Tracing information flow from Erk to target gene induction reveals mechanisms of dynamic and combinatorial control. Mus musculus

In prior work we developed an optogenetic system for delivering highly precise, time-varying inputs to Ras, termed OptoSOS (Toettcher et al., 2013). This system relies on a membrane-targeted photoswitchable protein (Phy-CAAX) and a cytoplasmic Ras activator (PIF-SOScat) whose localization to the membrane can be controlled with light. In this system, Phy/PIF heterodimerization can be triggered on and off by exposure to 650 and 750 nm light, respectively. We found that this system could be used to deliver highly precise levels and dynamics of Ras/Erk signaling both in vitro and in vivo. Here, we aimed to globally assess the transcriptional response to light-activated Ras and compare it to that induced by growth factor stimulation. We stimulated NIH3T3 OptoSOS cells with either constant activating red light or PDGF and measured transcriptional responses by RNAseq. Total mRNA was collected after 0, 30, 60 and 120 minutes and used to track the dynamics of transcript abundance in both conditions. Genes were defined as upregulated if they satisfied two criteria: (i) induced at least three-fold over unstimulated cells, and (ii) induced at least two consecutive timepoints. By these criteria we detected 118 genes that were upregulated within 2 h by either PDGF or light stimulation, a comparable number of Ras-responsive genes to that found in previous studies. We found that both PDGF and light induced nearly identical profiles of gene expression, with 100/118 genes induced by PDGF and 110/118 induced by light. At each time point we found excellent agreement between the levels of gene induction in response to both stimuli. This agreement also extended to response dynamics. where hierarchical clustering revealed three classes of dynamic response: an early response peaking within 30 min, an intermediate response peaking at ~1 h, and a late response where gene expression gradually increased over the full 2 h timecourse. In all three classes, we found that light and PDGF led to highly similar expression changes over time. We thus concluded that sole stimulation of the Ras/Erk pathway by light was sufficient to recapitulate at least the first two hours of the PDGF-induced transcriptional response. Overall design: RNA-seq to measure global transcript abundance at various timepoints after PDGF stimulation or direct optogenetic activation of Ras using the OptoSOS system in NIH3T3 cells (Toettcher et al, Cell 2013). 9 samples were collected using the TruSeq library preparation kit (Illumina), multiplexed, pooled and measured in 3 lanes of an Illumina Hi-Seq 2000. Library quality was assessed by Agilent Bioanalyzer. Roughly 30-50 million reads were measured per sample across all 3 lanes. Baseline transcript abundance was measured in triplicate (0 min controls) and each successive timepoint was measured in a single collection. Genes were considered upregulated if they were induced at least 5-fold in at least two consecutive timepoints relative to their baseline abundance.

既往研究中，我们开发了一种可向Ras传递高精度、时变输入的光遗传系统，命名为OptoSOS（Toettcher等人，2013）。该系统依赖于两种组分：膜靶向光开关蛋白Phy-CAAX，以及胞质Ras激活因子PIF-SOScat，后者向细胞膜的定位可通过光进行调控。在该系统中，Phy/PIF异二聚化可分别通过650nm和750nm的光照触发开启与关闭。我们发现，该系统可在体外与体内环境中，实现Ras/Erk信号通路的高精度水平与动态调控。
本研究旨在全面评估光激活Ras所引发的转录应答，并将其与生长因子刺激诱导的转录应答进行比较。我们分别以持续性激活红光或血小板衍生生长因子（PDGF）刺激NIH3T3 OptoSOS细胞，并通过RNA测序（RNA-seq）检测转录应答变化。分别在刺激后0、30、60和120分钟收集总mRNA，以追踪两种刺激条件下转录本丰度的动态变化。
基因的初始上调判定标准为同时满足以下两项：(i) 表达量较未刺激细胞至少上调3倍；(ii) 至少在两个连续时间点呈现诱导上调。基于上述标准，我们共检测到118个在2小时内可被PDGF或光照刺激上调的基因，该Ras应答基因数量与既往研究报道的结果相当。我们发现，PDGF与光照诱导的基因表达谱几乎完全一致：118个上调基因中有100个可被PDGF诱导，110个可被光照诱导。在每个时间点，两种刺激所引发的基因诱导水平均具有极佳的一致性，这种一致性同样体现在应答动态上：通过层次聚类分析，我们将动态应答分为三类：早期应答（30分钟内达到峰值）、中期应答（约1小时达到峰值）以及晚期应答（基因表达在完整2小时时间进程中逐渐升高）。在三类应答中，光照与PDGF刺激所引发的基因表达随时间的变化均高度相似。因此我们得出结论：仅通过光照激活Ras/Erk通路，即可复现PDGF诱导的转录应答至少前两小时的变化过程。
实验整体设计：在NIH3T3细胞中，分别通过PDGF刺激或OptoSOS系统直接光遗传激活Ras，于不同时间点采集样本，采用RNA-seq检测全局转录本丰度（Toettcher等人，《Cell》，2013）。共收集9个样本，使用Illumina TruSeq文库制备试剂盒构建文库，经多重标记、混合后，在Illumina HiSeq 2000的3个测序泳道中进行测序。通过Agilent Bioanalyzer评估文库质量；所有3个泳道中，每个样本的测序读数约为3000万至5000万。转录本丰度的基线水平设置为3次生物学重复（0分钟对照组），后续每个时间点的样本均单次收集检测。本研究最终的基因上调判定标准为：若基因相较于基线丰度，至少在两个连续时间点内出现至少5倍的表达上调，则将其判定为上调基因。