Genetic Analysis of a Metazoan Pathway using Transcriptomic Phenotypes

RNA-seq is commonly used to identify genetic modules that respond to a perturbation. Although transcriptomes have been mainly used for target gene discovery, their quantitative nature makes them attractive structures with which to study genetic interactions. To understand whether whole organism RNA-seq is suitable for genetic pathway reconstruction, we sequenced the transcriptome of four single mutants and two double mutants of the hypoxia pathway in C. elegans. By comparing the expression levels of double mutants with their corresponding single mutants, we were able to determine, on a genome-wide level, that EGL-9 acts along VHL-1-dependent and independent branches to inhibit HIF-1. We were also able to observe transcriptome-wide suppression of the egl-9(lf) phenotype in an egl-9(lf) hif-1(lf) double mutant. As a by-product of our analysis, we identified a core hypoxic response consisting of 355 genes, and 45 genes that have hif-1 independent, vhl-1-dependent expression. Finally, we are able to identify 31 genes that exhibit non-canonical epistasis: for these genes, vhl-1(lf) mutants show opposing effects to egl-9(lf) mutants, but the egl-9(lf);vhl-1(lf) exhibits the egl-9(lf) phenotype. We suggest that this non-canonical epistasis reflects unexplored aspects of the hypoxia pathway. We discuss the implications, benefits and advantages of using transcriptomic phenotypes to perform pathway analysis.