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

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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