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      A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-κB

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          Abstract

          The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors.

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          Most cited references 13

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          Haploid genetic screens in human cells identify host factors used by pathogens.

          Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.
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            The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation.

            Nuclear factor kappa B (NF-κB) transcription factors are evolutionarily conserved, coordinating regulators of immune and inflammatory responses. They also play a pivotal role in oncogenesis and metabolic disorders. Several studies during the past two decades have highlighted the key role of the IKK/NF-κB pathway in the induction and maintenance of the state of inflammation that underlies metabolic diseases such as obesity and type 2 diabetes. Recent reports, however, reveal an even more intimate connection between NF-κB and metabolism. These studies demonstrate that NF-κB regulates energy homeostasis via direct engagement of the cellular networks governing glycolysis and respiration, with profound implications beyond metabolic diseases, including cancer, ageing and anticancer therapy. In this review, we discuss these emerging bioenergetic functions of NF-κB and their significance to oncogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.
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              Global gene disruption in human cells to assign genes to phenotypes

              Insertional mutagenesis in a haploid background can lead to complete disruption of gene function 1 . Here we generate a population of human cells that contain insertions in >98% of their expressed genes. We established Phenotypic Interrogation via Tag Sequencing (PhITSeq) as a method to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of selected cells rather than individual clones provides a rapid assessment of the spectrum of genes involved in phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic gram-negative bacteria that cause tissue damage at distinct anatomical sites 2 . We identified 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. While related CDTs may share host factors, they also exploit unique host factors yielding a characteristic profile for each CDT.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                8 July 2013
                : 8
                : 7
                Affiliations
                [1 ]Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America
                [2 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                Tufts School of Medicine, United States of America
                Author notes

                Competing Interests: I have read the journal's policy and have the following conflicts: JEC and TRB are inventors on a patent on mutagenesis in haploid or near-haploid cells (U.S. Patent Application no: 2012/0190,011) and TRB is co-founder of Haplogen GmbH. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: CCL JEC TRB HLP. Performed the experiments: CCL JEC. Analyzed the data: CCL JEC TRB HLP. Contributed reagents/materials/analysis tools: CCL JEC TRB HLP. Wrote the manuscript: CCL HLP.

                [¤a]

                Current Address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America

                [¤b]

                Current Address: Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands

                Article
                PONE-D-13-10691
                10.1371/journal.pone.0070339
                3704534
                23861985

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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