ISGF3 and STAT2/IRF9 direct basal and IFN-induced transcription through genome-wide binding of phosphorylated and unphosphorylated complexes to commonly ISRE containing ISGs [ChIP-Seq]. ISGF3 and STAT2/IRF9 direct basal and IFN-induced transcription through genome-wide binding of phosphorylated and unphosphorylated complexes to commonly ISRE containing ISGs [ChIP-Seq]

To further understand the role of phosphorylation in ISGF3- and STAT2/IRF9-mediated constitutive and long-term IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and anti-viral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells IFNα-inducible transcription and anti-viral activity relied on the recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, ISG expression correlated with DNA-binding of phosphorylated STAT2/IRF9. This pointed to a dominant role of classical ISGF3 and STAT2/IRF9, and not U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection, in WT and STAT1-KO cells. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all ISGF3 components (ST1-ST2-IRF9-U3C), revealed a threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-treated ISG expression and anti-viral activity. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for 2fTGH, ST2-U3C, Huh7.5, and Huh STAT1KO cells treated with IFN alpha

为进一步阐明磷酸化在干扰素刺激因子3（ISGF3）以及STAT2/IRF9介导的组成型与长期I型干扰素（IFN-I）刺激转录应答中的作用，我们联合运用磷酸化抑制实验、抗病毒实验，结合RNA测序（RNA-Seq）与染色质免疫沉淀测序（ChIP-Seq）技术开展研究。首先，我们鉴定出一组携带干扰素刺激反应元件（ISRE）的干扰素刺激基因（ISGs），该类基因在经α干扰素（IFNα）处理的野生型（WT）与STAT1基因敲除（STAT1-KO）细胞中呈现共同的表达调控模式。据此，在2fTGH与Huh7.5野生型细胞中，IFNα诱导的转录激活与抗病毒活性，依赖于ISGF3复合物组分STAT1、STAT2与IRF9的招募，且该过程呈磷酸化依赖性与时间依赖性。同理，在STAT1缺失的ST2-U3C与Huh-STAT1KO细胞中，ISG的表达与磷酸化STAT2/IRF9复合物的DNA结合活性显著相关。上述结果表明，在野生型与STAT1-KO细胞中，经典ISGF3与STAT2/IRF9复合物而非U-ISGF3或U-STAT2/IRF9复合物，在调控早期及持续型ISG表达与病毒防护功能中发挥主导作用。此外，在过表达全部ISGF3复合物组分的U3C（STAT1-KO）细胞（即ST1-ST2-IRF9-U3C细胞）中开展的对照实验显示，U-ISFG3以及潜在的U-STAT2/IRF9复合物，在调控组成型及可能的长期IFNα处理后的ISG表达与抗病毒活性中，具有阈值依赖性的调控功能。实验总体设计：对经α干扰素处理的2fTGH、ST2-U3C、Huh7.5以及Huh-STAT1KO细胞开展染色质免疫沉淀测序（ChIP-Seq）。