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PiggyBac insertional mutagenesis screen identifies a role for nuclear RHOA in human ES cell differentiation.

ABSTRACT: The mechanisms regulating human embryonic stem (ES) cell self-renewal and differentiation are not well defined in part due to the lack of tools for forward genetic analysis. We present a piggyBac transposon gain of function screen in human ES cells that identifies DENND2C, which genetically cooperates with NANOG to maintain self-renewal in the presence of retinoic acid. We show that DENND2C negatively regulates RHOA activity, which cooperates with NANOG to block differentiation. It has been recently shown that RHOA exists in the nucleus and is activated by DNA damage; however, its nuclear function remains unknown. We discovered that RHOA associates with DNA and that DENND2C affects nuclear RHOA localization, activity, and DNA association. Our study illustrates the power of piggyBac as a cost-effective, efficient, and easy to use tool for forward genetic screens in human ES cells and provides insight into the role of RHOA in the nucleus.

SUBMITTER: Gayle S

PROVIDER: S-EPMC4437468 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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piggyBac insertional mutagenesis screen identifies a role for nuclear RHOA in human ES cell differentiation.

Gayle Sophia S Pan Yukun Y Landrette Sean S Xu Tian T

Stem cell reports 20150409 5

The mechanisms regulating human embryonic stem (ES) cell self-renewal and differentiation are not well defined in part due to the lack of tools for forward genetic analysis. We present a piggyBac transposon gain of function screen in human ES cells that identifies DENND2C, which genetically cooperates with NANOG to maintain self-renewal in the presence of retinoic acid. We show that DENND2C negatively regulates RHOA activity, which cooperates with NANOG to block differentiation. It has been rece ...[more]

PMID: 25866159

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Project description:In the last few decades, dengue virus, an arbovirus, has spread to over 120 countries. Although a vaccine has been approved in some countries, limitations on its effectiveness and a lack of effective antiviral treatments reinforce the need for additional research. The functions of several viral nonstructural proteins are essentially unknown. To better understand the functions of these proteins and thus dengue virus pathogenesis, we embarked on a genomewide transposon mutagenesis screen with next-generation sequencing to determine sites in the viral genome that tolerate 15-nucleotide insertions. Using this approach, we generated support for several published predicted transmembrane and enzymatic domains. Next, we created 7 mutants containing the 15-nucleotide insertion from the original selection and found 6 of them were capable of replication in both mammalian and mosquito tissue culture cells. Interestingly, one mutation had a significant impairment of viral assembly, and this mutation may lead to a better understanding of viral assembly and release. In addition, we created a fully infectious virus expressing a functionally tagged NS4B protein, which will provide a much-needed tool to elucidate the role of NS4B in viral pathogenesis.IMPORTANCE Dengue virus is a mosquito-borne virus distributed in tropical and subtropical regions globally that can result in hospitalization and even death in some cases. Although a vaccine exists, its limitations and a lack of approved antiviral treatments highlight our limited understanding of dengue virus pathogenesis and host immunity. The functions of many viral proteins are poorly understood. We used a previously published approach using transposon mutagenesis to develop tools to study these proteins' functions by adding insertions randomly throughout the viral genomes. These genomes were transferred into cells, and infectious progeny were recovered to determine sites that tolerated insertions, as only the genomes that tolerated insertions would be able to propagate. Using these results, we created viruses with epitope tags, one in the viral structural protein Capsid and one in the viral nonstructural protein NS4B. Further investigation of these mutants may elucidate the roles of Capsid and NS4B during dengue virus infections.

Dataset Information

PiggyBac insertional mutagenesis screen identifies a role for nuclear RHOA in human ES cell differentiation.

Publications

piggyBac insertional mutagenesis screen identifies a role for nuclear RHOA in human ES cell differentiation.

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