A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis

Abstract

BackgroundThe overall influence of gene interaction in human disease is unknown. In cystic fibrosis (CF) a single allele of the cystic fibrosis transmembrane conductance regulator (CFTR-ΔF508) accounts for most of the disease. In cell models, CFTR-ΔF508 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 CFTR-ΔF508. 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 CFTR-ΔF would manifest phenotypically.MethodsTo 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 CFTR-ΔF508-equivalent residue of a yeast homolog is mutated (Yor1-ΔF670), and where the genome is scanned quantitatively for interaction. We first confirmed that Yor1-ΔF undergoes protein misfolding and has reduced half-life, analogous to CFTR-ΔF. Gene interaction was then assessed quantitatively by growth curves for approximately 5,000 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.ResultsFrom a comparative genomic perspective, yeast gene interactions influencing Yor1-ΔF biogenesis were representative of human homologs previously found to modulate processing of CFTR-ΔF in mammalian cells. Additional evolutionarily conserved pathways were implicated by the study, and a ΔF-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 CFTR-ΔF508. 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.ConclusionWe propose phenomic analysis of Yor1-ΔF as a model for investigating gene interaction networks that can modulate cystic fibrosis disease severity. Although the clinical relevance of the Yor1-ΔF gene interaction network for cystic fibrosis remains to be defined, the model appears to be informative with respect to human cell models of CFTR-ΔF. 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.

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps

“…where K functionally represents the carrying capacity (maximum culture density), l is the time to reach half-carrying capacity, and r is the maximum specific cell proliferation rate (33). Phenotypic differences were visualized by comparing growth curves between mutants in a particular column of the cell array (a single concentration) or comparison of the change in a cell proliferation parameter across the cell array.…”

“…The yeast oligomycin exporter Yor1p was chosen for this analysis because it is an MRP4 homolog whose transport activity can be scored as cell growth on oligomycin media (33,45). In addition, Yor1p possesses the conserved proline at the base of TM6 that is a feature of the ABCC subfamily (Pro-485 in Yor1p), a lysine in the outer TM collar at the same position as Lys-978 in CFTR (Lys-997 in Yor1p), and an A-loop tyrosine in NBD2 at the same location as Tyr-1219 in CFTR (Tyr-1222 in Yor1p; see alignments in Fig.…”

Gene Expression in Transformed Lymphocytes Reveals Variation in Endomembrane and HLA Pathways Modifying Cystic Fibrosis Pulmonary Phenotypes

“…Identified pathways, related to the endomembrane system for synthesis and post-translational modification of membrane proteins (membranes, vesicles, ER/Golgi) and the ER stress response, have been previously implicated in CF pathophysiology, including p.Phe508del processing. 23,[33][34][35] Although the ER stress response is related to endomembrane function, the two signals (post-translational processing and ER stress response) are both pathologically relevant and are probably acting independently to modify lung disease phenotype. The strength of the genomic signatures in our results supports continued research directed at these processes for CF.…”

Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases

“…Despite these challenges, identification of secondary modulators has proven successful across a multitude of model organisms in which the prominent role of second-site suppressors that buffer or modify traits has been established [8][9][10][11] . For example, human genetic studies have identified rare mutations in CCR5 that confer resilience against HIV infection 12 , mutations in globin genes that modify the severity of sickle cell disease by buffering primary mutations in β-globin genes 13 , and LoF mutations in PCSK9 that protect carriers from high lipid levels and resulting heart disease 14 .…”