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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.

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.

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.

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.

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.

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.

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.

Yeast sporulation is a highly regulated developmental program by which diploid cells generate haploid gametes, termed spores. To better define the genetic pathways regulating sporulation, a systematic screen of the set of ~3300 nonessential Schizosaccharomyces pombe gene deletion mutants was performed to identify genes required for spore formation. A high-throughput genetic method was used to introduce each mutant into an h90 background, and iodine staining was used to identify sporulation-defective mutants. The screen identified 34 genes whose deletion reduces sporulation, including 15 that are defective in forespore membrane morphogenesis. In S. pombe, the total number of sporulation-defective mutants is a significantly smaller fraction of coding genes than in S. cerevisiae, which reflects the different evolutionary histories and biology of the two yeasts.

We have screened chromosome arm 3L for ethyl methanesulfonate−induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis.

The heat-shock response in cells, involving increased transcription of a specific set of genes in response to a sudden increase in temperature, is a highly conserved biological response occurring in all organisms. Despite considerable attention to the processes activated during heat shock, less is known about the role of genes in survival of a sudden temperature increase. Saccharomyces cerevisiae genes involved in the maintenance of heat-shock resistance in exponential and stationary phase were identified by screening the homozygous diploid deletants in nonessential genes and the heterozygous diploid mutants in essential genes for survival after a sudden shift in temperature from 30 to 50°. More than a thousand genes were identified that led to altered sensitivity to heat shock, with little overlap between them and those previously identified to affect thermotolerance. There was also little overlap with genes that are activated or repressed during heat-shock, with only 5% of them regulated by the heat-shock transcription factor. The target of rapamycin and protein kinase A pathways, lipid metabolism, vacuolar H+-ATPase, vacuolar protein sorting, and mitochondrial genome maintenance/translation were critical to maintenance of resistance. Mutants affected in l-tryptophan metabolism were heat-shock resistant in both growth phases; those affected in cytoplasmic ribosome biogenesis and DNA double-strand break repair were resistant in stationary phase, and in mRNA catabolic processes in exponential phase. Mutations affecting mitochondrial genome maintenance were highly represented in sensitive mutants. The cell division transcription factor Swi6p and Hac1p involved in the unfolded protein response also play roles in maintenance of heat-shock resistance.