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

DNA 结合转录抑制复合物 CIC-ATXN1/L 对先天抗病毒反应的调节

基本信息

批准号：
10671677
负责人：
Balaji Manicassamy
金额：
$ 54.75万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10671677
关键词：
A549 Antiviral Response Autoimmune Diseases Binding Binding Sites Biological Assay Bone Marrow CRISPR screen Cancer Model Cells ChIP-seq Chromatin Chronic Complex DNA DNA Binding Dendritic Cells Diabetes Mellitus Disease Distal Drosophila genus Elements Embryo Embryonic Development Encephalomyocarditis virus Enterobacteria phage P1 Cre recombinase Epithelial Cells Fibroblasts Gene Expression Genes Genetic Transcription Goals Homeostasis Human IRF3 gene ISG15 gene Immune Immunity Infection Inflammatory Influenza A virus Innate Immune Response Integration Host Factors Interferon Type I Interferon Type II Interferon-beta Interferons Knock-out Knockout Mice Luciferases Macrophage Malignant Neoplasms Mediating Modeling Mus Mutate Natural Immunity Nerve Degeneration Neurodegenerative Disorders Nucleic Acids Pathway interactions Pattern recognition receptor Post-Translational Protein Processing Production Promoter Regions RNA RNA Viruses Receptor Activation Receptor Protein-Tyrosine Kinases Regulation Reporter Repression Repressor Proteins Respiratory System Role STAT1 gene Spinocerebellar Ataxias Stimulus Transcription Repressor Transcriptional Regulation Transposase Up-Regulation Validation Vesicular stomatitis Indiana virus Viral Viral Genes Virus Virus Diseases Virus Replication Zika Virus airway epithelium ataxin-1 cancer type cell type chromatin immunoprecipitation comparison control cytokine derepression gene repression genetic corepressor genome-wide in vivo influenza infection insight loss of function mutation microbial mouse model multicatalytic endopeptidase complex neuron development novel prevent promoter sensor transcription factor transcriptome sequencing

项目摘要

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

抽象的干扰素 (IFN) 和干扰素刺激基因 (ISG) 是先天免疫反应的关键组成部分，有助于在病毒感染的早期控制中，IFN 和 ISG 的转录上调受到严格控制。基础条件下负调节因子维持体内平衡，如 IFN 和 ISG 的异常表达可以通过基因组促进慢性病毒感染、癌症、神经退行性疾病和糖尿病等疾病。我们通过广泛的 CRISPR/Cas9 筛选来鉴定原病毒宿主因子和先天免疫调节因子 Capicua (CIC) 作为先天免疫反应的新型调节剂，CIC 敲除 (CIC KO) 细胞证明了这一点基础条件下 IFN 和 ISG 的转录上调，并显示不同的复制受到限制 RNA 病毒。基于对果蝇和癌症模型的研究，CIC 是一种高度保守的 DNA 结合病毒。转录抑制因子，与目标基因附近的特定八聚体序列（CIC 结合位点或 CBS）结合通过对 CIC KO 中上调的 ISG 进行启动子基序分析，组成型抑制基因转录。通过 RNA-Seq 鉴定的细胞，我们观察到几个（但不是全部）上调的 ISG 中存在 CBS 基序，包括 RIG-I、MDA-5、IRF3/7/9、IFNβ、IFNγ、IFIT1、TRIM22、MX1 和 ISG15 的初步检测。转座酶可及染色质 (ATAC)-Seq 分析证实，转座酶可及染色质附近的染色质可及性增加与对照 A549 细胞相比，CIC KO 中推定的 CBS 含有 IFIT1 和 TRIM22 启动子。我们通过染色质验证了 CIC 与 IFNβ 和 ISG 启动子（STAT1、IFIT1、TRIM22）的直接结合此外，CIC-ATXN1/L 共阻遏物复合物可快速进行免疫沉淀 qPCR (ChIP-qPCR) 分析。在流感病毒感染期间降解，表明 CIC-ATXN1/L 复合物的降解可能是先天免疫反应途径的组成部分，重要的是，先天免疫调节功能。 Cic-Atxn1/l 共阻遏物复合物在小鼠模型中是保守的，基于这些发现，我们提出了一个。 IFN 和 ISG 转录调控的新模型：在基础条件下，CIC-ATXN1/L co- 阻遏复合物与 CBS 结合并抑制 ISG 转录；然而，病毒诱导的蛋白酶体 CIC-ATXN1/L 的降解可解除这些靶基因的主动抑制，从而实现稳健的通过 IRF 和 STAT 进行转录该提案的目标是获得对 IRF 和 STAT 的机制的了解。最终，CIC-ATXN1/L 共阻遏物复合体对 IFN 和 ISG 启动子的转录调节为细胞自主抗病毒反应的调节提供了新的范例。 PHS 398/2590（修订版 06/09）页面延续格式页面