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Genetic Analysis of a Metazoan Pathway using Transcriptomic Phenotypes

ABSTRACT: RNA-seq is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Linear models can quantify expression effects of individual mutants and identify epistatic effects in double mutants. However, interpreting these high-dimensional measurements is unintuitive. We developed a single coefficient to quantify transcriptome-wide epistasis which accurately reflects the underlying interactions. To demonstrate the power of our approach, we sequenced four single and two double Caenorhabditis elegans mutants. From these mutants, we successfully reconstructed the known hypoxia pathway. Using this approach, we uncovered a class of 31 genes that have opposing changes in expression in egl-9(lf) and vhl-1(lf) but for which the egl-9(lf);vhl-1(lf) mutant phenocopies egl-9(lf). These changes violate the classical model of HIF-1 regulation, but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE97355 | GEO | 2017/04/04

REPOSITORIES: GEO

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Project description:Gene expression data from VHL teratomas comparing genes differentially expressed based on apoptotic response to tumor microenvironment. Abstract BACKGROUND: The risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations. METHODS: VHL deficient (Vhl -/- ) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided. RESULTS: The P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation-induced apoptosis. CONCLUSIONS: The TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response. Three genotypes of VHL teratomas were examined: Three replicate wildtype VHL, Three replicate R167Q VHL, and Four replicate P81S VHL (representing two replicates of two different P81S clones)

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Genetic Analysis of a Metazoan Pathway using Transcriptomic Phenotypes

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