All ETDs from UAB

Advisory Committee Chair

John L Hartman

Advisory Committee Members

David M Bedwell

Kim M Keeling

Brett A McKinney

Eric J Sorscher

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Much remains unknown about gene interaction in the context of human disease. In cystic fibrosis (CF) a single mutation of the cystic fibrosis transmembrane conductance regulator (CFTR-deltaF508) accounts for most disease. In cell models, CFTR-deltaF508 causes defective protein biogenesis and degradation rather than proper trafficking to the plasma membrane where CFTR normally functions in ion transport. Numerous genes function in the biogenesis of CFTR and influence the fate of CFTR-deltaF508 in cell models. However it is not known whether natural genetic variation in such genes contributes to disease severity in patients. Moreover, there is no easy way to predict how numerous gene interactions involving CFTR-deltaF could manifest phenotypically. To gain insight into the function and evolutionary conservation of a gene interaction network that regulates biogenesis of a misfolded ABC-transporter, we utilize yeast genetics to develop a "phenomic" model, in which the CFTR-F508-equivalent residue of a yeast homolog is mutated (Yor1-deltaF670), and where the genome is scanned quantitatively for interaction. We first confirmed that Yor1-deltaF undergoes protein misfolding and has a reduced half-life, analogous to CFTR-deltaF. Gene interaction was then assessed quantitatively by growth curves for all ~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. Clustering of the data revealed phenomic modules; i.e., genes that similarly influence the phenotype and have shared functional annotations such as cooperation in the same pathway or complex. Yeast gene interaction with Yor1-deltaF resembled, by homology, modulation of processing of CFTR-deltaF in mammalian cells. Novel evolutionarily conserved pathways were implicated by the yeast study, and a recently discovered ER Membrane Complex (EMC) was discovered from the yeast screen to function in trafficking of misfolded Yor1-deltaF670 and validated by siRNA of CFTR-deltaF508 in a human cell line. Based on these findings, the Yor1-deltaF model appears to have translational potential for investigating the molecular basis of gene interaction networks and their functions in modulating cystic fibrosis disease severity.

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