A yeast phenomic model for the gene interaction network modulating F508del-CFTR protein biogenesis
BACKGROUND:The overall influence of gene interaction in human disease is unknown. In cystic fibrosis (CF) a single allele of the cystic fibrosis transmembrane conductance regulator (F508del-CFTR) accounts for most of the disease. In cell models, F508del-CFTR exhibits defective protein biogenesis and degradation rather than proper trafficking to the plasma membrane where CFTR normally functions. Numerous genes function in the biogenesis of CFTR and influence the fate of F508del-CFTR. However it is not known whether genetic variation in such genes contributes to disease severity in patients. Nor is there an easy way to study how numerous gene interactions involving F508del-CFTR would manifest phenotypically.METHODS:To gain insight into the function and evolutionary conservation of a gene interaction network that regulates biogenesis of a misfolded ABC-transporter, we employed yeast genetics to develop a "phenomic" model, in which the F508del-CFTR-equivalent residue of a yeast homolog is mutated (F670del-Yor1), and where the genome is scanned quantitatively for interaction. We first confirmed that F670del-Yor1 undergoes protein misfolding and had reduced half-life, analogous to F508del-CFTR. Gene interaction was then assessed quantitatively by growth curves for all ~5000 double mutants, based on alteration in the dose response to growth inhibition by oligomycin, a toxin extruded from the cell at the plasma membrane by Yor1.RESULTS:From a comparative genomic perspective, yeast gene interaction influencing F670del-Yor1 biogenesis was representative of human homologs previously found to modulate processing of F508del-CFTR in mammalian cells. Additional evolutionarily conserved pathways were implicated by the study, and an F670del-Yor1-specific pro-biogenesis function of the recently discovered ER Membrane Complex (EMC) was evident from the yeast screen. This novel function was validated biochemically by siRNA of an EMC ortholog in a human cell line expressing F508del-CFTR. The precision and accuracy of quantitative high throughput cell array phenotyping (Q-HTCP), which captures tens of thousands of growth curves simultaneously, provided powerful resolution to measure gene interaction on a phenomic scale, based on discrete cell proliferation parameters.CONCLUSION:We propose phenomic analysis of F670del-Yor1 as a model for investigating gene interaction networks that can modulate cystic fibrosis disease severity. Although the clinical relevance of the F670del-Yor1 gene interaction network for cystic fibrosis remains to be defined, the model appears to be informative with respect to human cell models of F508del-CFTR. Moreover, the general strategy of yeast phenomics can be employed in a systematic manner to model gene interaction for other diseases relating to pathologies that result from protein misfolding or potentially any disease involving evolutionarily conserved genetic pathways.