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 <i>Drosophila</i> 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.依赖于环境背景的遗传脆弱性变化，对于解析细胞通路的调控网络以及不同环境条件下的细胞变异机制具有重要意义。然而，当前仍缺乏可用于随时间动态或响应外界胁迫探究遗传网络可塑性的方法学框架。为分析遗传互作的可塑性，我们在多个时间节点以及Ras信号通路活性经药物抑制后，于果蝇（Drosophila）细胞中开展了排列式组合RNA干扰（RNA interference）筛选。我们采用基于成像的形态学检测来捕获多样化表型，对六种不同条件下的12768组成对RNA干扰扰动的效应进行了评估。研究发现遗传互作以不同动态轨迹形成，并据此开发了一款名为MODIFI的算法，用于解析遗传互作随时间的重连模式。借助该框架，我们相较于终点检测法鉴定出了更多统计学上显著的遗传互作，还进一步观察到多条信号通路间存在背景依赖的串扰实例，例如依赖于MEK活性的Ras与Rel之间的互作。