Louie et al. Genome Medicine 2012, 4:103 http://genomemedicine.com/content/4/12/103 RESEARCH Open Access A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis Raymond J Louie3†, Jingyu Guo1,2†, John W Rodgers1, Rick White4, Najaf A Shah1, Silvere Pagant3, Peter Kim3, Michael Livstone5, Kara Dolinski5, Brett A McKinney6, Jeong Hong2, Eric J Sorscher2, Jennifer Bryan4, Elizabeth A Miller3* and John L Hartman IV1,2* Abstract 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 (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. 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 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. Results: From 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. Conclusion: We 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 * Correspondence: [email protected]; [email protected] † Contributed equally 1Department of Genetics, University of Alabama at Birmingham, 730 Hugh Kaul Human Genetics Building, 720 20th Street South, Birmingham, AL 35294 USA 3Department of Biology, Columbia University, 1212 Amsterdam Ave. MC2456, New York, NY 10027 USA Full list of author information is available at the end of the article © 2013 Mckinney et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Louie et al. Genome Medicine 2012, 4:103 Page 2 of 20 http://genomemedicine.com/content/4/12/103 to model gene interaction for other diseases relating to pathologies that result from protein misfolding or potentially any disease involving evolutionarily conserved genetic pathways. Keywords: Gene interaction, Genetic buffering, Genotype-phenotype complexity, Phenomics, Quantitative high throughput cell array phenotyping (Q-HTCP), Cystic fibrosis transmembrane conductance regulator (CFTR), ER mem- brane complex (EMC), ATP binding cassette (ABC) transporter, Membrane protein biogenesis, Yeast model of human disease, Comparative functional genomics Background strains [10-12], and used Q-HTCP [13,14] to measure Since release of the human genome sequence, genome- the influence of gene-gene interactions on cell prolifera- wide association studies (GWAS) and other advances in tion in the presence of oligomycin, a toxin extruded from genomic technology have challenged simplistic notions of cells by Yor1. From a drug discovery perspective, protein the genetic basis of human disease. Even Mendelian dis- regulators of CFTR-ΔF biogenesis represent novel tar- ease phenotypes are now thought to be driven by complex gets, and cell culture experiments indicate such targets genetic relationships [1]. For example, modifier genes can are numerous [15,16]. Many of these regulators are evo- influence the severity of cystic fibrosis [2]. However, the lutionarily conserved, thus a quantitative systems level influence on disease contributed by multi-locus, combina- model of a gene interaction network model derived from tion-specific pairs of allelic variants remains largely yeast could complement human and animal studies [17]. unmapped and uncharacterized biologically. Moreover, From a systems biology perspective, the quantitative most disease traits are non-Mendelian (that is, ‘complex’ description of a gene network that modulates biogenesis traits), where expression of the phenotype involves multi- of a misfolded ABC transporter could provide useful ple different gene activities, none of which is individually insight for understanding the phenotypic complexity of required or accounts for a large fraction of heritability cystic fibrosis in association with human genetic data, [3,4]. Thus Mendelian and complex traits can be seen as and might similarly aid study of other diseases related to different ends of the same continuum in which multiple protein misfolding. If successful for cystic fibrosis, the genetic and environmental effects impact disease risk and/ same general strategy of yeast phenomic modeling should or severity in a combination-dependent manner. It is pre- be applicable to derive understanding about disease com- sumed that in some genetic or environmental contexts plexity involving any conserved cellular pathway. particular variant alleles are phenotypically expressed, and in other contexts they are buffered. However, whether Methods principles for disease variation can be deduced through Yeast strains systematic analysis of gene-gene interaction remains Deletion mutants were from the MATa collection, cre- unknown [5]. In this study we developed a yeast model of ated by the Saccharomyces Genome Deletion Project gene interaction for a clinically relevant disease mutation, [18], and obtained from Open Biosystems. The query CFTR-ΔF508, to investigate whether it can potentially strain background for double mutant construction was serve as a useful tool to better understand the genetic 15578-1.2b [10]. The R1116T mutation (Figure 1) was complexity underlying the human disease, cystic fibrosis introduced into pSM2056 (yor1-ΔF670-HA-GFP::URA3 [6]. Saccharomyces cerevisiae is a workhorse for funda- integrating vector) [19] by Quik Change mutagenesis mental biology, but the extent to which experimental (Stratagene) to create plasmid pRL026. This vector was models of gene-gene interaction employing an endogen- used as a template to amplify a PCR fragment corre- ous yeast cellular context could provide disease-relevant sponding to yor1-ΔF670/R1116T-HA-GFP-3’UTR which insight via gene homology is unknown [5]. To investigate was combined with another PCR fragment encoding the this question, we applied the Q-HTCP method to systema- NATMX cassette flanked by further YOR1 3’UTR tically query the yeast genome for modifiers of a specific sequence by splice overlap PCR. The full product (yor1- phenotype resulting from Yor1-ΔF670, and provide evi- ΔF670/R1116T-HA-GFP-3’UTR-NATMX-3’UTR)was dence validating this yeast phenomic (genome-wide analy- transformed into yeast and selected for on media con- sis of phenotypic modification due to gene interaction) taining nourseothricin (’ClonNat’, Werner BioAgents); model for CFTR-ΔF508, the most prevalent human allele thepresenceofthegenomicΔF670/R1116T mutation causing cystic fibrosis [7]. was confirmed by sequence analysis, creating strain RL4. To model the evolutionarily conserved network of gene The endogenous YOR1 promoter was replaced with a interaction involving CFTR-ΔF508, we introduced the Tet-OFF regulatable element by insertion of pJH023, as homologous yeast ABC transporter, Yor1-ΔF670 [8,9], previously described [20], at the YOR1 locus to create into the library of non-essential yeast gene deletion strain RL8. RL8 was mated to the MATa deletion strain Louie et al. Genome Medicine 2012, 4:103 Page 3 of 20 http://genomemedicine.com/content/4/12/103 A B YPEG + 0.2Rg/ml oligomycin T+-T+-T+- pRS316 F-HA YOR1 yor1-)F670 F-Sec22 yor1-)F670/R1116T Yor1 Yor1-)F/ Yor1-)F R1116T C Trypsin D X-linker: (Rg/mL) 0 5 10 20 0 5 10 20 0 5 10 20 kDa 180 Agg. 115 82 X-linked 64 kDa 49 170- Native 37 130-