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：
10539709
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/10539709
关键词：
A549 Antiviral Response Autoimmunity 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 Influenza A virus Innate Immune Response Integration Host Factors Interferon Type I Interferon Type II Interferon-beta Interferons Knock-out Knockout Mice Luciferases 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 Virus Virus Diseases Virus Replication Zika Virus airway epithelium ataxin-1 base cancer type cell type chromatin immunoprecipitation cytokine derepression gene repression genetic corepressor genome-wide in vivo influenza infection insight loss of function mutation macrophage 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IFN刺激基因（ISGS）是先天免疫复杂的关键组成部分，可帮助在病毒感染的早期控制中。 IFN和ISGS的转录上调通过基本条件下的负调节因素维持体内平衡，如IFN和ISG的异常表达可以促进诸如慢性病毒感染，癌症，神经变性和糖尿病等疾病。通过基因组 - 广泛的CRISPR/CAS9屏幕以识别提供者的宿主因素和先天免疫的监管因素，我们确定 Capicua（CIC）是一种新型的先天免疫调节器，因为CIC敲除（CIC KO）细胞证明了 IFN和ISG在基本条件下的转录上调，并显示出不同的复制 RNA病毒。基于果蝇和癌症模型的研究，CIC是一种高度保守的DNA结合转录表示，与目标基因附近的特定八接序列（CIC结合位点或CBS）结合并组成代表基因转录。通过CIC KO中更新的ISG的启动子图案分析通过RNA-Seq鉴定的细胞，我们观察到几个（但不是全部）的CBS基序的存在，包括RIG-I，MDA-5，IRF3/7/9，IFNβ，IFNγ，IFIT1，TRIM22，MX1和ISG15。初步测定转座酶可访问的染色质（ATAC） - seq分析证实了在附近的染色质可及性提高与对照A549细胞相比，CIC KO中含有IFIT1和TRIM22启动子的推定CB。此外，我们通过染色质验证了与IFNβ和ISG启动子（STAT1，IFIT1，TRIM22）的直接CIC结合免疫沉淀-QPCR（CHIP-QPCR）分析。此外，CIC-ATXN1/L共抑制剂复合物迅速在影响扎病毒感染期间降解，表明CIC-ATXN1/L复合物的降解可能是先天免疫反应途径的整体部分。重要的是，先天免疫调节功能 CIC-ATXN1/L共抑制剂复合物在鼠模型中是保守的。根据这些发现，我们提出了一个 IFN和ISGS转录调控的新型模型：在基本条件下，CIC-ATXN1/L共同复制子复合物与CBS和Replicas ISG转录结合；但是，病毒诱导的蛋白酶体 CIC-ATXN1/L的降解使这些靶基因从主动表达中拯救出来，从而允许健壮通过IRF和统计数据转录。该提议的目的是获得机械洞察力 CIC-ATXN1/L共抑制剂复合物对IFN和ISG启动子的转录调节，最终为调节细胞自主抗病毒反应的新范式提供了新的范式。 PHS 398/2590（修订版06/09）页面延续格式页面