Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

Context-dependent changes in genetic vulnerabilities are important to understand the wiring of cellular pathways and variations in different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic networks over time or in response to external stresses are lacking. To analyze the plasticity of genetic interactions, we performed an arrayed combinatorial RNAi screen in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, we assessed the effect of 12768 pairwise RNAi perturbations in six different conditions. We found that genetic interactions form in different trajectories and developed an algorithm, termed MODIFI, to analyze how genetic interactions rewire over time. Using this framework, we identified more statistically significant interactions compared to end-points assays and further observed several examples of context-dependent crosstalk between signaling pathways such as an interaction between Ras and Rel which is dependent on MEK activity.