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Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

ABSTRACT: Glutathione-S-transferases mu 2 (GSTM2), a kind of important Phase II antioxidant enzyme of eukaryotes, is degraded by nonsense mediated mRNA decay due to a C27T substitution in the fifth exon of pigs. As a reproductive performance-related gene, GSTM2 is involved in embryo implantation, whereas, functional deficiency of GSTM2 induces pre- or post-natal death in piglets potentially. To have some insight into the role of GSTM2 in embryo development, high throughput RNA sequencing is performed using the swine testis cells (ST) with the deletion of GSTM2. Some embryo development-related genes are observed from a total of 242 differentially expressed genes, including STAT1, SRC, IL-8, DUSP family, CCL family and integrin family. GSTM2 affects expression of SRC, OPN, and SLCs. GSTM2 suppresses phosphorylation of STAT1 by binding to STAT1. In addition, as an important transcription factor, STAT1 regulates expression of uterus receptive-related genes including CCLs, IRF9, IFITs, MXs, and OAS. The present study provides evidence to molecular mechanism of GSTM2 modulating embryo development.

SUBMITTER: Lv Y

PROVIDER: S-EPMC5131268 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

Lv Yuqi Y Jin Yi Y Zhou Yongqiang Y Jin Jianjun J Ma Zhenfa Z Ren Zhuqing Z

Scientific reports 20161201

Glutathione-S-transferases mu 2 (GSTM2), a kind of important Phase II antioxidant enzyme of eukaryotes, is degraded by nonsense mediated mRNA decay due to a C27T substitution in the fifth exon of pigs. As a reproductive performance-related gene, GSTM2 is involved in embryo implantation, whereas, functional deficiency of GSTM2 induces pre- or post-natal death in piglets potentially. To have some insight into the role of GSTM2 in embryo development, high throughput RNA sequencing is performed usin ...[more]

PMID: 27905550

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Project description:BackgroundProtein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other.MethodologyWe used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles.Results and conclusionReproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.

Dataset Information

Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

Publications

Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

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