MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response

NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response.     

Cellular and transcriptomic analysis of NS0 cell response during exposure to hypoxia

Mammalian cells have the ability to alter their gene expression in order to survive or adapt to a variety of environment stresses including hypoxic stress. Maintaining oxygen supply has been accepted as essential for cell survival and growth. To determine the cellular and molecular changes which take place under oxygen deprivation, an NS0 cell line producing a human-mouse chimeric antibody was cultured under hypoxic conditions (<1%). Various cellular parameters such as viability, productivity, metabolism, apoptosis and cell cycle were studied and notable changes were shown to be accompanied by changes in metabolic rates. When the cells where exposed to hypoxia for 48 h, cell growth was suppressed and cell death was detected. To better understand and explore the mechanisms underpinning these biological alterations and to identify the genes involved in the genetic reprogramming, genome-wide analyses were performed using GeneChip Mouse Genome arrays. The gene expression profiling generated by the microarray technique revealed that hypoxia, even in the early stages (12h), induces significant changes in gene expression in NS0 cells. The primary responses to hypoxia within the cells were: (1) the up-regulation of pathways such as glycolysis that ultimately lead to alternative routes of ATP generation and increased oxygen availability; and (2) the down-regulation of genes involved in purine/pyrimidine and one carbon pool metabolisms required for DNA and RNA synthesis. By combining gene expression and physiological changes under hypoxia, it was possible to explore the mechanisms of hypoxia-induced alterations in more depth.     

Identification of DISE-inducing shRNAs by monitoring cellular responses

Off-target effects (OTE) are an undesired side effect of RNA interference (RNAi) caused by partial complementarity between the targeting siRNA and mRNAs other than the gene to be silenced. The death receptor CD95 and its ligand CD95L contain multiple sequences that when expressed as either si- or shRNAs kill cancer cells through a defined OTE that targets critical survival genes. Death induced by survival gene elimination (DISE) is characterized by specific morphological changes such as elongated cell shapes, senescence-like enlarged cells, appearance of large intracellular vesicles, release of mitochondrial ROS followed by activation of caspase-2, and induction of a necrotic form of mitotic catastrophe. Using genome-wide shRNA lethality screens with eight different cancer cell lines, we recently identified 651 genes as critical for the survival of cancer cells. To determine whether the toxic shRNAs targeting these 651 genes contained shRNAs that kill cancer cell through DISE rather than by silencing their respective target genes, we tested all shRNAs in the TRC library derived from a subset of these genes targeting tumor suppressors (TS). We now report that only by monitoring the responses of cancer cells following expression of shRNAs derived from these putative TS it was possible to identify DISE-inducing shRNAs in five of the genes. These data indicate that DISE in general is not an undefined toxic response of cells caused by a random OTE but rather a specific cellular response with shared features that points at a specific biological function involving multiple genes in the genome.     

An RNA interference screen identifies a novel regulator of target of rapamycin that mediates hypoxia suppression of translation in Drosophila S2 cells

In addition to its central role in energy production, oxygen has pervasive regulatory actions. Hypoxia (oxygen limitation) triggers the shutdown of major cellular processes, including gene expression. We carried out a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells for functions required to down-regulate translation during hypoxia. RNAi knockdown of specific genes allowed induction of a green fluorescent protein (GFP) reporter gene and continued protein synthesis during hypoxia. Among the identified genes, Tsc1 and Tsc2, which together form the tuberose sclerosis complex that negatively regulates target of rapamycin (TOR) kinase, gave an especially strong effect. This finding is consistent with the involvement of TOR in promoting translation. Another gene required for efficient inhibition of protein translation during hypoxia, the protein tyrosine phosphatase 61F (Ptp61F), down-regulates TOR activity under hypoxia. Lack of Ptp61F or Tsc2 improves cell survival under prolonged hypoxia in a TOR-dependent manner. Our results identify Ptp61F as a novel modulator of TOR activity and suggest that its function during hypoxia contributes to the down-regulation of protein synthesis.     

NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cells.

The NDRG2 gene belongs to a family of N-Myc downstream-regulated genes (NDRGs) and is expressed in many normal tissues. NDRG2 gene expression has been shown to be regulated in the stress response of certain cells. However, its function is not yet fully understood. Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types. In the current study, we investigated NDRG2 involvement in hypoxia response and found that NDRG2 expression was markedly up-regulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein level. We also observed that the expression of NDRG2 was regulated by Hypoxia-inducible factor 1 (HIF-1) in tumor cells under hypoxia. Three hypoxia-responsive elements (HREs) in the NDRG2 promoter were identified. HRE1 could directly bind Hif-1 in vivo. Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase apoptosis. In addition, our data also showed that Ndrg2 was able to be translocated from the cytoplasm to the nucleus, and the segment from 101 to 178 amino acids of Ndrg2 is responsible for its translocation. Taken together, this study suggests that NDRG2 is a Hif-1 target gene and closely related with hypoxia-induced apoptosis in A549 cells.     

Knockdown of specific host factors protects against influenza virus-induced cell death

Cell death is a characteristic consequence of cellular infection by influenza virus. Mounting evidence indicates the critical involvement of host-mediated cellular death pathways in promoting efficient influenza virus replication. Furthermore, it appears that many signaling pathways, such as NF-κB, formerly suspected to solely promote cell survival, can also be manipulated to induce cell death. Current understanding of the cell death pathways involved in influenza virus-mediated cytopathology and in virus replication is limited. This study was designed to identify host genes that are required for influenza-induced cell death. The approach was to perform genome-wide lentiviral-mediated human gene silencing in A549 cells and determine which genes could be silenced to provide resistance to influenza-induced cell death. The assay proved to be highly reproducible with 138 genes being identified in independent screens. The results were independently validated using siRNA to each of these candidates. Graded protection was observed in this screen with the silencing of any of 19 genes, each providing >85% protection. Three gene products, TNFSF13 (APRIL), TNFSF12-TNFSF13 (TWE-PRIL) and USP47, were selected because of the high levels of protection conferred by their silencing. Protein and mRNA silencing and protection from influenza-induced cell death was confirmed using multiple shRNA clones and siRNA, indicating the specificity of the effects. USP47 knockdown prevented proper viral entry into the host cell, whereas TNFSF12-13/TNFSF13 knockdown blocked a late stage in viral replication. This screening approach offers the means to identify a large number of potential candidates for the analysis of viral-induced cell death. These results may also have much broader applicability in defining regulatory mechanisms involved in cell survival.     

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.     

A genome-wide resource of cell cycle and cell shape genes of fission yeast

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.     

A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance

Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.     

Integrative Analysis of DNA Methylation and Gene Expression Data Identifies EPAS1 as a Key Regulator of COPD

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a ‘causal’ role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.