeast recapitulates several pathological features of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, formation of cytoplasmic inclusions, and cytotoxicity. Genetic screens using the yeast model of FUS have identified yeast genes and their corresponding human homologs suppressing FUS induced toxicity in yeast, neurons and animal models. To expand the search for human suppressor genes of FUS induced toxicity, we carried out a genome-scale genetic screen using a newly constructed library containing 13570 human genes cloned in an inducible yeast-expression vector. Through multiple rounds of verification, we found 37 human genes that, when overexpressed, suppress FUS induced toxicity in yeast. Human genes with DNA or RNA binding functions are overrepresented among the identified suppressor genes, supporting that perturbations of RNA metabolism is a key underlying mechanism of FUS toxicity.

Metastatic colonisation, whereby a disseminated tumour cell is able to survive and proliferate at a secondary site, involves both tumour cell-intrinsic and -extrinsic factors. To identify tumour cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumour cells to effectively colonise a tissue, we performed a genome-wide screen utilising the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonisation (15 increased and 19 decreased;P+60). Whilst several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4andPik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30andFgd4), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonisation, which may represent potential therapeutic targets.

We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by phenotype selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. This method also depends on the use of CRISPR/Cas9 based mutagenesis to recover alleles of a GOI that are refractory to CRISPR/Cas9 mediated activation of gene expression but are otherwise wild type. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.

Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe. As opposed to many other alkylating agents, ENU only induces an SN1-type reaction with a low s constant (s = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields., This work was supported by Grupos Emergentes UPO grant APP2D1011 to V.A.T. and BFU2016-77297 grant from the Spanish MINECO to J.J. R.H.M. is supported by FPU grant FPU16/04283 from the Spanish MECD. S.V.C is supported by FPU grant FPU18/04507 from the Spanish MEFP.

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.

In light of the rising prevalence of antimicrobial resistance (AMR) and the slow pace of new antimicrobial development, there has been increasing interest in the development of adjuvants that improve or restore the effectiveness of existing drugs. Here, we use a novel small RNA (sRNA) screening approach to identify genes whose knockdown increases ciprofloxacin (CIP) sensitivity in a resistant strain of Escherichia coli. 5000 sRNA constructs were initially screened on a gyrA S83L background, ultimately leading to 30 validated genes whose disruption reduces CIP resistance. This set includes genes involved in DNA replication, repair, recombination, efflux, and other regulatory systems. Our findings increase understanding of the functional interactions of DNA Gyrase, and may aid in the development of new therapeutic approaches for combating AMR.

Prostaglandins (PGs) are lipid signaling molecules with numerous physiologic functions, including pain/inflammation, fertility, and cancer. PGs are produced downstream of cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs). In numerous systems, PGs regulate actin cytoskeletal remodeling, however, their mechanisms of action remain largely unknown. To address this deficiency, we undertook a pharmaco-genetic interaction screen during late-stageDrosophilaoogenesis.Drosophilaoogenesis is as an established model for studying both actin dynamics and PGs. Indeed, during Stage 10B, cage-like arrays of actin bundles surround each nurse cell nucleus, and during Stage 11, the cortical actin contracts, squeezing the cytoplasmic contents into the oocyte. Both of these cytoskeletal properties are required for follicle development and fertility, and are regulated by PGs. Here we describe a pharmaco-genetic interaction screen that takes advantage of the facts that Stage 10B follicles will mature in culture and COX inhibitors, such as aspirin, block thisin vitrofollicle maturation. In the screen, aspirin was used at a concentration that blocks 50% of the wild-type follicles from maturing in culture. By combining this aspirin treatment with heterozygosity for mutations in actin regulators, we quantitatively identified enhancers and suppressors of COX inhibition. Here we present the screen results and initial follow-up studies on three strong enhancers – Enabled, Capping protein, and non-muscle Myosin II Regulatory Light Chain. Overall, these studies provide new insight into how PGs regulate both actin bundle formation and cellular contraction, properties that are not only essential for development, but are misregulated in diseases.

The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport; however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. We conducted a screen to determine how the INM proteome differs between mitotic cells and gametes. In addition, we used a strategy that allowed us to determine if spores synthesize their INM proteins de novo, rather than inheriting their INM proteins from the parental cell. This screen used a split-GFP complementation system, where we were able to compare the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation, and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis. Taken together, our data provide evidence for INM changes during gametogenesis and shed light on mechanisms used to shape the INM proteome of spores.

Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma (GBM), which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell–cell adhesion proteins during their migration through tissues and organs. In this study, we first identified cell adhesion, cell matrix, cell junction, and associated regulatory genes that are expressed in human brain tumors. We performed RNAi knockdown of the Drosophila orthologs in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, we identified eight adhesion-related genes that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human GBM patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to GBM and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

The Drosophila genome contains approximately 14,000 protein-coding genes encoding all the necessary information to sustain cellular physiology, tissue organization, organism development, and behavior. In this manuscript, we describe in some detail the phenotypes in the adult fly wing generated after knockdown of approximately 80% of Drosophila genes. We combined this phenotypic description with a comprehensive molecular classification of the Drosophila proteins into classes that summarize the main expected or known biochemical/functional aspect of each protein. This information, combined with mRNA expression levels and in situ expression patterns, provides a simplified atlas of the Drosophila genome, from housekeeping proteins to the components of the signaling pathways directing wing development, that might help to further understand the contribution of each gene group to wing formation.

Protein kinases and phosphatases constitute a large family of conserved enzymes that control a variety of biological processes by regulating the phosphorylation state of target proteins. They play fundamental regulatory roles during cell cycle progression and signaling, among other key aspects of multicellular development. The complement of protein kinases and phosphatases includes approximately 326 members in Drosophila, and they have been the subject of several functional screens searching for novel components of signaling pathways and regulators of cell division and survival. These approaches have been carried out mostly in cell cultures using RNA interference to evaluate the contribution of each protein in different functional assays and have contributed significantly to assign specific roles to the corresponding genes. In this work, we describe the results of an evaluation of the Drosophila complement of kinases and phosphatases using the wing as a system to identify their functional requirements in vivo. We also describe the results of several modifying screens aiming to identify among the set of protein kinases and phosphatases additional components or regulators of the activities of the epidermal growth factor and insulin receptors signaling pathways., PGC2018-094476-B-100 from the Secretaría del Estado de Investigación, Desarrollo e Innovación. The CBMSO enjoys institutional support from the Fundación Ramón Areces and Fundación Banco de Santander.

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

Aberration in nuclear morphology is one of the hallmarks of cellular transformation. However, the processes that, when mis-regulated, result aberrant nuclear morphology are poorly understood. In this study we carried out a systematic, high-throughput RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans embryos. The screen employed over 1700 RNAi constructs against genes required for embryonic viability. Nuclei of early embryos are typically spherical and their NPCs are evenly distributed. The screen was performed on early embryos expressing a fluorescently tagged component of the nuclear pore complex (NPC), allowing visualization of nuclear shape as well as the distribution of NPCs around the nuclear envelope. Our screen uncovered 182 genes whose down-regulation resulted in one or more abnormal nuclear phenotypes, including multiple nuclei, micronuclei, abnormal nuclear shape, anaphase bridges and abnormal NPC distribution. Many of these genes fall into common functional groups, including some that were not previously known to affect nuclear morphology, such as genes involved in mitochondrial function, the vacuolar ATPase and the CCT chaperonin complex. The results of this screen add to our growing knowledge of processes that affect nuclear morphology and that may be altered in cancer cells that exhibit abnormal nuclear shape.

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however, the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the APCs exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of S. cerevisiae titratable-essential gene and haploid nonessential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). Fifty-eight strains out of ∼5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER-resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developingDrosophilabody wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendons. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, includingbsd, kon, ths, andtum, arguing our screening strategy was effective and efficient. We also identified and sequenced new alleles ofsalm,barr, andptcthat presumably disrupt independent pathways directing muscle morphogenesis. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be identified. This screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that determine skeletal muscle topology.

Metastasis is the spread of cancer cells to a secondary site within the body, and is the leading cause of death for cancer patients. The lung is a common site of metastasis for many cancer types, including melanoma. Identifying the genes involved in aiding metastasis of melanoma cells to the lungs is critical for the development of better treatments. As the accessibility of cell surface proteins makes them attractive therapeutic targets, we performed a CRISPR activation screen using a library of guide RNAs (gRNAs) targeting the transcription start sites of 2195 membrane protein-encoding genes, to identify genes whose upregulated expression aided pulmonary metastasis. Immunodeficient mice were subcutaneously injected in the flank with murine B16-F0 melanoma cells expressing dCas9 and the membrane protein library gRNAs, and their lungs collected after 14–21 days. Analysis was performed to identify the gRNAs that were enriched in the lungs relative to those present in the cells at the time of administration (day 0). We identified six genes whose increased expression promotes lung metastasis. These genes included several with well-characterized pro-metastatic roles (Fut7, Mgat5, and Pcdh7) that have not previously been linked to melanoma progression, genes linked to tumor progression but that have not previously been described as involved in metastasis (Olfr322 and Olfr441), as well as novel genes (Tmem116). Thus, we have identified genes that, when upregulated in melanoma cells, can aid successful metastasis and colonization of the lung, and therefore may represent novel therapeutic targets to inhibit pulmonary metastasis.

In order to identify new players modulating the circadian clock in the model Neurospora crassa, we adopted a reverse genetics strategy. Thus, we focused on transcription factors knockouts and crossed them to strains containing circadian luciferase reporters. Our screen covered close to 60% de the 302 genes encoding for such proteins in Neurospora, identifying that 23 of them appear to modulate period, while none of the tested ones (besides the classic core-clock components) are essential for daily rhythms., Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors (TFs) knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and -2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 TFs that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated TFs, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

Two people with the same lifespan do not necessarily have the same healthspan. One person may retain locomotor and cognitive abilities until the end of life, while another person may lose them during adulthood. Unbiased searches for genes that are required to maintain locomotor ability during adulthood may uncover key regulators of locomotor healthspan. Here, we take advantage of the relatively short lifespan of the nematode Caenorhabditis elegans and develop a novel screening procedure to collect mutants with locomotor deficits that become apparent in adulthood. After ethyl methanesulfonate mutagenesis, we isolated five C. elegans mutant strains that progressively lose adult locomotor ability. In one of the mutant strains, a nonsense mutation in elpc-2, which encodes Elongator Complex Protein Component 2, causes a progressive decline in locomotor ability during adulthood. Mutants and mutations identified in the present screen may provide insights into mechanisms of age-related locomotor impairment and the maintenance of locomotor healthspan.

The metabolic enzymes that compose glycolysis, the citric acid cycle, and other pathways within central carbon metabolism have emerged as key regulators of animal development. These enzymes not only generate the energy and biosynthetic precursors required to support cell proliferation and differentiation, but also moonlight as regulators of transcription, translation, and signal transduction. Many of the genes associated with animal metabolism, however, have never been analyzed in a developmental context, thus highlighting how little is known about the intersection of metabolism and development. Here we address this deficiency by using theDrosophilaTRiP RNAi collection to disrupt the expression of over 1,100 metabolism-associated genes within cells of the eye imaginal disc. Our screen not only confirmed previous observations that oxidative phosphorylation serves a critical role in the developing eye, but also implicated a host of other metabolic enzymes in the growth and differentiation of this organ. Notably, our analysis revealed a requirement for glutamine and glutamate metabolic processes in eye development, thereby revealing a role of these amino acids in promotingDrosophilatissue growth. Overall, our analysis highlights how theDrosophilaeye can serve as a powerful tool for dissecting the relationship between development and metabolism.

Living organisms encounter various perturbations, and response mechanisms to such perturbations are vital for species survival. Defective stress responses are implicated in many human diseases including cancer and neurodegenerative disorders. Phenol derivatives, naturally occurring and synthetic, display beneficial as well as detrimental effects. The phenol derivatives in this study, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and bisphenol A (BPA), are widely used as food preservatives and industrial chemicals. Conflicting results have been reported regarding their biological activity and correlation with disease development; understanding the molecular basis of phenol action is a key step for addressing issues relevant to human health. This work presents the first comparative genomic analysis of the genetic networks for phenol stress response in an evolutionary context of two divergent yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genomic screening of deletion strain libraries of the two yeasts identified genes required for cellular response to phenol stress, which are enriched in human orthologs. Functional analysis of these genes uncovered the major signaling pathways involved. The results provide a global view of the biological events constituting the defense process, including cell cycle arrest, DNA repair, phenol detoxification by V-ATPases, reactive oxygen species alleviation, and endoplasmic reticulum stress relief through ergosterol and the unfolded protein response, revealing novel roles for these cellular pathways.

During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development.

Metabolic state is a potent modulator of sleep and circadian behavior, and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Across phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes.

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole-hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR–dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs, and organize the body plan. Cell adhesion and the cytoskeleton are important for executing morphogenesis, but their regulation remains poorly understood. As genes required for embryonic morphogenesis may have earlier roles in development, temperature-sensitive embryonic-lethal mutations are useful tools for investigating this process. From a collection of ∼200 such Caenorhabditis elegans mutants, we have identified 17 that have highly penetrant embryonic morphogenesis defects after upshifts from the permissive to the restrictive temperature, just prior to the cell shape changes that mediate elongation of the ovoid embryo into a vermiform larva. Using whole genome sequencing, we identified the causal mutations in seven affected genes. These include three genes that have roles in producing the extracellular matrix, which is known to affect the morphogenesis of epithelial tissues in multicellular organisms: the rib-1 and rib-2 genes encode glycosyltransferases, and the emb-9 gene encodes a collagen subunit. We also used live imaging to characterize epidermal cell shape dynamics in one mutant, or1219ts, and observed cell elongation defects during dorsal intercalation and ventral enclosure that may be responsible for the body elongation defects. These results indicate that our screen has identified factors that influence morphogenesis and provides a platform for advancing our understanding of this fundamental biological process.

Vemurafenib is a BRAF kinase inhibitor (BRAFi) that is used to treat melanoma patients harboring the constitutively active BRAF-V600E mutation. However, after a few months of treatment patients often develop resistance to vemurafenib leading to disease progression. Sequence analysis of drug-resistant tumor cells and functional genomic screens has identified several genes that regulate vemurafenib resistance. Reactivation of mitogen-activated protein kinase (MAPK) pathway is a recurrent feature of cells that develop resistance to vemurafenib. We performed a genome-scale CRISPR-based knockout screen to identify modulators of vemurafenib resistance in melanoma cells with a highly improved CRISPR sgRNA library called Brunello. We identified 33 genes that regulate resistance to vemurafenib out of which 14 genes have not been reported before. Gene ontology enrichment analysis showed that the hit genes regulate histone modification, transcription and cell cycle. We discuss how inactivation of hit genes might confer resistance to vemurafenib and provide a framework for follow-up investigations.

Mitochondrial dynamics plays an important role in mitochondrial quality control and the adaptation of metabolic activity in response to environmental changes. The disruption of mitochondrial dynamics has detrimental consequences for mitochondrial and cellular homeostasis and leads to the activation of the mitochondrial unfolded protein response (UPRmt), a quality control mechanism that adjusts cellular metabolism and restores homeostasis. To identify genes involved in the induction of UPRmt in response to a block in mitochondrial fusion, we performed a genome-wide RNAi screen in Caenorhabditis elegans mutants lacking the gene fzo-1, which encodes the ortholog of mammalian Mitofusin, and identified 299 suppressors and 86 enhancers. Approximately 90% of these 385 genes are conserved in humans, and one-third of the conserved genes have been implicated in human disease. Furthermore, many have roles in developmental processes, which suggests that mitochondrial function and their response to stress are defined during development and maintained throughout life. Our dataset primarily contains mitochondrial enhancers and non-mitochondrial suppressors of UPRmt, indicating that the maintenance of mitochondrial homeostasis has evolved as a critical cellular function, which, when disrupted, can be compensated for by many different cellular processes. Analysis of the subsets “non-mitochondrial enhancers” and “mitochondrial suppressors” suggests that organellar contact sites, especially between the ER and mitochondria, are of importance for mitochondrial homeostasis. In addition, we identified several genes involved in IP3 signaling that modulate UPRmt in fzo-1 mutants and found a potential link between pre-mRNA splicing and UPRmt activation.

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgs1-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

Stem cells are tightly controlled in vivo. Both the balance between self-renewal and differentiation and the rate of proliferation are often regulated by multiple factors. The Caenorhabditis elegans hermaphrodite germ line provides a simple and accessible system for studying stem cells in vivo. In this system, GLP-1/Notch activity prevents the differentiation of distal germ cells in response to ligand production from the nearby distal tip cell, thereby supporting a stem cell pool. However, a delay in germline development relative to somatic gonad development can cause a pool of undifferentiated germ cells to persist in response to alternate Notch ligands expressed in the proximal somatic gonad. This pool of undifferentiated germ cells forms a proximal tumor that, in adulthood, blocks the oviduct. This type of “latent niche”-driven proximal tumor is highly penetrant in worms bearing the temperature-sensitive weak gain-of-function mutation glp-1(ar202) at the restrictive temperature. At the permissive temperature, few worms develop tumors. Nevertheless, several interventions elevate the penetrance of proximal tumor formation at the permissive temperature, including reduced insulin signaling or the ablation of distal-most sheath cells. To systematically identify genetic perturbations that enhance proximal tumor formation, we sought genes that, upon RNAi depletion, elevate the percentage of worms bearing proximal germline tumors in glp-1(ar202) at the permissive temperature. We identified 43 genes representing a variety of functional classes, the most enriched of which is “translation”. Some of these genes also influence the distal germ line, and some are conserved genes for which genetic interactions with Notch were not previously known in this system.

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1 resistance. Here, we profiled the yeast genome for AFB1 resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 μM AFB1 for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB1 resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1 resistance genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

To investigate the genetic diversity of Chinese indigenous horses and determine the genetic status of extant horse breeds, novel Y chromosomal microsatellite markers and known Y chromosomal SNPs and mtDNA loop sequences, were employed to study the genetic diversity levels of 13 Chinese indigenous horse populations and four introduced breeds. Sixteen Y-chromosomal microsatellite markers, including seven newly identified loci, were used in the genotyping. The results showed that 4 out of the 16 loci were highly polymorphic in Chinese indigenous horse populations, in which the polymorphisms of 3 loci, ECAYP12, ECAYP13, and ECAYCAU3, were first reported in the present study. The polymorphic Y chromosomal microsatellite markers result in 19 haplotypes in the studied horses and formed 24 paternal lines when merged with the 14 Y chromosomal SNPs reported previously. The haplotypes CHT18 and SS24 harboring AMELY gene mutation were the ancestral haplotypes, and other haplotypes were derived from them by one or more mutation steps. The horse populations in mountainous and remote areas of southwestern China have the most ancient paternal lines, which suggests that ancient paternal lines preserved in local populations attributed to less human interventions. Our results also showed that the northern local breeds had higher mtDNA diversity than the southern ones in China. The frequency of haplogroup B, F, and G of mtDNA in Chinese indigenous horses has declined in recent years, and some breeds are in endangered status mainly due to small population sizes. Urgent actions should be taken to conserve the genetic diversity of the indigenous horse populations, especially the rare paternal lines. Our findings help to elucidate the genetic diversity and evolutionary history of Chinese domestic horses, which will facilitate the conservation of the indigenous horses in the future.

Alteration of the lipid composition of biological membranes interferes with their function and can cause tissue damage by triggering apoptosis. Upon lipid bilayer stress, the endoplasmic reticulum mounts a stress response similar to the unfolded protein response. However, only a few genes are known to regulate lipid bilayer stress. We performed a suppressor screen that combined the auxin-inducible degradation (AID) system with conventional RNAi in C. elegans to identify members of the lipid bilayer stress response. AID-mediated degradation of the mediator MDT-15, a protein required for the upregulation of fatty acid desaturases, induced the activation of lipid bilayer stress-sensitive reporters. We screened through most C. elegans kinases and transcription factors by feeding RNAi. We discovered nine genes that suppressed the lipid bilayer stress response in C. elegans. These suppressor genes included drl-1/MAP3K3, gsk-3/GSK3, let-607/CREB3, ire-1/IRE1, and skn-1/NRF1,2,3. Our candidate suppressor genes suggest a network of transcription factors and the integration of multiple tissues for a centralized lipotoxicity response in the intestine. Thus, we demonstrated proof-of-concept for combining AID and RNAi as a new screening strategy and identified eight conserved genes that had not previously been implicated in the lipid bilayer stress response.

The fungal species Candida albicans is both a member of the human microbiome and a fungal pathogen. C. albicans undergoes several different morphological transitions, including one called white-opaque switching. Here, cells reversibly switch between two states, “white” and “opaque,” and each state is heritable through many cell generations. Each cell type has a distinct cellular and colony morphology and they differ in many other properties including mating, nutritional specialization, and interactions with the innate immune system. Previous genetic screens to gain insight into white-opaque switching have focused on certain classes of genes (for example transcriptional regulators or chromatin modifying enzymes). In this paper, we examined 172 deletion mutants covering a broad range of cell functions. We identified 28 deletion mutants with at least a fivefold effect on switching frequencies; these cover a wide variety of functions ranging from membrane sensors to kinases to proteins of unknown function. In agreement with previous reports, we found that components of the pheromone signaling cascade affect white-to-opaque switching; however, our results suggest that the major effect of Cek1 on white-opaque switching occurs through the cell wall damage response pathway. Most of the genes we identified have not been previously implicated in white-opaque switching and serve as entry points to understand new aspects of this morphological transition.

At the oocyte-to-embryo transition the highly differentiated oocyte arrested in meiosis becomes a totipotent embryo capable of embryogenesis. Oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest and the trigger for the oocyte-to-embryo transition) serve as prerequisites for this transition, both events being controlled posttranscriptionally. Recently, we obtained a comprehensive list of proteins whose levels are developmentally regulated during these events via a high-throughput quantitative proteomic analysis of Drosophila melanogaster oocyte maturation and egg activation. We conducted a targeted screen for potential novel regulators of the oocyte-to-embryo transition, selecting 53 candidates from these proteins. We reduced the function of each candidate gene using transposable element insertion alleles and RNAi, and screened for defects in oocyte maturation or early embryogenesis. Deletion of the aquaporin gene CG7777 did not affect female fertility. However, we identified CG5003 and nebu (CG10960) as new regulators of the transition from oocyte to embryo. Mutations in CG5003, which encodes an F-box protein associated with SCF-proteasome degradation function, cause a decrease in female fertility and early embryonic arrest. Mutations in nebu, encoding a putative glucose transporter, result in defects during the early embryonic divisions, as well as a developmental delay and arrest. nebu mutants also exhibit a defect in glycogen accumulation during late oogenesis. Our findings highlight potential previously unknown roles for the ubiquitin protein degradation pathway and sugar transport across membranes during this time, and paint a broader picture of the underlying requirements of the oocyte-to-embryo transition.

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.

Mutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a non-syndromic form of autosomal recessive intellectual disability. Studies inDrosophilahave defined roles for the ZC3H14 ortholog, Nab2 (akaDrosophilaNab2 or dNab2), in axon guidance and memory due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental pathways regulated by Nab2 remain poorly defined. Structural defects inNab2null brains resemble defects observed upon disruption of the planar cell polarity (PCP) pathway, which regulates planar orientation of static and motile cells. A kinked bristle phenotype in survivingNab2mutant adults additionally suggests a defect in F-actin polymerization and bundling, which is also a PCP-regulated processes. To test for Nab2-PCP genetic interactions, a collection of PCP loss-of-function alleles was screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye (GMR-Nab2) and subsequently for modification ofNab2null phenotypes. Multiple PCP alleles dominantly modifyGMR-Nab2eye roughening and a subset of these alleles also rescue low survival and thoracic bristle kinking inNab2zygotic nulls. Moreover, alleles of two X-linked PCP factors,dishevelled(dsh) andβ amyloid protein precursor-like(Appl), rescueGMR-Nab2eye roughening in male progeny derived from hemizygousdshorApplmutant fathers, suggesting an additional effect inherited through the male germline. These findings demonstrate a consistent pattern of Nab2-PCP genetic interactions that suggest molecular links between Nab2 and the PCP pathway in the developing eye, wing and germline.

Zinc is essential for almost all living organisms, since it serves as a crucial cofactor for transcription factors and enzymes. However, it is toxic to cell growth when present in excess. The present work aims to investigate the toxicity mechanisms induced by zinc stress in yeast cells. To this end, 108 yeast single-gene deletion mutants were identified sensitive to 6 mM ZnCl2 through a genome-wide screen. These genes were predominantly related to the biological processes of vacuolar acidification and transport, polyphosphate metabolic process, cytosolic transport, the process utilizing autophagic mechanism. A result from the measurement of intracellular zinc content showed that 64 mutants accumulated higher intracellular zinc under zinc stress than the wild-type cells. We further measured the intracellular ROS (reactive oxygen species) levels of 108 zinc-sensitive mutants treated with 3 mM ZnCl2. We showed that the intracellular ROS levels in 51 mutants were increased by high zinc stress, suggesting their possible involvement in regulating ROS homeostasis in response to high zinc. The results also revealed that excess zinc could generate oxidative damage and then activate the expression of several antioxidant defenses genes. Taken together, the data obtained indicated that excess zinc toxicity might be mainly due to the high intracellular zinc levels and ROS levels induced by zinc stress in yeast cells. Our current findings would provide a basis to understand the molecular mechanisms of zinc toxicity in yeast cells.

The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5′-end of transcripts. Notably 183 – 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5′-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5′-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5′-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5′-UTR introns in An. stephensi.

In 10–15% of cancers, telomere length is maintained by a telomerase-independent, recombination-mediated pathway called alternative lengthening of telomeres (ALT). ALT mechanisms were first seen, and have been best studied, in telomerase-null Saccharomyces cerevisiae cells called “survivors”. There are two main types of survivors. Type I survivors amplify Y′ subtelomeric elements while type II survivors, similar to the majority of human ALT cells, amplify the terminal telomeric repeats. Both types of survivors require Rad52, a key homologous recombination protein, and Pol32, a non-essential subunit of DNA polymerase δ. A number of additional proteins have been reported to be important for either type I or type II survivor formation, but it is still unclear how these two pathways maintain telomeres. In this study, we performed a genome-wide screen to identify novel genes that are important for the formation of type II ALT-like survivors. We identified 23 genes that disrupt type II survivor formation when deleted. 17 of these genes had not been previously reported to do so. Several of these genes (DUN1, CCR4, and MOT2) are known to be involved in the regulation of dNTP levels. We find that dNTP levels are elevated early after telomerase inactivation and that this increase favors the formation of type II survivors.