Systematic Genetic Screens Research Articles

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast

Heterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified. To gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins, which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1. This work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation.

Environmental Stresses Disrupt Telomere Length Homeostasis

Telomeres protect the chromosome ends from degradation and play crucial roles in cellular aging and disease. Recent studies have additionally found a correlation between psychological stress, telomere length, and health outcome in humans. However, studies have not yet explored the causal relationship between stress and telomere length, or the molecular mechanisms underlying that relationship. Using yeast as a model organism, we show that stresses may have very different outcomes: alcohol and acetic acid elongate telomeres, whereas caffeine and high temperatures shorten telomeres. Additional treatments, such as oxidative stress, show no effect. By combining genome-wide expression measurements with a systematic genetic screen, we identify the Rap1/Rif1 pathway as the central mediator of the telomeric response to environmental signals. These results demonstrate that telomere length can be manipulated, and that a carefully regulated homeostasis may become markedly deregulated in opposing directions in response to different environmental cues.

Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy

Although single gene loss of function analyses can identify components of particular processes, important molecules are missed due to the robustness of biological systems. Here we show that large scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 C. elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologs. We experimentally confirmed functional roles predicted by the network and characterised through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.

Characterization of Conserved Region 2-Deficient Mutants of the Cytomegalovirus Egress Protein pM53

Dominant-negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein pM53 identified the accumulation of inhibitory mutations within conserved region 2 (CR2) and CR4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dölken, C. Buser, P. Walther, and U. H. Koszinowski, J. Virol. 84:9035-9046, 2010); therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and relocalized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.

Recruitment of Rpd3 to the Telomere Depends on the Protein Arginine Methyltransferase Hmt1

In the yeast Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.

A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general “house-keeping” genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system.

Drosophila in cancer research: to boldly go where no one has gone before

Transformation and metastasis are complex processes that depend on integration of effects from multiple mutations. Modeling this complexity requires manipulating multiple genes in particular sub-populations of cells in vivo. This is technically challenging in mammalian model systems and has limited the rate of progress in understanding the effects of the complex aberrations present in cancer cells. In contrast, powerful genetic methods in the fruit fly Drosophila allow efficient generation and analysis of complex genotypes in defined cell populations. These methods are already fruitful in exploring the interactions among cancer mutations, and between cell populations that mimic the tumor microenvironment. In this issue of Oncogene, Willecke et al. (2011) describe the implementation of a novel genetic screen in Drosophila to identify genes required for tumor growth in vivo. This report illustrates the power of using Drosophila to perform systematic genome-wide genetic screens in complex genetic backgrounds and for the resulting data to inform our understanding of transformation and metastasis in human systems.

The nonsense-mediated decay pathway maintains synapse architecture and synaptic vesicle cycle efficacy

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function.

Vectors and parameters that enhance the efficacy of RNAi-mediated gene disruption in transgenic Drosophila

Whole-genome transgenic RNAi libraries permit systematic genetic screens in individual tissues of Drosophila. However, there is a high incidence of nonspecific phenotypes because of off-target effects. To minimize such effects, it is essential to obtain a deeper understanding of the specificity of action of RNAi. Here, in vivo assays are used to determine the minimum, contiguous nucleotide pairing required between an siRNA and a target mRNA to generate a phenotype. We observe that as few as 16 nucleotides of contiguous homology are sufficient to attenuate gene activity. This finding provides an explanation for the high incidence of off-target effects observed in RNAi-based genetic screens. Toward improving the efficacy of RNAi-induced phenotypes in vivo, we describe siRNA expression vectors that allow coexpression of one or more siRNAs with a fluorescent reporter gene in cultured cells or transgenic flies. This expression system makes use of the small intron from the ftz segmentation gene to provide efficient processing of synthetic siRNAs from a reporter transcript. These studies provide a foundation for the specific and effective use of gene silencing in transgenic Drosophila.

The Rpd3L HDAC complex is essential for the heat stress response in yeast

To ensure cell survival and growth during temperature increase, eukaryotic organisms respond with transcriptional activation that results in accumulation of proteins that protect against damage and facilitate recovery. To define the global cellular adaptation response to heat stress, we performed a systematic genetic screen that yielded 277 yeast genes required for growth at high temperature. Of these, the Rpd3 histone deacetylase complex was enriched. Global gene expression analysis showed that Rpd3 partially regulated gene expression upon heat shock. The Hsf1 and Msn2/4 transcription factors are the main regulators of gene activation in response to heat stress. RPD3-deficient cells had impaired activation of Msn2/4-dependent genes, while activation of genes controlled by Hsf1 was deacetylase-independent. Rpd3 bound to heat stress-dependent promoters through the Msn2/4 transcription factors, allowing entry of RNA Pol II and activation of transcription upon stress. Finally, we found that the large, but not the small Rpd3 complex regulated cell adaptation in response to heat stress.

Putting the Brake on FEAR: Tof2 Promotes the Biphasic Release of Cdc14 Phosphatase during Mitotic Exit

The completion of chromosome segregation during anaphase requires the hypercondensation of the approximately 1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Delta cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.

Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling

A principal question in MAP kinase (MAPK/MPK) cascade signalling is how similar components dictate different specificity in the information-processing machineries from yeast to humans and plants. In Arabidopsis, how MPK3/6 modulates distinct outputs in diverse signal transduction pathways remains elusive. By combining systematic cellular and genetic screens, here we uncover a previously unexpected MKK9-MPK3/MPK6 cascade promoting ethylene-insensitive 3 (EIN3)-mediated transcription in ethylene signalling. The mkk9 mutant exhibits a broad spectrum of moderate ethylene-insensitive phenotypes, and translocated MKK9 governs nuclear signalling downstream of receptors. Breaking a linear model and conventional MAPK signalling, ethylene inactivates the negative regulator constitutive triple response 1 (CTR1, a Raf-like MAPK kinase kinase (MAPKKK)) to activate the positive MKK9-MPK3/6 cascade. The bifurcate and antagonistic CTR1 and MKK9 pathways are both critical in determining ethylene-signalling specificity through two MAPK phosphorylation sites with opposite effects on EIN3 stability. The results suggest a new paradigm for linking intertwined MAPK cascades to control quantitative responses and specificity in signalling networks.

Genome-wide, as opposed to local, antisilencing is mediated redundantly by the euchromatic factors Set1 and H2A.Z.

In Saccharomyces cerevisiae, several nonessential mechanisms including histone variant H2A.Z deposition and transcription-associated histone H3 methylation antagonize the local spread of Sir-dependent silent chromatin into adjacent euchromatic regions. However, it is unclear how and where these factors cooperate. To probe this question, we performed systematic genetic array screens for gene deletions that cause a synthetic growth defect in an htz1Delta mutant but not in an htz1Delta sir3Delta double mutant. Of the four genes identified, three, SET1, SWD1, and SWD3, encode components of the Set1 complex, which catalyzes the methylation of histone H3 on lysine 4 (H3-K4), a highly conserved modification that occurs in the coding sequences of transcribed genes. Using microarray-based transcriptional profiling, we find that H2A.Z and Set1 cooperate to prevent Sir-dependent repression of a large number of genes located across the genome, rather than the local effects reported previously for the individual mechanisms. This global, redundant function appears to be direct: using a DamID chromatin profiling method, we demonstrate ectopic association of Sir3 and Sir4 in htz1Delta set1Delta mutants at loci distant from silent chromatin domains. Antisilencing mechanisms may therefore cooperate to play a considerably broader role in regulating genome-wide transcription than previously thought.

A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.

Genetic Association Meets RNA Interference: Large-Scale Genomic Screens for Causation and Mechanism of Complex Diseases

While the genomic era offers great promise for biomedicine in general and for biomarker discovery in particular, it has yet to significantly impact drug target discovery. Meanwhile, despite improvements over the past 20 years in reducing attrition in clinical trials due to adverse drug responses, the pharmaceutical industry continues to be beset by the high rate of attrition of compounds in late-stage development, primarily due to the lack of drug efficacy. Clearly, even highly potent drugs with ideal safety and pharmacokinetic profiles will fail to survive clinical trials if the drug target itself is not a key point of intervention for most patients. Genetic association studies and RNA interference are two scaleable genomic approaches that together can address the quality as well as quantity of candidate drug targets. Human genetic information has long been used to identify 'molecular bottlenecks' that can highlight the importance of a gene or pathway at the clinical level. The recent availability of the human HapMap and of high-throughput genotyping platforms now enables more systematic genetic screens for novel, clinically-relevant drug targets. In addition, RNA interference can help dissect the molecular role of a candidate drug target in preclinical model systems in vitro and in vivo. Wider applicability of RNA interference methods will closely follow continued progress on efficient delivery into appropriate cell models and target tissues.

GenomeRNAi: a database for cell-based RNAi phenotypes

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at

Systematic yeast synthetic lethal and synthetic dosage lethal screens identify genes required for chromosome segregation

Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches.

Xenopus as a model organism in developmental chemical genetic screens

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.

Systematic gene targeting on the X chromosome of Drosophila melanogaster.

The genome of the model organism Drosophila melanogaster has been sequenced and annotated. Based on this groundwork, we performed a systematic genetic screen of the D. melanogaster X chromosome, which carries about one sixth of the genes of the organism. We generated a collection of single P-element insertions to provide genetic and molecular access to virtually all X-chromosomal genes. The study complements earlier work designed to systematically identify vital genes on the X chromosome by targeting transcription units which are phenotypically silent. We describe single UAS sequence-bearing P-element insertions throughout the X chromosome, which allows one to express the tagged genes under control of tissue/organ-directed GAL4 activity. In addition, the present collection of single insertion lines provides a tool to generate chromosomal deletions which are on average less than 33 kb in size.

Genomic Scale Mutant Hunt Identifies Cell Size Homeostasis Genes in S. cerevisiae

Background: In most eukaryotic cells, there is a relationship between cell size and proliferative capacity. For example, in order to commit to cell division, the yeast Saccharomyces cerevisiae must attain a “critical cell size.” This mechanism coordinates growth with cell division to maintain cell size homeostasis. Because very few cell size control genes are known, the genetic pathways responsible for cell size homeostasis remain obscure. Furthermore, elucidation of the mechanism of cell size homeostasis has been recalcitrant to genetic analysis primarily due to the difficulty in cloning cell size control genes.Results: To identify new size control genes, the effect of 5958 single gene deletions (4792 homozygous and 1166 heterozygous gene deletions) on cell size in yeast grown to saturation was systematically determined. From these data, 49 genes were identified that dramatically altered cell size. Of these, 34 are involved in transcription, signal transduction, or cell cycle control; 88% of these genes have putative human homologs. Sixteen genes regulate cell size in a dosage-dependent manner, and the majority of mutants identified fail to correctly exit the cell cycle. Many of these genes are components of Ccr4-Not transcriptional complexes or function in the PKC-MAP kinase pathway. These genes may modulate cell size by altering the expression or activity of G1-phase cyclins.Conclusions: These results illustrate how systematic genetic screens can be used to dissect intricate biological processes that are refractory to classic genetic approaches. This genomic-wide genetic screen yielded 46 new cell size mutants and systematically assessed the effect of 5958 single gene deletions on cell size as cells exited the cell cycle.