Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Yao, Rong; Nakamura, Shota; Hirata, Tetsuya; Motooka, Daisuke; Liu, Yi-Shi; He, Zeng-An; Gao, Xiao‐Dong; Maeda, Yusuke; Kinoshita, Taroh; Fujita, Morihisa

doi:10.1371/journal.pone.0138553

Cited by 22 publications

(19 citation statements)

References 54 publications

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“…As a model of mammalian haploid cells, we first used the nearhaploid human HAP1 cell line, which was generated while trying to generate induced pluripotent stem cells from KBM7 cells (11) and has been widely used for forward genetic screenings (11,(16)(17)(18). While growing HAP1 cells, we noticed a rapid reduction in the percentage of haploid cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

A p53-dependent response limits the viability of mammalian haploid cells

Olbrich

Mayor‐Ruiz

Vega-Sendino

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include nearhaploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell-sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells.haploidy | embryonic stem cells | p53 | HAP1 | chromosome segregation T he main advantage of yeast as a model organism for genetic studies is the availability of haploid cells, so that the mutation of a single allele can suffice to reveal a phenotype. This approach has been of enormous importance for biomedical research in recent decades, as exemplified by the number of Nobel Prizes awarded to discoveries that used yeast as a model system, including the discovery of autophagy, telomeres, and the cell cycle (1). In any case, there are questions intrinsic to mammalian biology, such as stemness or differentiation, that are difficult to address using yeast as a model, and that could be answered by the availability of mammalian haploid cell lines.

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Section: Resultsmentioning

confidence: 99%

A p53-dependent response limits the viability of mammalian haploid cells

Olbrich

Mayor‐Ruiz

Vega-Sendino

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…To identify such target factors, we used genetic approaches in the HAP1 human haploid cell line ( Carette et al, 2011 ). Using gene-trapping methods, which disrupt genes by inhibiting normal splicing, on the HAP1 cells, mutant cells were obtained and then screened for phenotypes of interest ( Carette et al, 2011 ; Rong et al, 2015 ). We applied these methods to analyze the factors required for GPI-inositol deacylation.…”

Section: Resultsmentioning

confidence: 99%

N-Glycan–dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing

Liu

Guo

Hirata

et al. 2017

Journal of Cell Biology

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“…The products were purified with Amicon Ultra filter and subjected to the second-round PCR using splinkerette Spl-F3 (5=-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTGGACTCCAACGAAGCGAAGG-3=) and PB3-2 (5=-CGTACGTCACAATATGATTATCTTTCTAGGGT-3=). The second-round PCR products after size selection were subjected to deep sequencing (Illumina MiSeq) and analyzed using CLC Genomic Workbench software (Qiagen) as previously reported (92). The number of different sites at which the exon-trapping vector had inserted was determined in each gene.…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection

Tanaka

Tumkosit

Nakamura

et al. 2017

J Virol

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112

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The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis. Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.

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Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Cited by 22 publications

References 54 publications

A p53-dependent response limits the viability of mammalian haploid cells

A p53-dependent response limits the viability of mammalian haploid cells

N-Glycan–dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing

Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection

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