Gene knockout and knockdown approaches are powerful tools of functional genomics, but as chronic techniques the phenotypes they produce are susceptible to masking by secondary adaptations. Small-molecule inhibitors of specific gene products act much more quickly and avoid such secondary phenomena, but they cannot be obtained for all genes and rarely extend to other genomes. This proposal circumvents those limitations by developing a method to deplete almost any gene product in response to a single small molecule (auxin). The approach involves one-step tagging of each gene of interest with a C-terminal auxin-inducible degron (AID) while introducing an AID-specific E3 ubiquitin ligase (TIR1) expression cassette. Upon addition of auxin, the AID-tagged protein is recognized and poly-ubiquitylated by TIR1, and then degraded by the 26S proteasome, resulting in loss of gene function within minutes of induction. To study the performance this platform on genome scales, we will perform Auxin-Inducible Depletion Analyses (AIDA) on all 1,068 essential genes of the model yeast Saccharomyces cerevisiae first using quantitative growth assays. We will determine whether modifications to TIR1 can increase or decrease the susceptibility of particular genes located in various subcellular compartments, potentially extending the range of AIDA to more genes while mapping the locations within cells where each AIDA-susceptible gene performs its essential functions. We will next exploit the intrinsic speed of AIDA in two new applications that were not possible with the conventional approaches. First, by measuring cell death in auxin treated populations, we will identify the subset of essential genes that is specifically required for cell survival rather than cell proliferation. Identification of survival enes is crucial for development of new cidal drugs that kill pathogens or tumors, as opposed to static drugs that merely slow their proliferation by inhibiting pro-growth gene products. For each new pro-survival gene identified in yeast, we will determine whether auxin-induced cell death depends on any previously identified pro-death gene (e.g. metacaspase, endonuclease G, calpain protease, V-ATPase, etc.) using conventional techniques in order to demonstrate method compatibility. Second, we will use AIDA to distinguish early- and late-acting genes that function in a particular pro-death pathway that becomes activated in yeasts by certain fungistatic and fungicidal drugs. Lastly, we will determine whether the AIDA can be used to identify similar pro-survival and pro-death genes in Candida glabrata, an opportunistic fungal pathogen of humans. The results will advance our understanding of cell death mechanisms in fungi, and validate AIDA as both a general tool of functional genomics and an important step toward developing fully druggable genomes.
The research described here will produce a powerful new approach, termed AIDA, that will allow researchers to more easily define the primary functions of genes, to find the 'Achilles heels' of pathogens and cancerous cells, and to model the actions of future drugs that target these genes. It is the first step toward more generalized applications that could eventually reveal new ways of controlling pathogens, killing cancerous human cells, and preventing the death of neurons or other cells during injury or aging. AIDA makes most genes immediately druggable without the need for identifying any new drugs and without the complications caused by much slower genetic approaches. AIDA therefore serves as a bridge between basic and translational research.
| Aouacheria, Abdel; Cunningham, Kyle W; Hardwick, J Marie et al. (2018) Comment on ""Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death"". Science 360: |
| Snyder, Nathan A; Stefan, Christopher P; Soroudi, Camille T et al. (2017) H+ and Pi Byproducts of Glycosylation Affect Ca2+ Homeostasis and Are Retrieved from the Golgi Complex by Homologs of TMEM165 and XPR1. G3 (Bethesda) 7:3913-3924 |
| Kim, Adam; Cunningham, Kyle W (2015) A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress. Mol Biol Cell 26:4631-45 |
