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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.

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.

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

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.

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.

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.

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.

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.

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.

Locomotion is an ancient and fundamental output of the nervous system required for animals to perform many other complex behaviors. Although the formation of motor circuits is known to be under developmental control of transcriptional mechanisms that define the fates and connectivity of the many neurons, glia and muscle constituents of these circuits, relatively little is known about the role of post-transcriptional regulation of locomotor behavior. MicroRNAs have emerged as a potentially rich source of modulators for neural development and function. In order to define the microRNAs required for normal locomotion in Drosophila melanogaster, we utilized a set of transgenic Gal4-dependent competitive inhibitors (microRNA sponges, or miR-SPs) to functionally assess ca. 140 high-confidence Drosophila microRNAs using automated quantitative movement tracking systems followed by multiparametric analysis. Using ubiquitous expression of miR-SP constructs, we identified a large number of microRNAs that modulate aspects of normal baseline adult locomotion. Addition of temperature-dependent Gal80 to identify microRNAs that act during adulthood revealed that the majority of these microRNAs play developmental roles. Comparison of ubiquitous and neural-specific miR-SP expression suggests that most of these microRNAs function within the nervous system. Parallel analyses of spontaneous locomotion in adults and in larvae also reveal that very few of the microRNAs required in the adult overlap with those that control the behavior of larval motor circuits. These screens suggest that a rich regulatory landscape underlies the formation and function of motor circuits and that many of these mechanisms are stage and/or parameter-specific.

The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. The genetic programs dictating the development of ChO cells with unique morphologies and mechanical properties are largely unknown. Here we describe an RNAi screen that focused on the ChO’s accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The ‘cell elongation’ group included the transcription factors Delilah and Stripe, implicating them for the first time in regulating the response of ChO cells to developmental stretching forces. Other genes found to affect the pattern of ChO cell elongation, such as αTub85E, β1Tub56D, Tbce, CCT8, mys, Rac1 and shot, represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties.

In Caenorhabditis elegans, Notch signaling regulates developmentally timed sleep during the transition from L4 larval stage to adulthood (L4/A) . To identify core sleep pathways and to find genes acting downstream of Notch signaling, we undertook the first genome-wide, classical genetic screen focused on C. elegans developmentally timed sleep. To increase screen efficiency, we first looked for mutations that suppressed inappropriate anachronistic sleep in adult hsp::osm-11 animals overexpressing the Notch coligand OSM-11 after heat shock. We retained suppressor lines that also had defects in L4/A developmentally timed sleep, without heat shock overexpression of the Notch coligand. Sixteen suppressor lines with defects in developmentally timed sleep were identified. One line carried a new allele of goa-1; loss of GOA-1 Gαo decreased C. elegans sleep. Another line carried a new allele of gpb-2, encoding a Gβ5 protein; Gβ5 proteins have not been previously implicated in sleep. In other scenarios, Gβ5 GPB-2 acts with regulators of G protein signaling (RGS proteins) EAT-16 and EGL-10 to terminate either EGL-30 Gαq signaling or GOA-1 Gαo signaling, respectively. We found that loss of Gβ5 GPB-2 or RGS EAT-16 decreased L4/A sleep. By contrast, EGL-10 loss had no impact. Instead, loss of RGS-1 and RGS-2 increased sleep. Combined, our results suggest that, in the context of L4/A sleep, GPB-2 predominantly acts with EAT-16 RGS to inhibit EGL-30 Gαq signaling. These results confirm the importance of G protein signaling in sleep and demonstrate that these core sleep pathways function genetically downstream of the Notch signaling events promoting sleep.

The Drosophila melanogaster ovarian follicle cell lineage provides a powerful system for investigating how epigenetic changes contribute to differentiation. Downstream from an epithelial stem cell, follicle progenitors undergo nine mitotic cell cycles before transitioning to the endocycle and initiating differentiation. During their proliferative phase, follicle progenitors experience Lsd1-dependent changes in epigenetic stability that can be monitored using GAL4::UAS variegation. Eventually, follicle progenitors acquire competence to respond to Delta, a Notch ligand present in the environment, which signals them to cease division and initiate differentiation. The time required to acquire competence determines the duration of mitotic cycling and hence the final number of follicle cells. We carried out a screen for dominant modifiers of variegation spanning nearly 70% of Drosophila euchromatin to identify new genes influencing follicle progenitor epigenetic maturation. The eight genes found include chromatin modifiers, but also cell cycle regulators and transcription factors. Five of the modifier genes accelerate the acquisition of progenitor competence and reduce follicle cell number, however, the other three genes affect follicle cell number in an unexpected manner.

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint.

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming.

With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2. Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone.

Genetic analysis of LIN-12/Notch signaling in C. elegans has provided many insights into human biology. Activating missense mutations in the Negative Regulatory Region (NRR) of the ectodomain of LIN-12/Notch were first described in C. elegans, and similar mutations in human Notch were later found to cause T-cell acute lymphoblastic leukemia (T-ALL). The ubiquitin ligase sel-10/Fbw7 is the prototype of a conserved negative regulator of lin-12/Notch that was first defined by loss-of-function mutations that enhance lin-12 NRR-missense activity in C. elegans, and then demonstrated to regulate Notch activity in mammalian cells and to be a bona fide tumor suppressor in T-ALL. Here, we report the results of an RNAi screen of 248 C. elegans protein kinase-encoding genes with human orthologs for enhancement of a weakly activating NRR-missense mutation of lin-12 in the Vulval Precursor Cells. We identified, and validated, thirteen kinase genes whose loss led to increase lin-12 activity; eleven of these genes have never been implicated previously in regulating Notch activity in any system. Depleting the activity of five kinase genes (cdk-8, wnk-1, kin-3, hpo-11, and mig-15) also significantly enhanced the activity of a transgene in which heterologous sequences drive expression of the untethered intracellular domain of LIN-12, suggesting that they increase the activity or stability of the signal-transducing form of LIN-12/Notch. Precedents set by other regulators of lin-12/Notch defined through genetic interactions in C. elegans suggest that this new set of genes may include negative regulators that are functionally relevant to mammalian development and cancer.

Cdk1 (Cdc28 in yeast) is a cyclin-dependent kinase (CDK) essential for cell cycle progression and cell division in normal cells. However, CDK activity also underpins proliferation of tumor cells, making it a relevant study subject. While numerous targets and processes regulated by Cdc28 have been identified, the exact functions of Cdc28 are only partially understood. To further explore the functions of Cdc28, we systematically overexpressed ∼4800 genes in wild-type (WT) cells and in cells with artificially reduced Cdc28 activity. This screen identified 366 genes that, when overexpressed, specifically compromised cell viability under conditions of reduced Cdc28 activity. Consistent with the crucial functions of Cdc28 in cell cycle regulation and chromosome metabolism, most of these genes have functions in the cell cycle, DNA replication, and transcription. However, a substantial number of genes control processes not directly associated with the cell cycle, indicating that Cdc28 may also regulate these processes. Finally, because the dataset was enriched for direct Cdc28 targets, the results from this screen will aid in identifying novel targets and process regulated by Cdc28.

Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development.

The mitotic kinetochore forms at the centromere for proper chromosome segregation. Deposition of the centromere-specific histone H3 variant, spCENP-A/Cnp1, is vital for the formation of centromere-specific chromatin and the Mis17-Mis6 complex of the fission yeast Schizosaccharomyces pombe is required for this deposition. Here we identified extragenic suppressors for a Mis17-Mis6 complex temperature-sensitive (ts) mutant, mis17-S353P, using whole-genome sequencing. The large and small daughter nuclei phenotype observed in mis17-S353P was greatly rescued by these suppressors. Suppressor mutations in two ribonuclease genes involved in the mRNA decay pathway, exo2 and pan2, may affect Mis17 protein level, as mis17 mutant protein level was recovered in mis17-S353P exo2 double mutant cells. Suppressor mutations in EKC/KEOPS complex genes may not regulate Mis17 protein level, but restored centromeric localization of spCENP-A/Cnp1, Mis6 and Mis15 in mis17-S353P. Therefore, the EKC/KEOPS complex may inhibit Mis17-Mis6 complex formation or centromeric localization. Mutational analysis in protein structure indicated that suppressor mutations in the EKC/KEOPS complex may interfere with its kinase activity or complex formation. Our results suggest that the mRNA decay pathway and the EKC/KEOPS complex negatively regulate Mis17-Mis6 complex-mediated centromere formation by distinct and unexpected mechanisms.

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered ∼8,500 and ∼5,000 genes, respectively, of a total of ∼16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest.

Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader—a heterodimer composed of Scc4 and Scc2. However, very little is known about the loader’s mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2. Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism.

The mangrove killifish, Kryptolebias marmoratus, is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis. As a result, it constitutes a simplistic and desirable vertebrate model for developmental genetics as it is easily maintained, reaches sexual maturity in about 100 days, and provides a manageable number of relatively clear embryos. After the establishment and characterization of an initial mutagenesis pilot screen using N-ethyl-N-nitrosourea, a three-generation genetic screen was performed to confirm zygotic mutant allele heritability and simultaneously score for homozygous recessive mutant sterile F2 fish. From a total of 307 F2 fish screened, 10 were found to be 1° males, 16 were sterile, 92 wild-type, and the remaining 189, carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes similar to those previously described in zebrafish and in the aforementioned pilot screen, as expected. Interestingly, new phenotypes such as golden yolk, no trunk, and short tail were observed. The siblings of sterile F2 mutants were used to produce an F3 generation in order to confirm familial sterility. Out of the 284 F3 fish belonging to 10 previously identified sterile families, 12 were found to be 1° males, 69 were wild-type, 83 sterile, and 120 were classified as */+ (either wild-type or carriers) with undefined genotypes. This screen provides proof of principle that K. marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility.

The multifunctional Saccharomyces cerevisiae Pif1 DNA helicase affects the maintenance of telomeric, ribosomal, and mitochondrial DNAs, suppresses DNA damage at G-quadruplex motifs, influences the processing of Okazaki fragments, and promotes breakage induced replication. All of these functions require the ATPase/helicase activity of the protein. Owing to Pif1’s critical role in the maintenance of mitochondrial DNA, pif1Δ strains quickly generate respiratory deficient cells and hence grow very slowly. This slow growth makes it difficult to carry out genome-wide synthetic genetic analysis in this background. Here, we used a partial loss of function allele of PIF1, pif1-m2, which is mitochondrial proficient but has reduced abundance of nuclear Pif1. Although pif1-m2 is not a null allele, pif1-m2 cells exhibit defects in telomere maintenance, reduced suppression of damage at G-quadruplex motifs and defects in breakage induced replication. We performed a synthetic screen to identify nonessential genes with a synthetic sick or lethal relationship in cells with low abundance of nuclear Pif1. This study identified eleven genes that were synthetic lethal (APM1, ARG80, CDH1, GCR1, GTO3, PRK1, RAD10, SKT5, SOP4, UMP1, and YCK1) and three genes that were synthetic sick (DEF1, YIP4, and HOM3) with pif1-m2.

In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.

When subjected to rapid drops of temperature (cold shock), Neurospora responds with a temporary shift in its morphology. This report is the first to examine this response genetically. We report here the results of a screen of selected mutants from the Neurospora knockout library for alterations in their morphological response to cold shock. Three groups of knockouts were selected to be subject to this screen: genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes; transcription factors; and genes homologous to E. coli genes known to alter their expression in response to cold shock. A total of 344 knockout strains were subjected to cold shock. Of those, 118 strains were identified with altered responses. We report here the cold shock morphologies and GO categorizations of strains subjected to this screen. Of strains with knockouts in genes associated with hyphal growth or morphology, 33 of 131 tested (25%) showed an altered response to cold shock. Of strains with knockouts in transcription factor genes, 30 of 145 (20%) showed an altered response to cold shock. Of strains with knockouts in genes homologous to E. coli genes which display altered levels of transcription in response to cold shock, a total of 55 of 68 tested (81%) showed an altered cold shock response. This suggests that the response to cold shock in these two organisms is largely shared in common.

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter, so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila. Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB, show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits.

Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how receptor tyrosine kinase signaling is controlled. However, the mechanisms that underpin the control of Tor signaling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localizes Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, we conducted a genome-wide screen to identify new pathway components that operate downstream of Tsl. Using a set of molecularly defined chromosomal deficiencies, we screened for suppressors of ligand-dependent Tor signaling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which we mapped to the causative gene, and a further 29 that were mapped to

Spike density and processing quality are important traits in modern wheat production and are controlled by multiple gene loci. The associated genes have been intensively studied and new discoveries have been constantly reported during the past few decades. However, no gene playing a significant role in the development of these two traits has been identified. In the current study, a common wheat mutant with extremely compact spikes and good processing quality was isolated and characterized. A new allele (Qc1) of the Q gene (an important domestication gene) responsible for the mutant phenotype was cloned, and the molecular mechanism for the mutant phenotype was studied. Results revealed that Qc1 originated from a point mutation that interferes with the miRNA172-directed cleavage of Q transcripts, leading to its overexpression. It also reduces the longitudinal cell size of rachises, resulting in an increased spike density. Furthermore, Qc1 increases the number of vascular bundles, which suggests a higher efficiency in the transportation of assimilates in the spikes of the mutant than that of wild type. This accounts for the improved processing quality. The effects of Qc1 on spike density and wheat processing quality were confirmed by analyzing nine common wheat mutants possessing four different Qc alleles. These results deepen our understanding of the key roles of Q gene, and provide new insights for the potential application of Qc alleles in wheat quality breeding.

We have previously identified embryo-specific (emb) mutations that resulted in maize kernels containing abnormal embryos with normal-appearing endosperm among the progeny of active Robertson’s Mutator stocks. Our rationale for the mutant screen described here is that it should be possible to produce ethyl methane sulfonate (EMS)-induced emb mutations at a frequency higher than that obtained by transposon mutagenesis and with greater ease. This proved to be the case when we screened for mutations that are embryo-specific among progeny of materials generated with EMS-treated pollen. The EMS-induced emb mutation frequency reported here is nearly three times the 4.5% we obtained with the transposable element stocks. The 45 mutants reported here were all tested for germination capacity and nearly all were lethal. The embryo phenotypes of 34 mutations were examined by dissection of the mature embryos. All were found to be retarded in development and morphologically abnormal. Half of the mutants in this group were blocked in the proembryo and transition stages. They likely include mutations in nuclear genes coding for plastid proteins. The other 17 are mainly blocked in the coleoptilar stage, or in later stages with a low frequency. This group likely includes mutations in genes regulating the completion of shoot apical meristem (SAM) development and accompanying morphogenetic events. Most of the complementation tests using 19 of the mutations in 35 unique combinations complimented each other, except for two pairs of mutations with similar phenotypes. Our results provide additional evidence for the presence of many emb loci in the maize genome.

DNA damage response pathways are crucial for protecting genome stability in all eukaryotes. Saccharomyces cerevisiaeDna2 has both helicase and nuclease activities that are essential for Okazaki fragment maturation, and Dna2 is involved in long-range DNA end resection at double-strand breaks. Dna2 forms nuclear foci in response to DNA replication stress and to double-strand breaks. We find that Dna2-GFP focus formation occurs mainly during S phase in unperturbed cells. Dna2 colocalizes in nuclear foci with 25 DNA repair proteins that define recombination repair centers in response to phleomycin-induced DNA damage. To systematically identify genes that affect Dna2 focus formation, we crossed Dna2-GFP into 4293 nonessential gene deletion mutants and assessed Dna2-GFP nuclear focus formation after phleomycin treatment. We identified 37 gene deletions that affect Dna2-GFP focus formation, 12 with fewer foci and 25 with increased foci. Together these data comprise a useful resource for understanding Dna2 regulation in response to DNA damage.

Budding yeast begin the transition to quiescence by prolonging G1 and accumulating limited nutrients. They undergo asymmetric cell divisions, slow cellular expansion, acquire significant stress tolerance and construct elaborate cell walls. These morphologic changes give rise to quiescent (Q) cells, which can be distinguished from three other cell types in a stationary phase culture by flow cytometry. We have used flow cytometry to screen for genes that are required to obtain the quiescent cell fraction. We find that cell wall integrity is critical and these genes may help define quiescence-specific features of the cell wall. Genes required to evade the host innate immune response are common. These may be new targets for antifungal drugs. Acquired thermotolerance is also a common property, and we show that the stress-response transcription factors Msn2 and Msn4 promote quiescence. Many other pathways also contribute, including a subset of genes involved in autophagy, ubiquitin-mediated proteolysis, DNA replication, bud site selection, and cytokinesis.

In Caenorhabditis elegans, Notch signaling regulates developmentally timed sleep during the transition from L4 larval stage to adulthood (L4/A) . To identify core sleep pathways and to find genes acting downstream of Notch signaling, we undertook the first genome-wide, classical genetic screen focused on C. elegans developmentally timed sleep. To increase screen efficiency, we first looked for mutations that suppressed inappropriate anachronistic sleep in adult hsp::osm-11 animals overexpressing the Notch coligand OSM-11 after heat shock. We retained suppressor lines that also had defects in L4/A developmentally timed sleep, without heat shock overexpression of the Notch coligand. Sixteen suppressor lines with defects in developmentally timed sleep were identified. One line carried a new allele of goa-1; loss of GOA-1 Gαo decreased C. elegans sleep. Another line carried a new allele of gpb-2, encoding a Gβ5 protein; Gβ5 proteins have not been previously implicated in sleep. In other scenarios, Gβ5 GPB-2 acts with regulators of G protein signaling (RGS proteins) EAT-16 and EGL-10 to terminate either EGL-30 Gαq signaling or GOA-1 Gαo signaling, respectively. We found that loss of Gβ5 GPB-2 or RGS EAT-16 decreased L4/A sleep. By contrast, EGL-10 loss had no impact. Instead, loss of RGS-1 and RGS-2 increased sleep. Combined, our results suggest that, in the context of L4/A sleep, GPB-2 predominantly acts with EAT-16 RGS to inhibit EGL-30 Gαq signaling. These results confirm the importance of G protein signaling in sleep and demonstrate that these core sleep pathways function genetically downstream of the Notch signaling events promoting sleep.

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling.