Regulation of innate antiviral responses by the DNA-binding transcriptional repressor complex CIC-ATXN1/L

摘要 干扰素(干扰素)和干扰素刺激基因(ISGs)是先天性免疫反应的关键组成部分，有助于 在早期控制病毒感染方面。干扰素和ISGs的转录上调通过以下途径受到严格控制 基础条件下维持动态平衡的负性调节因子，如干扰素和ISGs的异常表达 可以促进慢性病毒感染、癌症、神经退行性变和糖尿病等疾病。通过基因组- 广泛的CRISPR/Cas9筛选以识别前宿主因子和天然免疫的调节因子，我们确定 Capicua(CIC)作为一种新的天然免疫反应调节器，正如CIC基因敲除(CIC KO)细胞所证明的那样 干扰素和ISGs在基础条件下转录上调并显示不同类型的限制性复制 RNA病毒。基于对果蝇和癌症模型的研究，CIC，一种高度保守的DNA结合 转录抑制物，与靶基因附近的特定八聚体序列(CIC结合位点或CBS)结合 并结构性地抑制基因转录。通过对CIC KO上调的ISGs启动子基序的分析 通过RNA-Seq鉴定细胞，我们观察到在几个但不是所有上调的ISG中存在CBS基序， 包括RIG-I、MDA5、IRF3/7/9、干扰素β、干扰素γ、IFIT1、TRIM22、MX1和ISG15。的初步检测 转座酶可及染色质(ATAC)-SEQ分析证实 CIC KO中含有Ifit1和TRIM22启动子的CBS与对照A549细胞的比较。此外, 我们通过染色质验证了CIC与干扰素β和isg启动子(STAT1、IFIT1、TRIM22)的直接结合 免疫沉淀-定量聚合酶链式反应(ChIP-qPCR)分析。此外，CIC-ATXN1/L共抑制物复合体迅速 在流感病毒感染过程中降解，提示CIC-ATXN1/L复合体的降解可能是一种 先天免疫反应途径的组成部分。重要的是，人的先天性免疫调节功能 Cic-Atxn1/L共抑制物复合体在小鼠模型中是保守的。基于这些发现，我们提出了一个 干扰素和胰岛素样生长因子转录调控的新模型：在基础条件下，CIC-ATXN1/L联合 抑制物复合体与CBS结合并抑制ISG转录；然而，病毒诱导的蛋白酶体 CIC-ATXN1/L的降解使这些靶基因从主动抑制中解脱出来，从而允许强大的 通过红外线和统计数据转录。这项提议的目标是获得对 CIC-ATXN1/L共抑制物复合体对干扰素和异硫氰酸甘油启动子的转录调控 为调节细胞自主抗病毒反应提供了一个新的范例。 PHS 398/2590(06/09版)页面续格式页面

Abstract Interferon (IFN) and IFN stimulated genes (ISGs) are a critical component of innate immune responses that help in the early control of viral infections. Transcriptional upregulation of IFN and ISGs is tightly controlled through negative regulators under basal conditions to maintain homeostasis, as aberrant expression of IFN and ISGs can promote diseases like chronic viral infection, cancers, neurodegeneration, and diabetes. Through a genome- wide CRISPR/Cas9 screen to identify proviral host factors and regulators of innate immunity, we identified Capicua (CIC) as a novel regulator of innate immune responses, as CIC knockout (CIC KO) cells demonstrated transcriptional upregulation of IFN and ISGs under basal conditions and showed restricted replication of different RNA viruses. Based on studies in drosophila and cancer models, CIC, a highly conserved DNA-binding transcriptional repressor, binds to a specific octameric sequence (CIC binding site or CBS) near target genes and constitutively represses gene transcription. Through promoter motif analysis of ISGs upregulated in CIC KO cells identified by RNA-Seq, we observed the presence of CBS motifs in several, but not all, upregulated ISGs, including RIG-I, MDA-5, IRF3/7/9, IFNβ, IFNγ, IFIT1, TRIM22, MX1, and ISG15. Preliminary Assay for Transposase-Accessible Chromatin (ATAC)-Seq analysis confirmed increased chromatin accessibility near the putative CBS containing IFIT1 and TRIM22 promoters in CIC KO as compared to control A549 cells. In addition, we validated direct CIC binding to IFNβ and ISG promoters (STAT1, IFIT1,TRIM22) by chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis. Moreover, the CIC-ATXN1/L co-repressor complex is rapidly degraded during influenza virus infection, suggesting that degradation of the CIC-ATXN1/L complex may be an integral part of the innate immune response pathway. Importantly, the innate immune regulatory function of the Cic-Atxn1/l co-repressor complex is conserved in the murine model. Based on these findings, we propose a novel model for the transcriptional regulation of IFN and ISGs: under basal conditions, the CIC-ATXN1/L co- repressor complex binds to CBS and represses ISG transcription; however, virus-induced proteasomal degradation of CIC-ATXN1/L relieves these target genes from active repression, thereby allowing for robust transcription through IRFs and STATs. The goal of this proposal is to gain mechanistic insights into the transcriptional regulation of IFN and ISG promoters by the CIC-ATXN1/L co-repressor complex, ultimately providing a new paradigm for the regulation of cell autonomous antiviral responses. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page