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

摘要 干扰素（IFN）和IFN刺激基因（ISG）是先天免疫反应的关键组成部分， 病毒感染的早期控制。IFN和ISG的转录上调通过以下途径被严格控制： 在基础条件下维持稳态的负调节因子，如IFN和ISGs的异常表达 可以促进慢性病毒感染、癌症、神经退化和糖尿病等疾病。通过基因组- 广泛的CRISPR/Cas9筛选，以确定先天免疫的前病毒宿主因子和调节因子，我们确定了 Capicua（CIC）作为先天免疫应答的新型调节剂，如CIC敲除（CIC KO）细胞所示 在基础条件下，IFN和ISG的转录上调，并显示不同的限制性复制 RNA病毒。基于在果蝇和癌症模型中的研究，CIC，一种高度保守的DNA结合蛋白， 转录阻遏物，结合到靶基因附近的特定八聚体序列（CIC结合位点或CBS 并组成性抑制基因转录。通过CIC KO中上调的ISG的启动子基序分析 通过RNA-Seq鉴定的细胞中，我们观察到CBS基序在几个但不是全部上调的ISG中的存在， 包括RIG-I、MDA-5、IRF 3/7/9、IFNβ、IFNγ、IFIT 1、TRIM 22、MX 1和ISG 15。初步试验 转座酶可降解染色质（ATAC）-Seq分析证实了在细胞核附近增加的染色质可及性。 与对照A549细胞相比，CIC KO中含有IFIT 1和TRIM 22启动子的推定CBS。此外，本发明还提供了一种方法， 我们验证了CIC与IFNβ和ISG启动子（STAT 1，IFIT 1，TRIM 22）的直接结合， 免疫沉淀-qPCR（ChIP-qPCR）分析。此外，CIC-ATXN 1/L共阻遏物复合物被快速地 在流感病毒感染期间降解，这表明CIC-ATXN 1/L复合物的降解可能是流感病毒感染的一个原因。 先天免疫反应途径的组成部分。重要的是，先天性免疫调节功能， Cic-Atxn 1/l共阻遏物复合物在鼠模型中是保守的。基于这些发现，我们提出了一个 IFN和ISGs转录调控的新模型：在基础条件下，CIC-ATXN 1/L共- 阻遏物复合物与CBS结合并阻遏ISG转录;然而，病毒诱导的蛋白酶体 CIC-ATXN 1/L的降解解除了这些靶基因的活性抑制，从而允许稳健的表达。 通过IRFs和STAT转录。本提案的目标是获得对 CIC-ATXN 1/L共阻遏物复合物对IFN和ISG启动子的转录调控，最终 为调节细胞自主抗病毒反应提供了新的范例。 PHS 398/2590（Rev.06/09）

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