Genes induced by IFN-beta, IFN-gamma or unphosphoraylted STAT1 in human fibroblasts. Homo sapiens

A single high dose of IFNβ activates powerful cellular responses in which many antiviral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells down-regulate this initial response rapidly. However, the expression of many antiviral proteins that do no harm is sustained, prolonging a substantial part of the initial antiviral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced antiviral genes that show prolonged expression are driven by distinct interferon stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase of other IFNβ-induced proteins, and to constitutive resistance to DNA damage. We classified genes into U-ISGF3-induced genes and classical ISGF3-induced genes using microarray data. We identified 150 genes that are up-regulated by IFNβ after 6 h. Among these IFNβ-induced genes, only 29 (20%) were induced by the up-regulation of Y701F-STAT1, indicating that these are induced by U-ISGF3. Among the remaining 121 IFNβ-induced genes (150 minus 29), 73 are induced by IFNγ as well as IFNβ, and 48 are induced only by IFNβ. Overall design: Total 5 RNA samples were analyzed in duplicate on microarray chips. Untreated cells and empty vector-transfected cells were the control of IFN treated cells and Y701F-STAT1-transfected cells, respectively.

单次高剂量干扰素β（IFNβ）可激活强效细胞应答，此时多种抗病毒、促凋亡及抗增殖蛋白均呈高表达状态。由于其中部分蛋白存在有害性，细胞会快速下调这一初始应答。不过，诸多无害的抗病毒蛋白的表达仍会得以维持，使得初始抗病毒应答的核心部分得以延续数日，同时还可赋予细胞DNA损伤抵抗能力。

转录因子干扰素刺激基因因子3（ISGF3，由干扰素调节因子9（IRF9）与酪氨酸磷酸化的信号转导与转录激活因子1、2（STAT1、STAT2）组成）介导了首个快速应答阶段；而由IFNβ诱导产生的高浓度IRF9与STAT1、STAT2（未发生酪氨酸磷酸化）所形成的相关因子——非磷酸化ISGF3（U-ISGF3），则驱动了第二个持久应答阶段。由U-ISGF3诱导、呈现持续表达的抗病毒基因，由独特的干扰素刺激应答元件（ISRE）所调控。

细胞持续暴露于低浓度IFNβ的状态常见于癌症中，这仅会使U-ISGF3依赖型蛋白的表达呈稳态升高，而不会使其他IFNβ诱导蛋白出现持续上调，同时还会使细胞获得持续性DNA损伤抵抗能力。

本研究通过基因芯片（microarray）数据，将基因分为U-ISGF3诱导基因与经典ISGF3诱导基因两类。我们共筛选出150个在IFNβ处理6小时后表达上调的基因。在这些IFNβ诱导基因中，仅有29个（占比20%）可通过酪氨酸701位点突变的信号转导与转录激活因子1（Y701F-STAT1）的过表达诱导产生，表明此类基因由U-ISGF3介导。剩余121个IFNβ诱导基因（150减去29）中，73个可同时被IFNβ与干扰素γ（IFNγ）诱导，另有48个仅可被IFNβ诱导。

整体实验设计：共5组RNA样品，每组均在基因芯片上进行重复检测。未处理细胞与空载转染细胞，分别作为IFN处理组细胞与Y701F-STAT1转染组细胞的对照。