EGF treated SCN versus intra-animal control at two circadian times. EGF treated SCN versus intra-animal control at two circadian times

Background: Identifying the gene regulatory networks governing physiological signal integration remains an important challenge in circadian biology. Epidermal growth factor receptor (EGFR) has been implicated in circadian function and EGFR is expressed in the suprachiasmatic nucleus (SCN), the core circadian pacemaker. The transcription networks downstream of EGFR in the SCN are unknown, but by analogy to other SCN inputs we expect the response to EGFR activation to depend on circadian timing and thus be “circadian context–dependent”. Results: We have undertaken a systems level analysis of EGFR circadian context–dependent signaling in the SCN. We collected gene expression profiles to study how the SCN response to EGFR activation depends on circadian timing. Mixed–model analysis of variance (ANOVA) was employed to identify genes with circadian context–dependent EGFR regulation. The expression data was integrated with transcription factor (TF) binding predictions through gene group enrichment analyses to generate robust hypotheses about TFs responsible for the circadian phase–dependent EGFR responses. Conclusions: The analysis results suggest that the transcriptional response to EGFR signaling in the SCN may be partly mediated by established EGFR signaling regulated TFs (AP1, Ets1), TFs involved in circadian clock entrainment (CREB), and by core clock TFs (Rorα). qRT-PCR measurements of several TF expression levels support a model in which circadian context-dependent EGFR responses are partly achieved by circadian regulation of upstream signaling components. Our study suggests an important role for EGFR signaling in SCN function and provides an example for gaining physiological insights through systems-level analysis. Keywords: dose response; repeat sample Overall design: A 2X2 factorial experimental design was used to investigate differences between "day" (8 hours after lights on) and "night" (2 hours after lights off) SCN gene expression responses to EGFR activation induced by EGF treatment (20 nM, 1 hr). Two SCN were obtained from each rat and served as EGF–treated and vehicle-treated samples. Pairing control and treated samples from the same rat permitted detection of EGF effects in the presence of substantial animal-to-animal variability. SCN from two rats were experimentally treated at each circadian time, yielding a total of eight biological samples. Since our goal was a preliminary characterization of EGFR response clock phase dependency, samples were hybridized to one microarray each. A universal reference design was used for the microarrays themselves.

背景：解析调控生理信号整合的基因调控网络，仍是昼夜节律生物学领域的重要研究难题。表皮生长因子受体（EGFR）已被证实与昼夜节律功能相关，且在核心昼夜节律起搏器——视交叉上核（SCN）中存在表达。目前尚不明确SCN中EGFR下游的转录调控网络，但参考其他SCN输入信号的调控规律，我们推测EGFR激活后的应答反应依赖于昼夜节律时间，因此呈现“昼夜情境依赖性”。

结果：本研究针对SCN中EGFR的昼夜情境依赖性信号通路开展了系统级分析。我们采集了基因表达谱，以探究SCN对EGFR激活的应答反应如何随昼夜节律时间变化。我们采用混合效应方差分析（ANOVA），筛选出受昼夜情境依赖性EGFR调控的基因。通过基因集富集分析，将表达数据与转录因子（TF）结合预测结果进行整合，从而为调控昼夜相位依赖性EGFR应答的转录因子构建了可靠的假说。

结论：分析结果显示，SCN中EGFR信号通路的转录应答，部分由已证实受EGFR信号调控的转录因子（AP1、Ets1）、参与昼夜节律钟同步的转录因子（CREB）以及核心钟控转录因子（Rorα）所介导。对若干转录因子表达水平的定量实时聚合酶链反应（qRT-PCR）检测结果，支持“昼夜情境依赖性EGFR应答，部分通过上游信号组分的昼夜节律调控得以实现”这一模型。本研究揭示了EGFR信号通路在SCN功能中的重要作用，并为通过系统级分析获取生理机制见解提供了范例。

关键词：剂量应答；重复样本

实验设计：本研究采用2×2析因实验设计，探究“日间”（光照开启后8小时）与“夜间”（光照关闭后2小时）的SCN，在经EGF处理（20 nM，处理1小时）诱导EGFR激活后的基因表达应答差异。每只大鼠采集2个SCN组织，分别作为EGF处理组与溶剂对照组样本。通过配对同一只大鼠的对照与处理样本，得以在个体间存在显著差异的情况下，精准检测EGF的作用效应。每个昼夜时间点均使用2只大鼠的SCN进行实验处理，最终共获得8个生物学重复样本。鉴于本研究旨在初步表征EGFR应答的钟相位依赖性，每个样本均单独进行1次芯片杂交。芯片实验采用通用参考设计方案。