Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation

AbstractWolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. To investigate Wolbachia-insect interactions we used the Wolbachia/Drosophila model that provides a genetically tractable system for studying host-pathogen interactions. We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship.Author summaryInsects such as mosquitos act as vectors to spread devastating human diseases such as Dengue, West Nile, and Zika. It is critical to develop control strategies to prevent the transmission of these diseases to human populations. A novel strategy takes advantage of an endosymbiotic bacterium Wolbachia pipientis. The presence of this bacterium in insect vectors prevents successful transmission of RNA viruses. The degree to which viruses are blocked by Wolbachia is dependent on the levels of the bacteria present in the host such that higher Wolbachia levels induce a stronger antiviral effect. In order to use Wolbachia as a tool against vector-borne virus transmission a better understanding of host influences on Wolbachia levels is needed. Here we performed a genome-wide RNAi screen in a model host system Drosophila melanogaster infected with Wolbachia to identify host systems that affect Wolbachia levels. We found that host translation can influence Wolbachia levels in the host.

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Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica

Parasitology ◽

10.1017/s0031182013000528 ◽

2013 ◽

Vol 140 (12) ◽

pp. 1523-1533 ◽

Cited By ~ 31

Author(s):

J. HODGKINSON ◽

K. CWIKLINSKI ◽

N. J. BEESLEY ◽

S. PATERSON ◽

D. J. L. WILLIAMS

Keyword(s):

Genetic Resources ◽

Fasciola Hepatica ◽

Genome Wide Analysis ◽

Extensive Analysis ◽

Genome Wide ◽

A Genome ◽

Genetic Mechanisms ◽

Triclabendazole Resistance

SUMMARYDespite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

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Abstract 360: Loss of Endothelial CXCR7 Exacerbates Wire-injury Induced Neointimal Formation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.360 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Huifeng Hao ◽

Sheng Hu ◽

Dawei Bu ◽

Xiaogang Sun ◽

Miao Wang

Keyword(s):

Vascular Remodeling ◽

Tamoxifen Treatment ◽

Neointimal Formation ◽

Genome Wide ◽

Endothelial Repair ◽

A Genome ◽

Artery Disease

CXCR7 is a non-classical chemokine receptor for CXCL12, whose gene represents a genome-wide association locus for coronary artery disease. Global deletion of CXCR7 increased experimentally induced neointimal formation and atherosclerosis in hyperlipidemic mice, with evidence that CXCR7 modified cholesterol uptake to adipose tissue. We found that CXCR7 was expressed in endothelial cells of mouse neointima and human aortic lesions. To examine a role of endothelial CXCR7 in vascular remodeling, endothelial CXCR7 inducible knockout mice were studied for their vascular response to wire injury in femoral arteries. Tamoxifen treatment of mice harboring floxed CXCR7 and Cdh5 -promoter driven CreERT2 , essentially abolished endothelial CXCR7 expression in vitro and in vivo. Postnatal deletion of endothelial CXCR7 exacerbated neointimal formation on normalipidemic background, four weeks after injury. Mechanistically, this was attributable to attenuated endothelial repair following endothelial injury. Collectively, endothelial CXCR7 is a key regulator of vascular remodeling, independent of lipid traits.

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Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D

Scientific Reports ◽

10.1038/s41598-019-53572-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Jianguo Huang ◽

Mark Chen ◽

Eric S. Xu ◽

Lixia Luo ◽

Yan Ma ◽

...

Keyword(s):

Kinase Inhibitor ◽

Gene Mutations ◽

Soft Agar ◽

Reading Frame ◽

Immunodeficient Mice ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

AbstractCooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

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The tumor suppressor MIR139 is silenced by POLR2M to promote AML oncogenesis

Leukemia ◽

10.1038/s41375-021-01461-5 ◽

2021 ◽

Author(s):

Christiaan J. Stavast ◽

Iris van Zuijen ◽

Elena Karkoulia ◽

Arman Özçelik ◽

Antoinette van Hoven-Beijen ◽

...

Keyword(s):

Tumor Suppressor ◽

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Regulatory Factor ◽

Molecular Control ◽

Polycomb Repressive Complex 2 ◽

Genome Wide ◽

A Genome

AbstractMIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

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Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

The Journal of Cell Biology ◽

10.1083/jcb.201804132 ◽

2018 ◽

Vol 217 (11) ◽

pp. 3817-3828 ◽

Cited By ~ 44

Author(s):

Keigo Morita ◽

Yutaro Hama ◽

Tamaki Izume ◽

Norito Tamura ◽

Toshihide Ueno ◽

...

Keyword(s):

Membrane Protein ◽

Degradation Process ◽

Autophagic Flux ◽

Autophagosome Formation ◽

Early Step ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

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Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound

Cell Death and Disease ◽

10.1038/s41419-021-04191-9 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Tatiana A. Giovannucci ◽

Florian A. Salomons ◽

Martin Haraldsson ◽

Lotta H. M. Elfman ◽

Malin Wickström ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Malignant Cells ◽

Genome Wide ◽

A Genome ◽

Ubiquitin Proteasome ◽

Dependent Cell ◽

Increased Sensitivity ◽

The Impact

AbstractMalignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

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Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1503535112 ◽

2015 ◽

Vol 112 (31) ◽

pp. 9686-9691 ◽

Cited By ~ 33

Author(s):

Dan Jane-wit ◽

Yulia V. Surovtseva ◽

Lingfeng Qin ◽

Guangxin Li ◽

Rebecca Liu ◽

...

Keyword(s):

Endothelial Cells ◽

Dependent Manner ◽

Sirna Screen ◽

Akt Activation ◽

Signaling Complex ◽

Iκb Kinase ◽

Genome Wide ◽

A Genome

Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB–inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5+endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC+ endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt+NIK+ signalosome on Rab5+ endosomes.

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CBMT-42. A GENOME-WIDE CRISPR-Cas9 SCREEN FOR GENES REGULATING QUIESCENT-LIKE STATES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noz175.164 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi42-vi42

Author(s):

Heather Feldman ◽

Anca Mihalas ◽

Christopher Plaisier ◽

Anoop Patel ◽

Patrick Paddison

Keyword(s):

Tumor Cells ◽

Standard Of Care ◽

Cell Types ◽

Phenotypic Characterization ◽

Current Standard ◽

Genome Wide ◽

A Genome ◽

G0 Phase

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a p27-mVenus reporter construct, which is stabilized when cells enter a G0-like state. By assaying p27 reporteractivity, we were able to identify sgRNAs enriched in p27hipopulations and, which upon retest, trigger a G0-like arrest in GSCs. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that downregulation of KAT5 in vitro and in vivo dramatically increases the pool of cells in G0-like states in GSC cultures and GSC-induced tumors. Using single cell RNA-sequencing, we show that this cell state is characterized by gene expression signatures similar to those found in non-dividing subpopulations of GBM tumors and quiescent neural stem cells. In addition, we perform in-depth molecular and phenotypic characterization of these induced G0-like states, including epigenetic and metabolic profiles. These suggest a key role for KAT5 in regulating genes related to protein synthesis. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors and may provide novel therapeutic opportunities.

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Functional proteogenomics reveals biomarkers and therapeutic targets in lymphomas

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1701263114 ◽

2017 ◽

Vol 114 (25) ◽

pp. 6581-6586 ◽

Cited By ~ 17

Author(s):

Delphine C. M. Rolland ◽

Venkatesha Basrur ◽

Yoon-Kyung Jeon ◽

Carla McNeil-Schwalm ◽

Damian Fermin ◽

...

Keyword(s):

Cell Lines ◽

Therapeutic Targets ◽

Cancer Biomarkers ◽

Large Cell ◽

Primary Lymphoma ◽

Anaplastic Lymphoma ◽

Genome Wide ◽

A Genome

Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.

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Genome-wide CRISPR-Cas9 screen in E. coli identifies design rules for efficient targeting

10.1101/308148 ◽

2018 ◽

Cited By ~ 1

Author(s):

Belen Gutierrez ◽

Jérôme Wong Ng ◽

Lun Cui ◽

Christophe Becavin ◽

David Bikard

Keyword(s):

Copy Number ◽

Large Scale ◽

Mixed Population ◽

Target Sequence ◽

Guide Rna ◽

E Coli ◽

Genome Wide ◽

A Genome

AbstractThe main outcome of efficient CRISPR-Cas9 cleavage in the chromosome of bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population of bacteria, which can be achieved both in vitro, e.g. to select mutants, or in vivo as an antimicrobial strategy. The efficiency with which Cas9 kills bacteria has been observed to be quite variable depending on the specific target sequence, but little is known about the sequence determinants and mechanisms involved. Here we performed a genome-wide screen of Cas9 cleavage in the chromosome of E. coli to determine the efficiency with which each guide RNA kills the cell. Surprisingly we observed a large-scale pattern where guides targeting some regions of the chromosome are more rapidly depleted than others. Unexpectedly, this pattern arises from the influence of degrading specific chromosomal regions on the copy number of the plasmid carrying the guide RNA library. After taking this effect into account, it is possible to train a neural network to predict Cas9 efficiency based on the target sequence. We show that our model learns different features than previous models trained on Eukaryotic CRISPR-Cas9 knockout libraries. Our results highlight the need for specific models to design efficient CRISPR-Cas9 tools in bacteria.

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