Genetic buffering represents a major obstacle in understanding the molecular basis of disease. Buffering occurs when the effects of mutations in one gene are masked by expression of another gene. As a result, the genetic variants that contribute to disease are more difficult to identify because their phenotypes are invisible in some genetic backgrounds. To contend with this obstacle, we will test whether chaperone inhibition reduces genetic buffering thereby increasing the power of mapping studies to detect quantitative trait loci (QTL) contributing to cell morphology and growth. Our study stands to (1) identify QTLs contributing to these disease-relevant traits and (2) introduce a technique that will facilitate discovery of genes underlying disease. We will perform QTL screening using Saccharomyces cerevisiae, a widely used model organism for researching the molecular basis of human disease. The QTLs we identify are likely to contain yeast genes with human homologs because basic processes, like cell growth, are conserved between yeast and humans. We will reduce genetic buffering in this mapping family through inhibition of heat shock proteins (HSPs), a type of protein-folding chaperone. HSPs can help mutant proteins to fold and function normally, thereby masking loss of function phenotypes associated with these mutants. We will limit HSP function by treating cells with geldanamycin (GdA), a small-molecule inhibitor of a specific heat shock protein, HSP90. Studies in flies, plants, and yeast have shown that HSP90 inhibition reveals previously masked phenotypic variation that varies between genetically distinct strains. It is not surprising that many different strains harbor mutations with phenotypes that are HSP90 sensitive. Firstly, many gene products interact with HSP90. Secondly, the majority of amino acid substitutions destabilize protein folding, creating proteins that depend on HSP90 to achieve a functional conformation. We hypothesize that hindering HSP function in our QTL screen will reveal the phenotypic effects of some destabilizing mutations, uncovering their loss of function phenotypes and enabling detection of additional QTLs. We will test this hypothesis by performing identical QTL screens with and without chaperone inhibition and comparing the results. If successful, our technique can be utilized to enhance QTL detection in other organisms because HSPs are evolutionarily conserved. We implement several additional features to enhance detection of QTLs for cell morphology and growth. We study wild yeast strains in order to explore natural genetic variation that has not already been surveyed in studies of laboratory strains. We also use a high throughput microscopy platform that allows tracking of 220 morphological phenotypes per cell and measures growth curves for ~50,000 microcolonies per single-day experiment. This method can distinguish tiny phenotypic differences in growth and morphology, and may allow discovery of previously undetectable QTLs. Identifying QTLs contributing to cell morphology and growth is an important goal in understanding the genetic basis of diseases, cancers in particular.
Genetic buffering represents a major obstacle in understanding the molecular basis of disease. We hypothesize that inhibiting cell chaperones, a well-studied type of genetic buffer, will enhance detection of quantitative trait loci contributingto cell morphology and growth phenotypes. Our study stands to (1) identify genes contributing to these disease-relevant traits and (2) to introduce a broadly applicable technique that will facilitate discovery of genes underlying complex phenotypes and disease.
|Geiler-Samerotte, Kerry A; Zhu, Yuan O; Goulet, Benjamin E et al. (2016) Selection Transforms the Landscape of Genetic Variation Interacting with Hsp90. PLoS Biol 14:e2000465|
|Geiler-Samerotte, K A; Bauer, C R; Li, S et al. (2013) The details in the distributions: why and how to study phenotypic variability. Curr Opin Biotechnol 24:752-9|