Parimal Samir1, Rahul2, James C. Slaughter3, Andrew J. Link1,4,5, * Quantitative proteomic analysis reveals environmental interactions and epistasis in the responses to complex stimuli in Saccharomyces cerevisiae. Parimal Samir1, Rahul2, James C. Slaughter3, Andrew J. Link1,4,5, * 1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA, 2Department of Applied Mathematics, University of Waterloo, Waterloo, ON, Canada, 3Department of Biostatistics, Vanderbilt University School of Medicine, Nashville, TN, USA, 4Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 5Department of Chemistry, Vanderbilt University, Nashville, TN, USA Introduction Results Results The genotype of a cell and its interactions with the environment determine the cell’s biochemical state. Although, the cellular responses to a single stimulus have been extensively studied, the response to concurrent stimuli is important and often neglected. There is need for a unifying conceptual framework to model the effects of multiple environmental stimuli applied concurrently. To build such a concept, we hypothesized that, as an abstraction, environmental stimuli can be treated as analogous to genetic elements. This would allow modeling of the effects of multiple stimuli using the concepts and tools developed for studying gene interactions. We defined an environmental interaction as the interaction between different environmental stimuli that affect the same observable characteristic or trait. Similar to the statistical definition of genetic epistasis, environmental epistasis is an environmental interaction in which the effects of the individual stimuli are not independent of each other. We devised a methodology using dominance in environmental interactions to identify proteins and pathways important for responding to a stimulus. We tested our hypothesis using quantitative proteomic analysis of cellular response of yeast S. cerevisiae to concurrent environmental stimuli. HT#1 G+HT#1 G#1 HT#2 HT#3 G#2 G#3 G+HT#2 G+HT#3 A Fig. 3: Proteomic responses to complex environmental stimuli. A) Complete filtered proteomic dataset of 466 proteins for high temperature stimulus (HT), glycerol stimulus (G), and concurrent glycerol and high temperature stimuli (HT+G) (Red: Up, Green: Down, Black: No change). B) 283 proteins differentially expressed (DE) in response to HT stimulus. C) 379 DE proteins in response to the G stimulus. E) Top 5 pathways for the G stimulus. D) Top 5 pathways for the HT stimulus. F) Circos plot of fold changes and genomic locations. Outermost circle-chromosomes, Second circle-HT stimulus, Third circle-G stimulus, Fourth circle-HT+G stimuli, innermost circle-Environmental epistasis (Purple: Affected by epistasis, Orange: Not affected by epistasis). A C B F D E HT#1 G+HT#1 G#1 HT#2 HT#3 G#2 G#3 G+HT#2 G+HT#3 B HT#1 HT#2 HT#3 C G#1 G#2 G#3 D HT G HT+G HT G HT+G HT G HT+G E Experimental Design Cell Biochemical State Biological signaling molecules Nutrients Oxygen Temperature pH Fig. 1: The biochemical state of a cell is determined by the integration of the response to complex environmental information. Fig. 5. Environmental interaction and the enriched pathways . A) 175 Non-specific environmental response (NER) proteins. B) 41 proteins affected by discordant environmental interaction. C) 58 proteins affected by suppression. D) Top 5 pathways for the NER proteins. E) Top 5 pathways for discordance. F) Top 5 pathways for suppression. F 0 1 Fig. 6. Environmental epistasis. A) Top 10 pathways affected by epistasis. B) Top 10 pathways not affected by epistasis. –log q-value of a pathway with epistasis is shown in purple while and with no epistasis in orange. A B HT#1 G+HT#1 G#1 HT#2 HT#3 G#2 G#3 G+HT#2 G+HT#3 C D A B HT G HT+G HT G HT+G Starter Culture in glucose at 30 oC Glucose at 30 0C (control) Glucose at 37 0C (HT) Glycerol at 37 0C (G+HT) Glycerol at 30 0C (G) Prepare protein extract iTRAQ/MudPIT Statistical analysis Environment Gene 1 Gene Biochemical State 2 Biochemical State 1 Environmental Interactions Gene Interactions Phenotypic Plasticity Fig. 4: Dominance of an environmental stimulus used to identify proteins that are important for responding to the environmental stimulus. The theoretical expression patterns are depicted in the top panel (Red, upregulation; green, downregulation; and black no statistically significant change in expression). A) HT stimulus was dominant over G stimulus for 30 proteins. B) G stimulus was dominant for 121 proteins . C) Top five pathways when HT stimulus was dominant. D) Top five pathways when G stimulus was dominant. Fig. 7: Summary Gene and environmental interactions in concert with phenotypic plasticity determine the biochemical state of the cell. Environmental interactions can serve as filter to select and identify proteins that respond specifically to the dominant environmental stimulus and are important in the response. Fig. 2: Experimental design workflow. Two environmental stimuli were used, high temperature (HT) and glycerol (G).