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Published in final edited form as: Cell Host Microbe. 2025 Mar 24;33(4):512–528.e7. doi: 10.1016/j.chom.2025.02.017 Search in PMC Search in PubMed View in NLM Catalog Add to search Transcriptional Repressor Capicua is a Gatekeeper of Cell Intrinsic Interferon Responses Senthamizharasi Manivasagam Senthamizharasi Manivasagam 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA Find articles by Senthamizharasi Manivasagam 1 , Julianna Han Julianna Han 2 Department of Microbiology, University of Chicago, Chicago, IL, USA Find articles by Julianna Han 2 , Athmane Teghanemt Athmane Teghanemt 3 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA Find articles by Athmane Teghanemt 3 , Henry Keen Henry Keen 4 Bioinformatics Division of the Iowa Institute of Human Genetics, University of Iowa, Iowa City, IA, USA Find articles by Henry Keen 4 , Boopathi Sownthirarajan Boopathi Sownthirarajan 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA Find articles by Boopathi Sownthirarajan 1 , Boyang Cheng Boyang Cheng 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA Find articles by Boyang Cheng 1 , Abhiraj Singh Abhiraj Singh 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA Find articles by Abhiraj Singh 1 , Abigail Lewis Abigail Lewis 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA Find articles by Abigail Lewis 1 , Olivia Vogel Olivia Vogel 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA 2 Department of Microbiology, University of Chicago, Chicago, IL, USA Find articles by Olivia Vogel 1, 2 , Gayathri Loganathan Gayathri Loganathan 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA Find articles by Gayathri Loganathan 1 , Lei Huang Lei Huang 5 Center for Research Informatics, The University of Chicago, Chicago, IL 60637, USA Find articles by Lei Huang 5 , Maryline Panis Maryline Panis 6 Department of Microbiology, New York University, New York, NY, USA Find articles by Maryline Panis 6 , David K Meyerholz David K Meyerholz 7 Department of Pathology, University of Iowa, Iowa City, IA, USA Find articles by David K Meyerholz 7 , Benjamin tenOever Benjamin tenOever 6 Department of Microbiology, New York University, New York, NY, USA Find articles by Benjamin tenOever 6 , Jasmine T Perez Jasmine T Perez 2 Department of Microbiology, University of Chicago, Chicago, IL, USA Find articles by Jasmine T Perez 2 , Santhakumar Manicassamy Santhakumar Manicassamy 8 Georgia Cancer Center, Department of Medicine, Augusta University, Augusta, GA, USA Find articles by Santhakumar Manicassamy 8 , Priya D Issuree Priya D Issuree 3 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA Find articles by Priya D Issuree 3, * , Balaji Manicassamy Balaji Manicassamy 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA 9 Lead Contact Find articles by Balaji Manicassamy 1, 9, * Author information Article notes Copyright and License information 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, USA 2 Department of Microbiology, University of Chicago, Chicago, IL, USA 3 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA 4 Bioinformatics Division of the Iowa Institute of Human Genetics, University of Iowa, Iowa City, IA, USA 5 Center for Research Informatics, The University of Chicago, Chicago, IL 60637, USA 6 Department of Microbiology, New York University, New York, NY, USA 7 Department of Pathology, University of Iowa, Iowa City, IA, USA 8 Georgia Cancer Center, Department of Medicine, Augusta University, Augusta, GA, USA 9 Lead Contact AUTHOR CONTRIBUTIONS Conceptualization: S.M., J.H., J.T.P., P.D.I., and B.M.; Methodology: S.M., J.H., J.T.P., P.D.I., and B.M.; Investigation: S.M., J.H., A.T., B.S., B.C., A.S., A.L., O.V., G.L., M.P., D.K.M, B.t.O., J.T.P., S.M., P.D.I., and B.M.; Software: L.H., H.K., and P.D.I.; Writing – Original Draft: J.T.P., P.D.I., and B.M.; Writing – Review & Editing: all authors; Funding Acquisition, P.I., and B.M.; Supervision: P.D.I., and B.M. * Correspondence: [email protected] (P.I.) or [email protected] (B.M.) Issue date 2025 Apr 9. PMC Copyright notice PMCID: PMC11985295 NIHMSID: NIHMS2062688 PMID: 40132591 The publisher's version of this article is available at Cell Host Microbe SUMMARY Early detection of viral infection and rapid activation of host antiviral defenses through transcriptional upregulation of interferons (IFNs) and interferon-stimulated genes (ISGs) are critical for controlling infection. However, aberrant production of IFN in the absence of viral infection leads to auto-inflammation and can be detrimental to the host. Here, we show that the DNA binding transcriptional repressor complex composed of Capicua (CIC) and Ataxin-1 like (ATXN1L) binds to an 8-nucleotide motif near IFN and ISG promoters and prevents erroneous expression of inflammatory genes under homeostasis in humans and mice. In contrast, during respiratory viral infection, activation of the mitogen-activated protein kinase (MAPK) pathway results in rapid degradation of the CIC-ATXN1L complex, thereby relieving repression and allowing for robust induction of IFN and ISGs. Together, our studies define a new paradigm for host regulation of IFN and ISGs through the evolutionarily conserved CIC-ATXN1L transcriptional repressor complex during homeostasis and viral infection. Graphical Abstract eTOC blurb Manivasagm et al. describe a novel mechanism by which the evolutionarily conserved CIC-ATXN1L transcriptional repressor complex regulates IFN and ISGs under both homeostatic and viral infection conditions. The CIC-ATXN1L complex binds to CBS motifs near ISG promoters to repress transcription, which is removed through degradation of the complex during viral infection. INTRODUCTION Cell intrinsic innate immune responses against viruses are initiated upon detection of viral nucleic acid by host encoded pattern recognition receptors (PRRs) and the activation of downstream signaling cascades, resulting in the transcriptional upregulation of type I interferons (IFNs) 1 – 3 . In the context of RNA virus infection, retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA-5) (RIG-I like receptors - RLRs) recognize viral RNA as foreign and activate downstream signaling through the adaptor protein mitochondrial antiviral-signaling (MAVS), which organizes the signaling platform to activate the transcription factor interferon regulatory factor 3 (IRF3) or IRF7 (in specialized cell types) via phosphorylation 4 . Upon activation, IRF3/7 binds to IRF elements in type I IFN promoters and orchestrates transcriptional induction of IFN transcripts. IFN secreted from activated cells amplifies host antiviral responses by signaling via IFN receptors (IFNRs) in a feed-forward mechanism. IFNR signaling instructs Janus kinases (JAKs) to phosphorylate and activate signal transducer and activator of transcription 1 (STAT1) and/or STAT2. Activated STAT1/2 heterodimers associate with IRF9 to form the interferon-stimulated gene factor 3 (ISGF3) complex that binds to IFN-stimulated response elements (ISREs) and orchestrates transcriptional upregulation of hundreds of genes referred to as interferon stimulated genes (ISGs). Several of these induced ISGs participate in restricting viral replication and spread. Upon clearance of the viral threat, some ISGs restore homeostasis by negatively regulating PRR and IFNR signaling components. While robust induction of antiviral responses is critical for timely restriction of viral replication, aberrant activation of RLRs and production of IFN and ISGs under sterile conditions can lead to inflammatory diseases 5 , 6 . Under homeostasis, aberrant activation of RLR pathways is prevented through negative regulators that either directly suppress components of the RLR signaling cascade or function indirectly through post-translational modifications (phosphorylation or ubiquitinylation) 7 – 9 . Recent studies demonstrate that RLR pathways can be triggered by host-derived double-stranded RNA (endogenous dsRNA), and such dsRNAs are promptly removed by the host RNA editing enzyme ADAR1 10 – 13 . Except for FOXO3, negative regulation of RLR pathways occurs prior to transcription of IFN and ISGs 9 , 14 . FOXO3 has been reported to specifically bind to and repress transcription from the Irf7 promoter, and loss of FOXO3 increased IRF7 expression under homeostasis, resulting in increased IFN production and higher expression of ISGs through IFNR signaling 14 . As per the current paradigm, transcription from IFN and ISG promoters remains in a default off-state under homeostasis until activated by IRFs and/or the ISGF3 complex 3 , 15 . Here, we report that the DNA binding transcriptional repressor complex containing Capicua (CIC) and Ataxin-1 like (ATXN1L) is a critical negative regulator of transcription from IFN and ISG promoters in humans and mice. Our studies show that, under homeostasis, the CIC-ATXN1/L co-repressor complex binds to an 8-nucleotide CIC binding site (CBS) motif in the distal or promoter regions of IFN and ISGs, and represses basal transcription. However, during respiratory viral infection, activation of the receptor tyrosine kinase (RTK) – mitogen activated protein kinase (RTK-MAPK) signaling cascade induces rapid degradation of the CIC-ATXN1/L repressor complex, allowing for robust transcriptional upregulation of antiviral responses. Thus, our studies define a new paradigm for the host regulation of cell-autonomous antiviral responses in which the CIC-ATXN1/L complex safeguards against the aberrant expression of IFN and ISGs under homeostasis and is promptly degraded upon respiratory virus infection. RESULTS CIC or ATXN1L deficient cells express higher levels of IFN and ISGs Previously, through a genome-wide CRISPR/Cas9 knockout screen for influenza A virus (IAV) host factors, we identified CIC as necessary for regulating antiviral responses, as CIC KO cells showed elevated levels of IFN and ISGs under both homeostasis and viral infection 16 . Importantly, CIC KO cells showed restricted replication of several RNA viruses. To determine if CIC is involved in the regulation of IFN induction via RLR signaling or ISG induction by JAK-STAT signaling, we stimulated control or CIC KO A549 cells (human lung epithelial cell line) by either IAV viral RNA (vRNA) transfection or IFN treatment, and assessed the expression of ISGs at various times post-treatment ( Figures S1A and S1B ). We observed a higher magnitude of IFNB1 and ISG expression in CIC KO cells as compared to control cells under mock conditions as well as upon stimulation via IAV vRNA transfection or recombinant IFNI, respectively. ETV4, a known CIC target gene, was also highly upregulated in CIC KO cells. Interestingly, CIC transcript levels in control cells increased in a temporal manner upon both vRNA and IFN stimulations. These results indicate that CIC likely regulates inflammatory genes induced via both RLR and JAK-STAT signaling. Concordant with elevated IFN signaling, IAV replication was blunted in CIC KO cells as compared to Control A549 cells; as anticipated, complementation of CIC KO cells with either the CIC- long (CIC-L) or CIC-short (CIC-S) isoform restored IAV replication and lowered the expression of IFNB1 and ISGs, confirming that the observed phenotype in CIC KO cells is specifically due to loss of CIC ( Figures S2A – C ). As CIC requires ATXN1 or its homolog ATXN1L to form a stable transcriptional repressor complex, we generated ATXN1L CRISPR KO A549 cells and assessed IAV replication and antiviral responses ( Figures 1A and 1B ). In the absence of CIC or ATXN1L, the other binding partner becomes unstable and is targeted for degradation. Like CIC KO cells, ATXN1L KO cells showed restricted IAV replication and increased levels of IFNB1 and ISG transcripts ( Figures 1C and 1D ). To recapitulate these findings in primary cells, we performed siRNA knockdown of CIC and/or ATXN1L in primary human airway basal cells and observed elevated expression of IFN and ISGs in siCIC KD and siATXN1L KD cells upon IAV infection as compared to siCON KD cells, validating our findings in A549 KO cells ( Figures S2D and S2E ). Together, these findings demonstrate that the CIC-ATXN1L repressor complex is a critical regulator of IFN and ISG expression under both homeostasis and IAV infection conditions. Figure 1. CIC-ATXN1L complex suppresses aberrant expression of IFN and ISGs. Open in a new tab (A) Schematic representation of target gene repression by the CIC-ATXN1/L complex via binding to CIC binding site (CBS, green box; generated using Biorender). (B) Western blot analysis of CIC and ATXN1L expression in different A549 KO cells. β-Actin levels are shown as loading controls. (C) Comparison of IAV replication in Control, CIC KO, and ATXN1L KO A549 cells. Indicated cell lines were infected with IAV (H5N1) at MOI=0.001 and viral loads in the supernatants were measured by plaque assay. Data are shown as mean titer (PFU/mL) of triplicate samples ± SD. (D) qRT-PCR analysis of ISGs in different A549 KO cells. Cells were either mock treated or infected with IAV (H1N1) at MOI=5 and at 16 hpi, mRNA levels for the indicated genes were measured by qRT-PCR. Data are represented as fold expression relative to uninfected A549 cells (mock) ± SD. (E) Volcano plot of RNA-Seq data comparing differentially expressed genes between Control and CIC KO A549 cells. Infections were performed as described in panel D (n=2/group) and total RNA was subjected to Illumina sequencing. A select number of differentially expressed ISGs are indicated. (F) Cluster heat map comparison of representative gene ontology (GO) terms. Defense response to virus (GO:0051607; p-adj from Deseq2 <0.05) and cellular response to cytokine stimulus (GO:0071345; p-adj from Deseq2 <0.05) in CIC KO versus control A549 cells under mock and IAV infection conditions are shown. (G) Molecular Signatures Databases (MsigDB) C3 transcription factor binding sites in DEGs identified in RNA-Seq under mock conditions. See also Figures S1 , S2 and S3 . CIC KO cells show global upregulation of IFN and ISGs To understand if loss of CIC results in upregulation of individual ISGs or causes global changes to the ISG landscape, we performed RNA-Seq analysis of Control and CIC KO A549 cells and identified 800 and 3608 differentially expressed genes (DEGs; log 2 fold change 0.5, p-adj value <0.1) under either mock or IAV infection conditions, respectively ( Figure 1E , Figure S3A , and Table S1 ). Over 60% of these DEGs exhibited elevated expression in CIC KO cells as compared to Control cells for both mock conditions and during IAV infection, consistent with the established role of CIC as a transcriptional repressor. We observed a global upregulation of innate immune response genes in CIC KO cells as compared to Control cells, including several well-known innate immune restriction factors (MX1, ISG15, OAS1-3, IFIT1-3, IFITM1-3, BST2, IFI6, IFI16, IFI27, GBP1-3, TRIMs), antiviral transcription factors (IRF1, IRF2, IRF7, IRF9, STAT1/2), foreign RNA sensors (DDX58 (RIG-I), IFIH1 (MDA-5)), foreign DNA sensors (cGAS, IFI16), cytokines (IL1, IL6, TGFB1), and chemokines (CCL2, CCL8, CXCL1, CXCL2, CXCL5, CXCL11). We validated our RNA-Seq data by performing qRT-PCR analysis on a subset of identified ISGs ( Figure S3B ). Gene ontology (GO) assessment of DEGs in mock treated Control versus CIC KO cells showed significant enrichment for genes associated with host antiviral response pathways ( Figure S3C , Figure 1F , and Table S2 ). We observed a similar gene enrichment in IAV infected conditions. Importantly, we conducted gene set enrichment analysis of DEGs for common transcription factor (TF) motifs and observed enrichment of regulatory sequences containing IRF and ISRE TF motifs in CIC KO cells both under mock and IAV infection conditions, including IFN (IFNB1, IFNL1) and ISGs (IRF7, IRF9, STAT1, MX1, ISG15, IFIT1, IFI6, IFI16, TRIM22) ( Figure 1G , Figure S3D , and Table S3 ). Together, these findings illustrate the role of CIC as a comprehensive regulator of numerous innate inflammatory response genes rather than a regulator of individual ISGs. Endogenous dsRNAs trigger ISG expression in CIC KO cells under sterile conditions As ISG transcription is mediated by IRFs and ISGF3, we hypothesized that the largescale upregulation of numerous ISGs in CIC KO cells under basal (mock) conditions is due to activation of IRFs and ISGF3. To this end, we performed immunofluorescence assays for various IRFs and visualized nuclear localization in Control and CIC KO cells under sterile conditions. We observed increased expression and nuclear localization of IRF2 and IRF9 in CIC KO cells, indicating that activated IRFs and ISGF3 drive expression of IFN and ISGs under sterile conditions ( Figure 2A ). Antibodies against the remaining IRFs failed to show any signal in immunofluorescence analysis. Recent studies demonstrate that in addition to viral RNA, host-derived dsRNAs transcribed from retroelements can activate the sensor MDA-5 (RLR pathways) in the absence of negative regulation ( Figure 2B ) 10 , 12 , 13 . To investigate if IRF activation under sterile conditions in CIC KO cells is initiated by triggering of the RLR pathway by endogenous dsRNA ligands, we generated double KO (DKO) cells concurrently lacking both CIC and the RLR adaptor MAVS ( C/MAVS DKO) and assessed basal IFN and ISG expression. In addition, we generated cells lacking CIC and STAT1 ( C/STAT1 DKO) to abrogate IFNR signaling ( Figure 2C ). Concurrent deletion of MAVS and CIC lowered IFNB1 to levels similar to Control A549 cells, indicating that elevated IFNB1 expression in CIC KO cells under homeostasis is driven by triggering of the RLR-MAVS pathway, likely by endogenous dsRNA ligands ( Figure 2D ). Furthermore, basal IFNB1 levels remained elevated in C/STAT1 DKO cells, albeit at lower levels as compared to CIC KO cells. Exogenous type I IFN treatment induced significantly higher expression of MX1 in C/MAVS DKO cells as compared to Control A549 cells at levels similar to CIC KO cells, indicating that CIC also regulates ISGs induced by IFNR signaling ( Figure 2E ). Both C/MAVS DKO and C/STAT1 DKO cells showed increased IAV and IAV NS1-Mutant replication as compared to CIC KO cells ( Figure 2F ). These results demonstrate that the global upregulation of IFN and ISGs observed in CIC KO cells under homeostasis is not due to erroneous transcription of individual genes but rather driven by the activation of bona fide antiviral transcription factors IRFs and ISGF3. Importantly, these data indicate that CIC is critical for blocking inflammatory responses to endogenous dsRNAs through the RLR pathway. Figure 2. Endogenous dsRNAs trigger ISG expression in CIC KO cells under sterile conditions. Open in a new tab (A) Immunofluorescence analysis for expression of IRFs in CIC KO cells without stimulation. Control A549 and CIC KO cells without any treatments were subjected to immunofluorescence analysis using various anti-IRF antibodies (Scale bar, 100 μm). (B) Depiction of endogenous dsRNA activation of MDA-5/MAVS pathway. Endogenous dsRNAs transcribed from retroelements bind and activate MDA-5/MAVS pathway resulting in IRF mediated expression of IFNs. (C) Western blot analysis of MAVS and STAT1 protein expression in double KO cells. Control, CIC KO, CIC/MAVS DKO or CIC/STAT1 DKO cells were treated with IFN-I (1000 U/mL) and at 24 h, cell lysates were collected for western blot analysis. Nuclear KU86 levels are shown as loading controls. (D and E) qRT-PCR analysis of IFNB1 and MX1 in DKO cells. Indicated cells were either mock treated or transfected with vRNA or treated with IFN-I (1000 U/mL) and at 24 h, expression levels of indicated genes were analyzed by qRT-PCR. Expression in mock treated cells is shown in Panel D and vRNA/IFN treated cells are shown in Panel E. Data are represented as fold expression relative to uninfected Control A549 cells (mock) ± SD. (F) IAV and IAV NS1 Mutant replication in DKO cells. Cells were infected with IAV (H5N1, MOI=0.001) or NS1 R38A Mutant (H3N2, MOI=0.01) and viral loads in the supernatants were measured. Data are shown as mean titer (PFU/mL) of triplicate samples ± SD. CIC regulates the chromatin accessibility of genes essential for IFN responses As CIC is known to function as a DNA transcriptional repressor, we next sought to determine if the global upregulation of innate inflammatory genes in CIC KO cells (RNA-Seq) is initiated by changes in chromatin accessibility. We performed ATAC-Seq (assay for transposase-accessible chromatin using sequencing) analysis to determine the chromatin accessibility landscape of CIC KO versus Control A549 cells under both mock and IAV infection conditions. We detected 6992 differential chromatin accessibility peaks annotated to 4400 genes between CIC KO and Control cells under mock conditions ( Table S4 ). Of these, 3904 peaks showed significantly increased accessibility at gene loci of several key players in the intrinsic immune response pathways in CIC KO cells, including RLRs (DDX58, IFIH1), NLRs, transcription factors (IRFs, STATs), IFNs (IFNB1, IFNLs, IFNG), IFNRs (IFNAR1, IFNAR2, IFNGR), cytokines (IL6, IL1A, IL1B, TGFB1), and viral restriction factors (MX1/2, OAS, IFITs, IFITMs, TRIMs) as compared to Control A549 cells ( Figures 3A and 3B , and Table S4 ). Differentially accessible peaks were prominently situated in intronic and distal intergenic regions (39% and 40% respectively), although many were also present in promoter regions ( Figure 3C ). In the context of IAV infection, we identified 11004 differentially accessible peaks annotated to 5698 genes between CIC KO and Control A549 cells (log 2 FC 0.5, p-adj value <0.1). Interestingly, only 43% and 33% of DEGs identified in RNA-Seq showed a corresponding change in chromatin accessibility by ATAC-Seq analysis under mock and IAV infection conditions, respectively. However, among the DEGs that showed differential chromatin accessibility, a striking majority were upregulated in CIC KO cells (87% in mock and 65% in IAV infection conditions). In both mock and IAV infection conditions, we observed significant upregulation in chromatin accessibility and gene expression for several ISGs such as MX1, ISG15, TRIM22, STAT1, and IFIT1, as well as IRF1, IRF2, and IRF9 ( Figures 2A and 2B , Table S4 ). Taken together, these findings suggest that CIC is critical for the regulation of chromatin accessibility and expression of a large subset of ISGs. Figure 3. CIC-ATXN1L complex represses chromatin accessibility and transcription of ISGs via CBS motif. Open in a new tab (A) MA plot of ATAC-Seq data comparing Control and CIC KO A549 cells. Monolayers of cells were either mock or IAV infected (H1N1 at MOI=5) and at 24 hpi, cells were subjected to ATAC-Seq (n=2/group). (B) Genome browser track of ATAC-Seq datasets at IRF1, ISG15, MX1, and TRIM22 loci. (C) Pie chart depiction of genomic localization of differential ATAC-Seq peaks identified by comparing mock CIC KO versus Control A549 samples. (D) Venn diagram comparison of DEGs identified in RNA-Seq, ATAC-Seq, and ATAC-Seq peaks with putative CBS motif(s). Light blue circle represents genes with differentially accessible chromatin regions in ATAC-Seq, yellow circle represents genes identified in ATAC-Seq peaks with CBS motif(s), and red circle represents DEGs in RNA-Seq. Analysis of mock and IAV infected samples are shown at the top and bottom, respectively. (E) Homer analysis of transcription factor binding sites in 324 common genes identified in integrated analysis of mock samples. Identification of potential CBS motifs near ISG promoters As Drosophila Cic mediates transcriptional repression through binding at CIC binding site(s) (CBS), we assessed whether genes with differential chromatin accessibility were enriched for CBS motifs, indicating plausible direct regulation of these genes by CIC. Using a previously defined canonical CBS motif for Drosophila Cic (TSAATGRR) 17 – 21 , we found >93% of these genes had CBS motifs in both mock and IAV infection conditions, including MX1, ISG15, TRIM22, IFIT1, IRF1, and IRF9 ( Table S5 ). Next, to identify IFNs and ISGs that are regulated by CIC repression, we performed integrated data analyses with (1) DEGs identified in RNA-Seq, (2) genes with differential chromatin accessibility in ATAC-Seq, and (3) ATAC-Seq genes with potential canonical CBS motifs 17 – 21 . In our integrated analyses, we identified 324 common genes under mock conditions and 1108 genes under IAV infection conditions that were common for all three criteria ( Figure 3D , Table S6 ). Several of these CBS-containing genes were components of cell intrinsic antiviral response pathways (DDX58, IFIH1), transcription factors (IRF2, IRF7, IRF9, STAT2), and ISGs (IFIT1, MX1, IFI16, IFITM1, TRIM22, etc.). It should be noted that several type-I IFN genes and ISGs not identified as DEGs in RNA-Seq analysis showed increased chromatin accessibility and carried CBS motif(s) at their gene loci. Next, we conducted HOMER analysis on 324 common genes to determine if other putative TFs were co-enriched ( Table S7 ). Under mock conditions, IRF and ISRE motifs were significantly co-enriched with CBS motifs, suggesting that loss of CIC likely amplifies ISG induction in an IRF-dependent manner ( Figure 3E ). Together, these findings indicate that CIC functions as a transcriptional repressor at IFN and ISG promoters potentially via binding to the identified putative CBS motifs. CIC represses transcription from ISG promoters with CBS motifs To demonstrate that CIC functions as a transcriptional repressor of antiviral response gene promoters containing CBS motifs, we designed and tested a synthetic IFNB1 promoter-reporter with six copies of a CBS motif (IFNβ-6XCBS) downstream of TSS (transcription start site) followed by a firefly luciferase reporter ( Figure 4A ). In these reporter assays, co-expression of CIC and ATXN1 repressed luciferase expression from the IFNβ-6XCBS promoter stimulated with either a constitutively active RIG-I-2CARD (RIG-I (2C)) or Sendai virus (SeV), demonstrating that CIC-ATXN1 can repress expression from CBS motif containing antiviral response gene promoters ( Figures 4A and 4B ). This repression was sequence-specific, as CIC-ATXN1 was unable to repress luciferase expression from the synthetic IFNB1 with 6 copies of mutant CBS (IFNβ-6XCBS Mut ). As a control, when stimulated with RIG-I (2C), IFNβ-6XCBS Mut reporter showed robust luciferase expression. Next, as our integrated analyses revealed many ISG promoters with CBS motifs including MX1, IFIT1, and TRIM22, we evaluated the ability of CIC-ATXN1 to repress these endogenous ISG promoters containing 1-3 copies of native CBS motifs and observed suppression of luciferase expression from all three ISG promoters ( Figure 4C ). In contrast, mutation of native CBS motif(s) in these ISG promoters abrogated CIC-ATXN1 repression of luciferase expression ( Figure 4D ). As a control, CBS Mut ISG promoters showed robust luciferase expression in response to RIG-I (2C) stimulation, indicating that mutations at CBS motifs did not affect IRF3 and/or ISGF3 driven transcription (Data not shown). These results demonstrate that the transcriptional repressor function of CIC is mediated via binding to CBS motifs at ISG promoters. Figure 4. CIC represses transcription from ISG promoters via CBS motifs. Open in a new tab HEK 293T cells were transfected with ISG promoter-reporter, SV40-Renilla reporter, CIC and ATXN1 expressing plasmids or a control GFP, and a constitutively active RIG-2CARD (RIG-I (2C)). At 48 h, firefly and renilla luciferase activities were measured. For each ISG promoter construct, solid or open green boxes represent the relative location of CBS or CBS Mut motifs to transcription start site (TSS), respectively. For CBS Mut , 5 nucleotides were mutated in each of the CBS motif(s) in the ISG promoter. Data are represented as fold normalized luciferase activity upon stimulation with RIG-I (2C) or Sendai virus (SeV) relative to control GFP transfected cells ± SD. (A) IFNβ-6XCBS or CBS Mut promoter activity upon stimulation with RIG-I (2C). (B) IFNβ-6XCBS promoter activity upon stimulation with SeV. (C) Reporter activity of various ISG promoters with native CBS motif(s). (D) Reporter activity of various ISG promoters with CBS Mut motif(s). (E) Transcriptional activation of CBS motif(s) containing ISG promoters by a synthetic CIC-VP64. A 41 amino acid region in the C-terminal repressor domain of CIC was replaced with 4 copies of the HSV-1 VP16 transactivator motif. HEK 293T cells were co-transfected with CIC-VP64, ATXN1, and ISG promoter luciferase reporter constructs, and luciferase activity was measured at 48 h post-transfection. Data are represented as fold normalized luciferase activity relative to control GFP transfected cells ± SD. In some Ewing-like sarcomas, due to chromosomal translocation, CIC is expressed as a fusion protein with double homeobox 4 (DUX4) (CIC-DUX4), where the C-terminal repressor region of CIC is replaced with the DUX4 sequence 22 , 23 . Unlike the canonical CIC-ATXN1L repressor complex, the CIC-DUX4 fusion protein has been suggested to activate transcription of CIC target genes. To further demonstrate that CIC directly binds to CBS motifs and regulates transcription, we generated a synthetic CIC with a transcriptional activator by replacing its C-terminal repressor region with four copies of an HSV-1 VP16 transactivator motif based on the CIC-DUX4 gene rearrangement (CIC-VP64; Figure 4E ). In IFNβ reporter assays, synthetic CIC-VP64 induced robust luciferase expression from the IFNβ-6XCBS promoter at levels similar to constitutively active RIG-I (2C) (Data not shown). In contrast, CIC-VP64 was unable to induce luciferase expression from the IFNβ-6XCBS Mut promoter (Data not shown). In ISG promoter assays, CIC-VP64 induced robust luciferase expression from native CBS containing IRF (IRF1, IRF7) and ISG (MX1, IFIT1, TRIM22) promoters without any RLR stimuli ( Figure 4E ). In contrast, CIC-VP64 was unable to induce luciferase expression from IRF and ISG promoters with mutated CBS motif(s). Taken together, these results demonstrate that CIC directly interacts with CBS motifs and represses transcription from these ISG promoters. CIC-ATXN1L repressor complex is rapidly degraded during IAV infection Although the CIC-ATXN1L complex safeguards against aberrant antiviral responses under homeostasis, timely removal of CIC repression at IFN and ISG promoters is critical for robust transcriptional upregulation of antiviral responses during viral infection. To this end, we assessed CIC levels during IAV infection in A549 cells stably expressing a GFP-tagged CIC (GFP-CIC) and observed complete loss of GFP-CIC in IAV infected cells ( Figure 5A ). We also validated the loss of both endogenous CIC and ATXN1L during IAV infection in A549 cells using specific antibodies ( Figure 5B ). Next, to establish a timeline for degradation during IAV infection, we assessed CIC and ATXN1L levels between 20 to 60 min post synchronized IAV (H1N1) infection in A549 cells (MOI=3) ( Figure 5C ). We observed decreased CIC and ATXN1L levels starting at 40 min post H1N1 infection in a proteasome dependent manner, as treatment with proteasome inhibitors (MG132, Bortezomib) blocked CIC-ATXN1L degradation ( Figure 5D ). Similarly, in primary human airway basal cells, H1N1 IAV infection resulted in rapid degradation of the CIC-ATXN1L complex at 40 min post-infection, likely prior to viral genome replication ( Figure 5E ). As CIC mRNA expression increased in A549 cells upon IFN treatment or vRNA stimulation ( Figure S1 ), we assessed CIC and ATXN1L protein levels at later times post-IAV infection and observed a gradual increase for both between 8-24 h post H1N1 infection, indicating that the CIC-ATXN1L complex may participate in restoring homeostasis by ramping down IFN responses after viral clearance ( Figure 5F ). Interestingly, exogenous IFN treatment induced transient loss of CIC and ATXN1L at 40-60 min post-treatment with levels recovering at 120 min, potentially allowing for transient de-repression of ISG promoters ( Figure 5G ),. These results demonstrate that CIC repression at IFN and ISG promoters is rapidly removed during IAV infection through proteasomal dependent degradation of the complex. Figure 5. CIC-ATXN1L repressor complex is rapidly degraded during IAV infection. Open in a new tab (A) Immunofluorescence analysis of GFP-CIC expression during IAV infection. A549 cells stably expressing GFP-CIC-S construct were either mock or IAV (H1N1) infected at MOI=3, and at 16 hpi, cells were fixed and stained with anti-CIC and anti-NP (IAV) antibodies. (B) Immunofluorescence analysis of endogenous CIC and ATXN1L levels during IAV infection. Representative fluorescence micrographs of mock and IAV infected samples are shown for Panel A-B (Scale bar, 100 μm). (C) Western blot analyses of CIC and ATXN1L levels at different times post-IAV infection. A549 cells were incubated with IAV (H1N1) at MOI=3 on ice for 60 min to allow for synchronized virus binding and placed in a 37°C incubator to initiate virus infection. At indicated times, cell lysates were prepared and analyzed for levels of CIC and ATXN1L. (D) Western blot analyses of CIC and ATXN1L levels in proteasome inhibitor treated A549 cells. (E) Western blot analyses of CIC and ATXN1L levels in primary airway basal cells during IAV infection. (F) Western blot analyses of CIC and ATXN1L levels in IAV infected A549 cells at longer durations. (G) Western blot analyses of CIC and ATXN1L levels in type I IFN treated A549 cells. β-Actin levels are shown as loading controls. CIC-ATXN1L complex is degraded during respiratory viral infection via activation of the EGFR-MAPK pathway Next, we sought to determine if CIC-ATXN1L degradation is specific to IAV infection or if other RNA viruses induce degradation. We observed decreased CIC and ATXN1L protein levels in A549 cells after infection with different IAV subtypes ( Figure 6A ). Importantly, CIC and ATXN1L levels were significantly lower in A549 cells infected with various respiratory viruses, including IAV, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV), and SeV ( Figure 6B ). Interestingly, however, CIC and ATXN1L levels remained unchanged in A549 cells infected with non-respiratory viruses, such as Zika virus, encephalomyocarditis virus (EMCV), and Ebola glycoprotein (EGP) pseudotyped vesicular stomatitis virus (EGP-VSV). Figure 6. CIC-ATXN1L complex is degraded during respiratory viral infection via activation of the EGFR-MAPK pathway. Open in a new tab (A) Western blot analyses of CIC and ATXN1L levels during infection with different IAV strains. A549 cells were infected with indicated IAV strain (MOI=3). (B) Western blot analyses of CIC and ATXN1L levels during infection with diverse RNA viruses. Infections in A549 cells and western blot analyses were performed similar to IAV infection conditions. RSV - Respiratory syncytial virus, HPIV - human parainfluenza virus type 3, EMCV - encephalomyocarditis virus, Zika virus, and EGP-VSV - Ebola glycoprotein pseudotyped vesicular stomatitis virus were infected at MOI=5. SeV - Sendai virus stock was used at 1:1000 dilution. (C) Western blot analyses of CIC and ATXN1L levels during IAV (H1N1) infection in the presence of MAPK inhibitors. (D) Western blot analyses of CIC and ATXN1L levels in A549 cells incubated with recombinant HA protein. (E) IFNB1 expression in Control A549 or CIC KO cells pre-treated with MEK inhibitor. Indicated cells were stimulated with either IFN-I, SeV, or vRNA and at 24 h, expression of IFNB1 transcripts was measured by qRT-PCR. (F) Western blot analyses of CIC and ATXN1L after treatment with recombinant EGF. (G) qRT-PCR analysis of ISG expression during viral infection performed in the presence of EGF. Monolayers of A549 cells were incubated with indicated viruses (EMCV or EGP-VSV, MOI=10) either with or without recombinant EGF (250 ng). IFNB1, MX1, IFIT1 and ISG15 mRNA levels were measured at 36 hpi. Data are represented as fold (log) expression relative to Control treated (mock) cells ± SD for Panels E and G. See also Figure S4 . Respiratory viruses such as IAV and RSV are known to activate RTK-MAPK pathways during the virus entry process 24 , 25 . Specifically, epidermal growth factor receptor (EGFR)-MAPK signaling is activated during IAV and RSV entry into host cells 26 , 27 . During Drosophila development, Cic-regulated genes are derepressed through Cic degradation via activation of the Egfr-Mapk pathway 17 , 18 , 28 . To establish if IAV infection induced degradation of CIC-ATXN1L occurs via the RTK-MAPK pathway, we performed IAV infection in A549 cells in the presence of MEK inhibitor (MEKi, CI-1040) or ERK inhibitor (ERKi, Ravoxertinib). Treatments with MEKi or ERKi completely blocked IAV infection induced degradation of the CIC-ATXN1L complex ( Figure 6C ). As prior studies indicate that IAV binding to host cell receptors trigger the EGFR-MAPK pathway, we next tested if binding of recombinant viral hemagglutinin (HA) protein is sufficient to trigger degradation of CIC-ATXN1L ( Figure 6D ) 27 . We observed decreased CIC and ATXN1L levels in A549 cells incubated with recombinant HAs from two different IAV subtypes (H1 and H5), indicating that engagement of viral HA with host cell receptors triggers degradation. Taken together, these findings demonstrate that activation of EGFR-MAPK signaling during respiratory viral entry triggers degradation of the CIC-ATXN1L complex. Removal of CIC repression is necessary for robust antiviral responses As robust induction of antiviral responses requires removal of CIC repression at IFN and ISG promoters, we assessed if inhibition of CIC-ATXN1L degradation with MEKi or ERKi would affect the magnitude of ISG induction. Pre-treatment with MEKi or ERKi significantly diminished the levels of IFNB1 and MX1 transcripts in A549 cells in response to SeV, type I IFN, and vRNA stimulations as compared to DMSO treated A549 cells ( Figure 6E , Figures S4A and S4C ). In contrast, pre-treatment with MEKi or ERKi did not affect the magnitude of ISG induction in CIC KO cells by all three stimuli as compared to Control (DMSO) treated CIC KO cells ( Figures S4B and S4D ). These results suggest that MAPK pathway induced CIC degradation is critical for robust transcriptional upregulation of antiviral responses. To test if EGFR (RTK) activation results in degradation of the CIC-ATXN1L complex, we treated A549 cells with exogenous EGF and assessed levels of CIC and ATXN1L proteins. Treatment with recombinant EGF induced rapid degradation of the CIC-ATXN1L complex, which occurred within 5 min post temperature shift ( Figure 6F ). As EMCV or EGP-VSV infection did not trigger CIC and ATXN1L degradation ( Figure 6B ), we assessed if addition of exogenous EGF during viral infection would enhance expression of IFN and ISGs. EGF treatment increased the magnitude of IFNB1 and ISG induction against EMCV and EGP-VSV infection in A549 cells as compared to control treatment ( Figure 6G ). In addition, exogenous EGF treatment increased the magnitude of ISG induction by vRNA and Poly IC stimulation ( Figure S4E ). Taken together, these findings demonstrate that timely removal of CIC repression at IFN and ISG promoters via activation of the EGFR-MAPK pathway is critical for robust initiation of antiviral responses. Conservation of IFN and ISG promoter repressor functions in murine Cic As CIC is an evolutionarily conserved gene, we next determined if murine CIC is able to regulate antiviral response pathways in a manner similar to human CIC. To this end, we utilized a tamoxifen inducible Cic KO model ( Rosa26-Cre ERT2 -Cic f/f ) and generated three different primary murine cell types lacking CIC: mouse embryonic fibroblasts (MEFs), bone marrow derived dendritic cells (BMDCs), and bone marrow derived macrophages (BMDMs) 29 . Similar to our findings in human lung cells, IAV infection in MEFs resulted in degradation of the CIC-ATXN1 complex ( Figures S5A – B ). In addition, in all three cell types, loss of murine CIC resulted in higher induction of Ifnb1 and ISG transcripts under both mock and IAV infection conditions ( Figures S5C – E ). Next, we performed RNA-Seq analysis and compared transcriptional changes in the lungs of Cic KO and Control mice under mock and IAV infection conditions ( Figures S6A – B ). We identified 5296 DEGs (log 2 fold change 0.5, p-adj value <0.1) under mock conditions ( Figure 7A , Table S8 ). Importantly, similar to our findings in CIC KO human lung cells (A549), we observed elevated expression of IFN, ISGs, and cytokines/chemokines in Cic KO murine lungs under mock conditions, including Ifnb1, Ifnl2, Ifng, Mx1-2, Ifit1-3, Ifitm3, Irfs, Stat1/2, Jak2 etc. Gene ontology assessment of DEGs in mock Cic KO versus Control murine lungs showed significant enrichment for genes (GO terms) associated with host antiviral responses under mock conditions, including positive regulation of cytokine production (GO:000119), positive regulation of defense responses (GO:0031349), innate immune response (GO:0045087), response to virus (GO:0009615) etc. ( Figures 7B – C and Table S9 ). Upon IAV infection, however, only 61 DEGs were identified between Control and Cic KO murine lungs on day 5 post-infection, as most ISG genes were upregulated following 5 days of viral infection in Control mice ( Figure 7A and Figure S6C ). These results demonstrate that, similar to human CIC, murine CIC is a comprehensive regulator of numerous innate inflammatory response genes. Figure 7. Murine CIC represses ISG promoters in vivo . Open in a new tab Rosa26-Cre ERT2 - Cic f/f mice were intraperitoneally injected with either vehicle Control or tamoxifen (4 mg; six doses) to activate CRE recombinase. Treated mice were either mock or IAV infected (H1N1 PR8 strain) at a dose of 10 PFU and at various times post-infection, lungs were harvested for measurement of viral loads and ISGs (days 2, 5 and 8), and for RNA-Seq analysis (day 5). (A) Volcano plot of RNA-Seq data comparing DEGs between uninfected (Mock) Control (n=3) and Cic KO mice (n=2). A select number of differentially expressed ISGs are indicated. (B) Gene ontology (GO) assessment of DEGs in uninfected Control versus Cic KO mice. GO assessment was performed using Metascape. Shown are the most significantly enriched GO terms. (C) Cluster heat map comparison of representative GO terms. Defense response to virus (GO:0051607; p-adj from Deseq2 <0.05) and cellular response to cytokine stimulus (GO:0071345; p-adj from Deseq2 <0.05) in Cic KO versus Control murine lungs under mock conditions are shown. (D) Comparison of body weight changes in Control and Cic KO mice. Control (n=15) or tamoxifen treated mice (n=15) were infected with IAV (H1N1) PR8 strain at a sublethal dose of 10 PFU (Circle). Mock infected Control (n=7) and Cic KO (n=3) mice are denoted as square. Body weights of mice were measured daily for 28 days and changes in body weight are shown as relative percentage of day 0 weight (mean ± SEM). (E) Viral burden in the lungs of IAV infected mice. Lungs from IAV infected mice were harvested on days 2, 5 and 8 post-infection and viral burden was measured by plaque assay (PFU/lung). Each data point represents an individual mouse (n=5-6) (mean ± SD). (F) qRT-PCR analysis of ISG expression in the lungs of IAV infected mice. Total RNA from IAV infected or Control murine lungs was isolated at indicated days and subjected to qRT-PCR analysis. Each data point represents an individual mouse (Control groups (n=3) and IAV groups (n=5-6)). Data are represented as fold (log) expression relative to mock infected Control mice (mean ± SD). (G) Histological analysis of IAV infected mouse lungs. Control or tamoxifen treated mice were infected with 10 PFU of IAV (H1N1) PR8 strain and lungs were subjected to histopathological analysis on days 3 and 6 post-infection. H&E images of mock and IAV infected lungs are also shown. Asterisks and arrows indicate regions of immune cell infiltration/inflammation. See also Figures S5 and S6 . Next, we assessed the in vivo significance of Cic in a sublethal IAV challenge murine model. Cic KO mice showed reduced weight loss and recovered from IAV infection earlier than IAV infected Control mice ( Figure 7D ). These favorable clinical outcomes of IAV infection in Cic KO mice correlated with a 5-50 fold lower viral burden in the lungs as compared to Control mice ( Figure 7E ). qRT-PCR analysis of antiviral genes in IAV infected lungs showed 5-10 fold higher expression of Ifnb1 and ISGs in Cic KO mice as compared to Control mice ( Figure 7F ). In addition, histopathological analysis of IAV infected lungs showed smaller areas of inflammation in Cic KO mice as compared to Control mice ( Figure 7G and Figure S6D ). These findings demonstrate that murine CIC is critical for regulating inflammatory responses against respiratory viral infection in vivo . Next, we assessed the in vivo importance of the MAPK pathway in CIC mediated regulation of antiviral responses by treating Control or Cic KO mice with MEK inhibitor and stimulating with Poly IC ( Figures S6E – S6F ). Pre-treatment of Control (C57BL/6J) mice with MEKi significantly decreased the magnitude of ISGs induced by Poly IC ( Figure S6E ). In contrast, treatment of Cic KO mice with MEKi did not significantly change the levels of ISGs induced by Poly IC ( Figure S6F ). These results demonstrate that MAPK pathway induced CIC degradation is critical for robust transcriptional upregulation of antiviral responses in vivo . Taken together, our studies in Cic KO mice show that murine CIC is critical for homeostatic repression of Ifn and Isg promoters, and this repression is relieved during IAV infection via degradation of the CIC repressor complex, demonstrating the conservation of CIC-mediated transcriptional repression of antiviral response pathways between humans and mice. DISCUSSION Here, we report a novel role for the DNA binding transcriptional repressor CIC in safeguarding against aberrant transcriptional activation of IFN and ISGs by host derived endogenous dsRNA ligands under normal conditions ( Figure S7 ). However, during respiratory viral infection, this safeguard is swiftly turned-off by degradation of the CIC-ATXN1L repressor complex via activation of the EGFR-MAPK cascade. This degradation primes host cells for robust activation of antiviral responses in a cell-intrinsic manner. Importantly, our studies further demonstrate that murine CIC similarly represses IFN and ISG expression under homeostasis, highlighting the evolutionary conservation of Capicua-mediated transcriptional regulation of host IFN responses. Taken together, our studies define a new paradigm in the regulation of cell intrinsic antiviral responses, mediated by the CIC-ATXN1L complex under both homeostasis and respiratory viral infection conditions. Capicua is an evolutionarily conserved gene from Drosophila to humans. The transcriptional repressor function of Cic was initially identified in the Drosophila model, where timely de-repression of Cic target genes is crucial for proper development 17 , 18 , 30 . This de-repression is mediated by rapid degradation of the Cic repressor complex via activation of the Egfr-Mapk pathway. In human lung cells, we observed rapid degradation of the CIC repressor complex during respiratory viral infection, which also occurred via activation of the EGFR-MAPK pathway. Like Drosophila, CIC in humans has been implicated in regulating promoters through transcriptional repression in the context of different types of cancers, neurodegeneration, and other diseases 21 , 31 – 33 . Our study reveals a novel role for CIC in the negative regulation of innate antiviral response genes in mammals. In Drosophila, Cic mediates transcriptional repression by binding to CBS motifs (TSAATGRR) near target gene loci 17 – 21 . Remarkably, our studies show that mammalian CIC binds to similar CBS motif(s) located near the gene loci of numerous IFNs and ISGs to repress transcription. Although antiviral defense through IFN and ISGs is specific to vertebrates, it is interesting to note that the transcriptional repressor function of CIC has been co-opted to regulate IFN based antiviral defense pathways in mammalian cells. Despite the repression of distinct classes of genes by CIC homologs in Drosophila versus humans, de-repression of CIC target genes is achieved through activation of the EGFR-MAPK pathway in both species. Aside from the induction of IFN and ISGs, innate immune cell activation in vertebrates leads to the secretion of cytokines and chemokines, which promotes recruitment of innate and adaptive immune cells to sites of infection. As we also observed increased expression of cytokines and chemokines in cells lacking CIC, our studies further suggest that CIC plays a broader role in the regulation of immune homeostasis. Prior to this study, the prevailing model for activation of host antiviral responses indicated that IFN and ISG promoters remained in a default off-state under homeostasis until bound by IRF/STAT transcription factors activated via PRRs or IFNR signaling. Except for FOXO3, homeostatic negative regulation was thought to occur via posttranslational modification of RLR or IFNR pathway components; importantly, negative regulation through a transcriptional repressor binding to specific DNA elements has not been previously reported 14 . Our findings support a new paradigm for the regulation of inflammatory response genes, whereby transcription from IFN and ISG promoters is negatively regulated by the CIC-ATXN1/L transcriptional repressor complex via binding to CBS motif(s). Recent studies indicate that the dsRNA editing enzyme ADAR1 edits endogenous dsRNAs to prevent MDA-5 activation 10 – 13 . In the absence of CIC, basal transcription of IFNB1 and ISGs increased in CIC KO A549 cells due to tonic activation of the RLR pathway by endogenous dsRNA ligands ( Figure 2D ). Thus, our studies reveal that the CIC-ATXN1L transcriptional repressor complex provides an additional layer of protection against sterile inflammation by repressing inflammatory response pathway genes. As such, transcriptional regulation of inflammatory genes via CIC-ATXN1/L may also have important implications for the development or progression of autoimmune disease. Indeed, the ATXN1 gene loci has been linked to an increased risk of developing multiple sclerosis (MS) 34 . Mice deficient in Atxn1 showed increased susceptibility to autoimmune demyelination in a pre-clinical murine model of MS, primarily driven by induction of proinflammatory genes in the B cell compartment. Viruses usurp several host pathways to create an intracellular environment conducive for viral replication. Several RNA (IAV, RSV, coronavirus) and DNA viruses activate the MAPK signaling cascade to promote various stages of the viral life-cycle, including viral internalization, gene transcription, replication and translation, viral protein/genome trafficking, etc 24 – 27 , 35 – 37 . Our studies demonstrate that CIC-ATXN1L degradation occurs during the initial stages of viral entry between 40-60 min post infection ( Figures 5C and 5F ). Specifically, the binding of viral HA to host cell receptors is sufficient to trigger CIC-ATXN1L degradation ( Figure 6D ). Interestingly, exogenous type I IFN treatment induced transient degradation of CIC and ATXN1L albeit less pronounced as compared to respiratory viral infection, likely allowing for transient de-repression of ISG promoters. While CIC-ATXN1L repression is necessary for tight regulation of IFN and ISGs under homeostasis, our studies show that host cells utilize MAPK signaling to promptly inactivate CIC-ATXN1L repression at these promoters early during respiratory viral infection. As such, host shutdown of CIC-ATXN1L repression enables a strong antiviral response against invading viruses that activate the MAPK pathway to promote viral life-cycle. It should be noted that the extent of CIC-ATXN1L degradation during viral entry varies amongst respiratory viruses, likely due to differences in interactions with components of the MAPK pathway ( Figure 6B ). In contrast, non-respiratory viruses that do not trigger CIC-ATXN1L degradation can establish infections more efficiently, due to delayed and lowered expression of IFN and ISGs under CIC-ATXN1L repression. Eventually, accumulation of viral PAMPs during replication will trigger IFN responses via PRRs. Prior studies indicate that signaling through TLRs and RLRs can activate MAPK pathways, augmenting PRR-induced antiviral responses 38 , 39 . Future studies are required to establish whether delayed degradation of CIC by non-respiratory viruses is important for host-pathogen responses. Sustained activation of EGFR and MAPK pathways has been shown to exacerbate disease severity caused by highly virulent respiratory viruses, such as avian H5N1 and SARS-CoV via hyperinduction of proinflammatory cytokines (‘cytokine storm’) 37 , 40 – 42 . Interestingly, treatments with MEK or ERK inhibitors have been shown to limit inflammatory responses in such infections, raising the possibility that MEK/ERK inhibitors may prevent CIC-ATXN1L degradation. In agreement, in our studies, treatment of mice with MEKi significantly decreased expression of Poly IC induced inflammatory genes in a Cic dependent manner, providing a scientific rationale for the use of MAPK inhibitors to dampen the hyperinflammation associated with respiratory viral infection. As hyperactivation of the MAPK pathway can promote tumorigenesis and metastasis, MEK and ERK inhibitors are routinely used to treat cancers 43 . Interestingly, loss-of-function mutations of CIC are a major mechanism by which cancer cells gain resistance or reduce sensitivity to MAPK inhibitors 33 , 44 . Moreover, genetic alterations in CIC promote tumor progression and metastasis, primarily through de-repression of the PEA3 ( ETV1/4/5 ) family of ETS transcription factors 31 . In addition, loss of CIC in mice results in the induction of T-cell lymphoma as well as defects in B-cell lineage development 45 , 46 . Based on our findings, we speculate that the progression of some CIC-associated cancers may be promoted by chronic low-grade inflammation. In summary, our study establishes a new paradigm for the regulation of IFN and ISGs by the CIC-ATXN1L DNA binding repressor complex, with implications that extend beyond viral immunity. These include relevance to cancer, autoimmune diseases, and developmental disabilities, as these conditions are often associated with dysregulation of immune homeostasis. RESOURCE AVILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by Balaji Manicassamy ( [email protected] ) Materials availability Plasmids and cell lines described in this study, together with documenting information, will be made available upon request and following execution of a UBMTA between the University of Iowa (as appropriate, depending on the resource being requested) and the recipient institution. Data Availability RNA-seq data comparing control and CIC KO A549 cells are available at GEO ( GSE271898 ). ATAC-Seq analysis in Control and CIC KO A549 cells are available under accession number GSE271897 . Mouse lung RNA-Seq data are available at GEO ( GSE271899 ). Summary data from RNA-Seq and ATAC-Seq are included as Extended Data Tables S1-S9. There are no restrictions on data availability. STAR METHODS EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Cell lines Human lung cell line A549 (ATCC CCL-185), Lenti-X 293T cells (HEK 293T, Clontech, 632180), Vero cells (ATCC CCL-81), L929 cells (ATCC CCL-1), and HEp-2 cells were maintained in DMEM (Gibco) supplemented with 10% FBS (R&D Systems) and 1% Penicillin/streptomycin (Corning). Madin-Darby canine kidney (MDCK, ATCC CCL-34) cells were cultured in MEM (Gibco) supplemented with 10% FBS and 1% P/S. Primary cells Primary airway basal cells were obtained from the Cell and Tissue Core at the University of Iowa and cultured in PneumaCult-Ex Plus Basal Media (STEMCELL Technologies) as previously described 47 . Viruses Influenza A virus strains described here were obtained from various sources: recombinant A/Puerto Rico/8/1934 (H1N1, Mount Sinai), A/Hong Kong/1/1968 (H3N2), and a low pathogenic version of recombinant A/Vietnam/1203/2004 (H5N1-low pathogenic) were kindly provided by Dr. Adolfo Garcia-Sastré (Icahn School of Medicine). A low pathogenic version of A/Netherlands/213/2003 (H7N7-low pathogenic) was provided by Dr. Ron Fouchier (Erasmus Medical Center). All IAV strains were grown in 10 day old embryonated specific pathogen-free (SPF) eggs (Charles River), aliquoted and stored at −80°C before titeration by plaque assay on MDCK cells using 0.8% Avicel RC-581 (a kind gift from FMC BioPolymer, Philadelphia, PA). Viral plaques were quantified 2-3 days post infection by crystal violet staining 48 , 49 . Respiratory syncytial Virus A2 (RSV-A2) (Dr. Steve Varga) and HPIV-3 (BEI Resources) were grown and titered in HEp-2 cells using 0.8% Avicel RC-581 media. Vesicular stomatitis virus expressing GFP (VSV) was kindly provided by Dr. Glenn Barber at the University of Miami, FL. Recombinant Ebola GP-VSV was provided by Dr. Kartik Chandran (Albert Einstein Medical School). EMCV (BEI Resources), Zika virus MR766 (BEI Resources), and VSV strains were grown and titered in Vero cells using 1% methylcellulose (Sigma). Mouse Strains B6(SJL)- Cic tm1c(KOMP)Wtsi /HzoJ ( Cic f/f , stock #030555) mice were kindly made available by Dr. Huda Zoghbi through Jackson Laboratory and were maintained at the University of Iowa specific pathogen free animal facility 29 . R26-CreER(Gt(ROSA)26Sor tm1(cre/ERT2)Tyj ) strain ( R26-Cre ERT2 ) backcrossed into C57BL6 background was kindly provided by Dr. Carl Walkley (University of Melbourne). R26-Cre ERT2 -Cic f/f strain was generated by crossing Cic f/f mice with R26-Cre ERT2 strain. METHOD DETAILS Lentivirus production and transduction Lentivirus particles were produced in HEK 293T cells by co-transfecting expression vector (sgRNA, cDNA) with packaging constructs (pPX2) and pVSV-G (Addgene #138479) at a ratio of 2:1.5:0.5. Briefly, a total of 4 μg of plasmid mixture was transfected into 2x10 6 cells using PEI transfection reagent (Polysciences; DNA:PEI ratio 1:5) in DMEM/10%FBS media without Pen/Strep. At 5-6 h post transfection, media containing transfection mixture was replaced with fresh media (DMEM/10%FBS/ P/S, 1% BSA (MP Biomedicals). At 48 h post transfection, lentivirus containing supernatants were clarified by centrifugation at 3000 RPM for 10 min and filtered through a 0.45 μm PES filter. A549 cells seeded at a density of 5x10 5 cells per well (6-well) a day prior were transduced with 1-2 mL of lentivirus supernatant supplemented with 8 μg/mL of polybrene (Millipore) via spinoculation (2000RPM, at 25C). At 8-16 h post transduction, cells were placed in fresh DMEM/10%FBS/1%PS media and at 48 h, cells were placed in antibiotic selection media (puromycin or blasticidin). Generation of CRISPR KO Cells CIC KO A549 cells were generated using two sgRNAs cloned into lentiCRISPR v2 (#52961, Addgene) and lentiCRISPR v2-GFP as previously described 16 . ATXN1L KO A549 cells were generated using pXPR-BRD003 sgATXN1L-1 (Addgene, #74970) single vector system as previously described with Puromycin selection 50 , 51 . On day 2 post transduction, A549 cells were subjected to drug selection for ~14 days at 2 μg/mL of puromycin (Invitrogen). Clonal KO cells were isolated by single cell sorting in a 96-well plate and successful KO clones were identified by western blot analysis. CIC KO A549 cells were complemented with a lentivirus vector expressing V5-tagged CIC-S (pLX-V5-CIC-S) or CIC-L (pLX-V5-CIC-L) under blasticidin resistance marker (10 μg/mL). CIC complemented cells were single cell sorted in 96-well plates and clones were identified by western blot analysis with an anti-V5 antibody (Bio-Rad). Measurement of IAV replication A549 Control, KOs, or complemented cells were seeded at a density of 8x10 5 cells per well in a 6-well plate. The next day, adhered cells were washed twice with PBS and placed in a serum free media (DMEM/0.2% BSA). After serum starvation for 24 h, cells were washed twice (PBS) and infected with H5N1 (VN04 strain) at MOI=0.001 in infection media (DMEM/ 0.2% BSA supplemented with 1 μg/mL TPCK-treated trypsin (T1426, Sigma)). After 1 h, viral inoculum was removed, washed twice with PBS and cells were placed in fresh infection media. An aliquot of supernatants were collected at 0, 24, 48, 72 and 96 hpi and viral titers were determined by standard plaque assay on MDCK cells 52 . siRNA transfection in primary human airway cells Dharmacon ON-TARGETplus siRNA SMARTpool against human CIC (Gene ID: 23152, L-015185-01), human ATXN1L (Gene ID: 342371, L-022594-02), and Non-targeting pool (D-001810-10) were purchased from Horizontal Discovery. Primary human airway cells were transfected with either siRNA-CIC or siRNA-ATXN1L individually or in combination at a concentration of 10 nM using RNAiMax transfection reagent (Thermo Fisher, #13778150) and at 48 h post-transfection, cells were placed in DMEM/0.2% BSA media. All virus infections were performed in DMEM/0.2% BSA media and at 48 hpi, total cellular RNA was extracted and used for qPCR analysis. Western blotting Whole cell extracts were prepared using RIPA lysis buffer containing protease and phosphatase inhibitors (Roche). Lysates were cleared by centrifugation and protein concentrations were determined using Bradford assay (Bio-Rad). Equal amounts of total protein (60 μg) were loaded onto SDS gels and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked using 5% (w/v) bovine serum albumin (Sigma) in 1XTBST (50 mM Tris buffered saline containing 0.1% Tween 20, pH 7.4) for 1 h at room temperature and then incubated with primary antibodies (CIC, ATXN1L, beta-Actin, STAT1 or MAVS) overnight at 4°C. Subsequently, membranes were extensively washed 3 times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000 dilution) in blocking buffer. Immunoreactive bands were visualized with SuperSignal West Pico PLUS Chemiluminescent substrate (Thermo Scientific, #34580). CIC-ATXN1L degradation assay Indicated cell types were seeded and serum starved as described above. Prior to infection, cells were washed and pre-chilled on ice for 15 min in 750 μl infection media for synchronized infection. Subsequently, cells were infected with different viruses at the indicated MOI and incubated on ice for an additional hour to allow for virus biding. After 1 h, cells were placed at 37°C for indicated times and cell lysates were prepared in RIPA buffer for western blot analysis. In experiments using recombinant HA, cells were incubated with indicated concentration of recombinant HA (H1 or H5) instead of infectious virus. For proteasomal inhibition experiments, A549 cells were seeded and serum starved as mentioned above. Cells were pre-treated with either DMSO or MG132 (10 μM) or Bortezomib (10 μM) for 4 h followed by H1N1 infection in the presence of inhibitors as indicated above. For EGF stimulation experiments, A549 cells were seeded and subjected to serum starvation as above. Prior to addition of EGF (Peprotech), cells were placed on ice for 15 min and incubated with 250 ng/mL EGF on ice for an additional 1 h. At indicated times post-temperature shift, cell lysates were prepared for western blot analysis. For MAPK inhibitor studies, monolayers of A549 cells were pre-treated with either DMSO (Control) or MEK inhibitor (MEKi, CI-1040, 10 μM) or ERK inhibitor (ERKi, Ravoxertinib, 10 μM) for 2 h and infected with IAV in the presence of MAPK inhibitors as described above For type IFN-I experiments, monolayers of A549 cells were incubated with IFN-I (1000 U/mL) on ice for 60 min to allow for synchronized binding and subsequently, placed in a 37°C incubator and at indicated times, cell lysates were collected for western blot analysis. Quantitative RT-PCR analysis of ISG expression Total RNA was extracted from indicated cell types using PureLink RNA Mini kit (Invitrogen, #12183018) according to the manufacturer’s instructions. Purified RNA (1μg) was reverse transcribed with oligo(dT) 12-18mer using Superscript IV Reverse Transcriptase (Cat #18090010). qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, #4367659) on the QuantStudio 3 Real-Time PCR system 16 . Delta delta cycle thresholds (2 −ΔΔ C t ) were calculated using tubulin as the endogenous housekeeping gene and data are represented as fold expression relative to uninfected (mock) vector Control cells ± SD or H1N1 infected vector Control cells ± SD. Primer sequences are listed in Supplementary Data Table 10 . To analyze ISG expression upon IAV infection, Control and KO A549 cells were seeded at a density of 1.2x10 6 cells per well in a 6-well plate. After 24 h serum starvation, cells were mock treated or infected with H1N1 at MOI=1 in DMEM/0.2% BSA/1% P/S at 37°C. At the indicated time points, total cellular RNA was extracted for qRT-PCR analysis. IAV viral RNA used for stimulation was isolated from egg grown stocks of H1N1 (PR8 strain) using QIAmp Viral RNA Mini Kit following manufacturer’s instructions (Qiagen). Control and KO A549 cells were transfected with vRNA using PEI reagent (1:5 ratio) prepared in OptiMem (OMEM). After 48 h post-transfection, total cellular RNA was extracted for qRT-PCR analysis. For MEKi and ERKi studies, Control and KO cells were seeded and serum starved as above. At 2 h prior to IAV infection, cells were pretreated with MEK inhibitor (MEKi, CI-1040) or ERK inhibitor (ERKi, Ravoxertinib) at 10 μM concentration. For EGF stimulation of ISG induction during EMCV or Ebola-VSV infection, Control A549 cells were seeded at a density of 1.2x10 6 cells per well of a 6-well plate and serum starved as above. Pre-chilled cells were infected with EMCV or Ebola-VSV (MOI=10) in media containing 250 ng/mL of EGF (PeproTech, AF10015). At 24 hpi, total cellular RNA was extracted for qPCR analysis of ISG expression. For Poly IC and SeV stimulation, Control or KO A549 cells were seeded and serum starved as above. Cells were transfected with 1ug of poly IC or infected with a SeV stock containing higher amounts of defective interfering particles at 1:1000 dilution. Confocal microscopy Indicated cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. To block non-specific antibody binding, permeabilized cells were placed in blocking buffer (PBS, 1% BSA, 0.5% fish gelatin) for 1 h at room temperature. Cells were then incubated with relevant primary antibodies in blocking buffer for 3 h, followed by incubation with appropriate fluorophore-conjugated secondary antibodies (1:2000 dilution) in blocking buffer for 1 h at room temperature. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI, Gibco), and cells were imaged on a Leica confocal laser microscope. Images were processed and analyzed using Fiji software. ISG promoter reporter assays HEK 293T cells seeded in a 24-well plate (2x10 5 cells/well) were transfected with a plasmid mixture containing ISG promoter reporter (100 ng; IFNβ-6x-CBS, IFNβ-6x-CBS mutant, MXA or IFIT1 or TRIM22 promoter), SV40-Renilla (50 ng), and with or without RIG-I-2CARD (RIG-I (2C), 50 ng, Dr. Adolfo Garcia-Sastre) along with varying concentrations of CIC-S (MRC, University of Dundee) and ATXN1 (Addgene, #48189) expression plasmids. For controls, a GFP plasmid was used as a substitute for RIG-I-(2C), CIC-S, and ATXN1 plasmids. Transfections were performed with PEI reagent with DNA:PEI at a ratio of 1:5. For SeV stimulation, SeV stock containing a higher amount of defective interfering particles was added to media at a 1:1000 dilution. After 6 h, cells were placed in fresh DMEM/0.2% BSA media. At 48 h post-transfection or SeV infection, cell lysates were prepared in 1x Passive Lysis Buffer (Promega) and luciferase expression levels were measured with the Dual Luciferase Reporter Assay Kit (Promega, cat# E1960) using the GloMax 20/20 Luminometer instrument (Promega). Data are represented as mean percent of normalized luciferase activity relative to Control in triplicates ± SD. RNA-Seq analysis For RNA-Seq analysis of A549 cells, Control and CIC KO A459 cells were infected with PR8 at MOI=5 for 16h. RNA extraction was performed using TRIzol extraction method. NGS library was prepared using the TruSeq RNA Library Prep Kit v2 (Illumina). RNA sequencing (paired end reads, 150-bp) was performed using Illumina NextSeq500 and quality control of the resulting fastq files was assessed using the FastQC program (version 0.11.5). A nextflow pipeline (version 20.01.0) was used to process the fastq files and gene expression was quantitated by alignment of reads with the STAR aligner (version 2.5.3a) to the human (build GRCh37) genome. Raw counts were then imported into R (version 3.5.2) and were normalized and transformed using a variance stabilizing transformation (vst). Principal components analysis (PCA) was performed to visualize sample clusters and to identify outlier samples. For statistical analysis of the data, the DESeq2 package (version 1.22.2) was used. In brief, a model incorporating all the experimental factors was created and Wald tests were used to compute statistical metrics. A gene was considered to have a statistically significant change in expression if the False Discovery Rate < 1%. Results from statistical analyses were visualized using heatmaps (created with pheatmap, version 1.0.12) and volcano plots (created with ggplot2, version 3.4.4). For RNA-Seq analysis of murine lungs, RNA from IAV infected murine lungs was isolated using TRIzol reagent. RNA sequencing (paired end reads, 100-bp) was performed using Illumina NovaSeq6000 and quality control of the resulting fastq files was assessed using the FastQC program (version 0.11.5). A nextflow pipeline (version 20.01.0) was used to process the fastq files and gene expression was quantitated by alignment of reads with the STAR aligner (version 2.5.3a) to the mouse (build mm10) genome. Data was analyzed and visualized as described above. Preparation of ATAC-Seq libraries and analysis ATAC-Seq was performed using 90,000 cells/sample. Briefly, IAV infected or uninfected CIC KO and Control A549 cells were harvested and pelleted at 500g for 10 min. Cells were washed in 1x DPBS, counted and processed using an ATAC-Seq Kit (Active Motif) following the manufacturer’s protocol. After purification of tagmented DNA, quantitative PCR on the tagmented DNA product determined that an average of 7 cycles were required. The library was purified, and size selected two times with Agencourt AMPure beads to remove >2,000-bp fragments and excess primers. Samples underwent quality control and quantification on an Agilent BioAnalyzer by the Iowa Genomics Core at the University of Iowa before pooling and sequencing on a NovaSeq6000. Sequencing adapters were removed from paired-end reads using Trimmomatic version 0.39 53 . Trimmed reads were aligned to human reference genome GRCh37/hg19 using Bowtie 2 version 2.4.1 with options-very-sensitive-X 2000. Following genome alignment, Picard was used to mark PCR duplicates, and samtools version 1.3.1 was used to remove PCR duplicates, discordant pairs, and alignments to the mitochondrial genome. Peak calling was performed using MACS2 version 2.2.7.1, and differential accessibility analyses of MACS2 peaks were performed using the R package DiffBind version 2.10.0. Differentially accessible peaks were annotated to genome regions of interest using the R package ChIPseeker version 1.18.0, and sequence motif enrichment analyses were performed using HOMER version 4.11.1. Gene ontology analysis was performed using Enrichr. CBS motif analysis was performed using the CBS sequence TSAATGRR. Animal Experiments All animal studies were performed in accordance with the principles described by the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of laboratory animals in biomedical research. The protocols for performing mouse studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa (Protocol number 1062127). Generation of mouse embryonic fibroblasts Mouse embryonic fibroblasts (MEFs) were obtained from 13.5 days post-coitum Cic f/f or R26-Cre ERT2 Cic f/f mouse embryos by standard protocols. Briefly, after removing the head and red tissues, the remaining tissues were minced and incubated with 0.25% trypsin for 45 min at 37°C. Trypsin was neutralized by adding DMEM/10% FBS/1% Glutamine to the cell suspension and cells were further disaggregated. Cell suspensions were left untouched for 5 min to allow for larger tissue fragments to settle down at the bottom of the tube and supernatants were placed in a 150 mm dish with 25 mL media. After 24 h, MEFs were placed in fresh media. Deletion of Cic was achieved by infecting MEFs with an adenovirus (MOI=100) expressing GFP (Ad-GFP) or Cre recombinase (Ad-Cre, Viral Vector Core, University of Iowa), and cells were used for indicated experiments at 4 days post Ad infection. Alternatively, MEFs isolated from R26-Cre ERT2 -Cic f/f mice were treated with 4-hydroxy tamoxifen (1μM; Sigma) for 72 h to induce Cic deletion. IAV infection, qRT-PCR analysis, and CIC-ATXN1L degradation in MEFs were performed as described for A549 cells. Bone marrow derived cells Bone marrow derived dendritic cells (BMDCs) and macrophages (BMDMs) were cultured as described previously 48 , 54 . Bone marrow cells were harvested from the femurs of R26-Cre ERT2 -Cic f/f mice, passed through a 70 μm filter to remove the fibrous tissue, and red blood cells (RBC) were lysed using red cell lysis solution (ACK lysis buffer, BioWhittaker, 10-548E). BMDCs were generated by culturing BM cells in RPMI-1640 media with 10% heat inactivated FBS, Pen/Strep in the presence of GM-CSF (20 ng/mL; PeproTech, #315-03) and IL-4 (20 ng/mL; PeproTech, #214-14). On day 2, half of the culture medium was replaced with fresh media to remove the floating granulocytes, and subsequently media was changed every 48 h. Adherent BMDCs were cultured for a total of 8-10 days. For isolating BMDMs, after RBC lysis, BM cells were cultured for 6 days in RPMI-1640 media supplemented with 10% heat inactivated FBS and 10% L929 cell conditioned media (M-CSF). To generate Cic KO cells, Rosa26-Cre ERT2 -Cic f/f mice derived BMDCs and BMDMs were treated with 4-hydroxy tamoxifen (1 μM) for 72 h. For IAV infection and qRT-PCR analysis, adherent BMDCs and BMDMs were infected with IAV H1N1 (PR8 strain) at MOI=1 as described for human cell lines. Mouse Infection Six to eight week old (both sexes) Cic f /f (Control) and Rosa26-Cre ERT2 -Cic f/f mice were used for IAV infection. To induce Cic deletion, mice were administered with tamoxifen (4mg) dissolved in corn oil (Sigma) 6 times via intraperitoneal (IP) injections. On the last day of tamoxifen injection, mice were anesthetized with ketamine-xylazine (dosage - 87.5 mg/kg of ketamine/12.5 mg/kg of xylazine) IP injection and intranasally instilled with 10 PFU of H1N1 (PR8 strain) diluted in 40 μl of PBS. IAV infected mice were monitored daily for body weight changes for 28 days and any mice showing more than 25% body weight loss were considered to have reached the experimental end point and were humanely euthanized. At indicated days, lungs from infected mice were harvested for assessment of viral titers, ISG expression, and RNA-Seq analysis (Day 5). For viral titer determination, whole lungs were homogenized in 1 mL DMEM/0.2% BSA using a bead homogenizer and viral titers in clarified lung homogenates were determined by plaque assay in MDCK cells. For ISG expression analysis, lungs from mock and IAV infected mice were homogenized in 1 mL of TRIzol (Invitrogen) using a bead homogenizer and total RNA was extracted according to the manufacturer’s instructions. After cDNA synthesis, real time PCR was performed as described for human cells. Changes in ISG expression were determined using the ΔΔCt method and values are shown as fold (log) change relative to mock samples, after normalization to 18S RNA gene expression. For histological analysis, whole lungs were fixed with 10% neutral formalin solution for 24 h (Pathology core, University of Iowa). Fixed lung tissues were dehydrated through a series of alcohol and xylene baths, paraffin-embedded, sectioned (~4 μm) and stained with hematoxylin and eosin (HE) stain. Tissues were examined by a boarded pathologist using a post-examination method of masking 55 . Scores were assigned to each lung for parameters of severe disease including edema and consolidation based on distribution in the lung. Scores ranged from 0, none; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, >75% of lung fields affected. For in vivo MEK inhibitor studies, C57BL6/J mice were pre-treated with 2 doses of MEKi (CI-1040, 150mg/kg) via IP injection at 24 h and 8 h prior to Poly IC administration. Subsequently, mice were anesthetized and intranasally instilled with 40 μg of Poly IC diluted in PBS. At 24 h post Poly IC administration, total RNA from Mock or Poly IC treated murine lungs was isolated and subjected to qRT-PCR analysis as described for in vivo IAV infection studies. For MEK inhibition studies in Rosa26-Cre ERT2 -Cic f/f strain, adult mice were first treated with tamoxifen to induce Cic deletion as described above prior to MEKi studies. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical significance was determined by one-way analysis of variance (ANOVA) using GraphPad Prism 10 software. A value of P < 0.05 was considered to indicate statistical significance (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001). Non-significant values are denoted as ns. The details of the statistical analyses were depicted in the figure legends. Supplementary Material 1 NIHMS2062688-supplement-1.pdf (6.1MB, pdf) 2 Table S1: DEGs identified in RNA-Seq Analysis of CIC KO A549 cells, related to Figure 1 and Figure S3 . NIHMS2062688-supplement-2.xlsx (407.8KB, xlsx) 3 Table S2: Gene ontology analysis of DEGs identified in RNA-Seq Analysis of CIC KO A549 cells, related to Figure 1 and Figure S3 . NIHMS2062688-supplement-3.xlsx (2MB, xlsx) 4 Table S3: MSigDB transcription factor analysis of DEGs identified in RNA-Seq analysis of CIC KO A549 cells, related to Figure 1 and Figure S3 . NIHMS2062688-supplement-4.xlsx (15.9KB, xlsx) 5 Table S4: DE peaks identified in ATAC-Seq analysis of CIC KO A549 cells, related to Figure 3 . NIHMS2062688-supplement-5.xlsx (1.4MB, xlsx) 6 Table S5: CBS motif containing DE identified in ATAC-Seq analysis of CIC KO A549 cells, related to Figure 3 . NIHMS2062688-supplement-6.xlsx (511.1KB, xlsx) 7 Table S6: Genes identified in the integrated analysis of RNA-Seq, ATAC-Seq and CBS motif mining, related to Figure 3 . NIHMS2062688-supplement-7.xlsx (403.4KB, xlsx) 8 Table S7: Homer transcription factor analysis of 324 common genes identified in integrated analysis of mock samples, related to Figure 3 . NIHMS2062688-supplement-8.xlsx (121KB, xlsx) 9 Table S8: DEGs identified in RNA-Seq Analysis of Cic KO mouse lungs, related to Figure 7 and Figure S6 . NIHMS2062688-supplement-9.xlsx (506.1KB, xlsx) 10 Table S9: Gene ontology analysis of DEGs identified in RNA-Seq analysis of Cic KO mouse lungs, related to Figure 7 and Figure S6 . NIHMS2062688-supplement-10.xlsx (584.6KB, xlsx) KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-CIC Sigma-Aldrich Cat# HPA044341 RRID: AB_2678901 Rabbit polyclonal anti-ATXN1L Thermo Fisher Scientific Cat# PA549084 RRID: AB_2634540 Rabbit polyclonal anti-ATXN1 Cell Signaling Technology Cat# 2177 RRID: AB_2061047 Mouse monoclonal anti-MAVS Santacruz Biotechnology Cat# Sc-166583 RRID: AB_2012300 Mouse monoclonal anti-STAT1 Santacruz Biotechnology Cat# Sc-417 RRID: AB_675902 Mouse monoclonal anti-beta actin Santacruz Biotechnology Cat# Sc-47778 RRID: AB_626632 Mouse monoclonal anti-KU86 Santacruz Biotechnology Cat# Sc-5280 RRID: AB_672929 Mouse monoclonal anti-V5 Thermo Fisher Scientific Cat# MCA1360GA RRID: AB_567249 Mouse monoclonal anti-Ubiquitin (P4D1) Cell Signaling Technology Cat# 3936 RRID: AB_331292 Rabbit polyclonal anti-PR8 (NP) Dr. Adolfo Garcia-Sastré (Icahn School of Medicine) N/A Mouse monoclonal anti-IRF2 BioLegend Cat# 13B2A38 RRID: AB_2562588 Mouse monoclonal anti-IRF9 BioLegend Cat # 5A3A39 RRID: AB_2563575 Bacterial and virus strains Recombinant A/Puerto Rico/8/1934 (H1N1, Mount Sinai) Dr. Adolfo Garcia-Sastré (Icahn School of Medicine) N/A A/Hong Kong/1/1968 (H3N2) Dr. Georg Kochs (University of Freiburg) N/A A/Hong Kong/1/1968 (H3N2), NS1 (R38A) Mutant Dr. Georg Kochs (University of Freiburg) N/A Recombinant A/Vietnam/1203/2004 (H5N1-low pathogenic) Dr. Adolfo Garcia-Sastré (Icahn School of Medicine) N/A A/Netherlands/213/2003 (H7N7-low pathogenic) Dr. Ron Fouchier (Erasmus Medical Center) N/A Respiratory syncytial Virus A2 (RSV-A2) Dr. Steven Varga (St.Jude Children’s Research Hospital, Memphis) N/A EMCV BEI resources NR-19846 Zika virus MR766 BEI resources NR-50065 VSV-GFP Dr. Glenn Barber (University of Miami, FL) N/A Recombinant Ebola GP-VSV Dr. Kartik Chandran (Albert Einstein Medical School) N/A Chemicals, peptides, and recombinant proteins ERK inhibitor (Ravoxertinib) Selleckchem Cat# S-7554 MEK inhibitor (CI-1040) Selleckchem Cat# S-1020 MG132 Selleckchem Cat# S-2619 Bortezomib Selleckchem Cat# PS-341 EGF PeproTech Cat# AF10015 GM-CSF Peprotech Cat# 315-030-100UG IL-4 Peprotech Cat# 214-14-100UG Universal Type I IFN PBL assay science Cat# 11200-2 Protease inhibitor cocktail Roche Cat# 11836170001 Tamoxifen Sigma Cat# T5648 4-Hydroxytamoxifen Sigma Cat# SML-1666 Cremophor EL MCE Cat# HY-Y1890 Prolong antifade mountant Invitrogen Cat# 92148 Chemiluminescence substrate Thermo Scientific Cat# 34580 DMEM Gibco Cat# 11965-092 RPMI-1640 Gibco Cat# 11875-093 Opti-MEM Gibco Cat# 31985-070 0.05% Trypsin Corning Cat# 25-051-CI 0.25% Trypsin Corning Cat# 25-053-CI Penn/Strep Corning Cat# 30-002-CI Purelink DNase Invitrogen Cat# 12185010 Superscript IV Invitrogen Cat# 18090010 RNaseOUT Invitrogen Cat# 10777019 SYBR Power Green Applied Biosystem Cat# 4367659 Purelink RNA Minikit Invitrogen Cat# 12183018A Bovine Serum Albumin Sigma Cat# A9647 Bradford reagent Bio-rad Cat# 5000006 Recombinant HA (H1N1 A/Puerto Rico/08/1934) - Baculovirus BEI Resources Cat# NR-19240 Recombinant HA (H5N1 A/Vietnam/1203/04) - Baculovirus BEI Resources Cat# NR-10510 Critical commercial assays Dual luciferase Reporter Assay kit Promega Cat# E1960 Deposited data ATAC-Seq (A549 cells) This paper GSE271897 RNA-Seq (A549 cells) This paper GSE271898 RNA-Seq (primary murine lung cells) This paper GSE271899 Experimental models: Cell lines A549: Human lung epithelial cell line ATCC CCL-185 A549 CIC KO Han et al N/A A549 ATXN1L KO This paper N/A Primary Cic KO MEFs This paper N/A Vero: Monkey kidney epithelial cell line ATCC CCL-81 L929: Mouse fibroblast cell line ATCC CCL-1 MDCK: Dog kidney epithelial cell line ATCC CCL-34 Lenti-X 293T: Human embryonic kidney cell line Clontech 632180 Hep-2: Dr. Steven Varga (St.Jude Children’s Research Hospital, Memphis) Primary human airway epithelial cells Dr. Paul McCray (University of Iowa) N/A Experimental models: Organisms/strains Mouse: B6(SJL)- Cic tm1c(KOMP)Wtsi /HzoJ ( Cic f/f, Dr. Huda Zoghbi (Jackson Laboratory) Stock #030555) Mouse: R26-CreER(Gt(ROSA)26Sor tm1(cre/ERT2)Tyj ) ( R26-Cre ERT2 ) Dr. Carl Walkley (University of Melbourne). N/A Oligonucleotides siRNA SMARTpool human CIC Horizon Discovery L-015185-01 siRNA SMARTpool human ATXN1L Horizon Discovery L-022594-02 siRNA Non-targeting pool Horizon Discovery D-001810-10 Primer sequences, see Table S10 This paper N/A Recombinant DNA pXPG vector Addgene Cat# 71248 RIG-I-2CARD Dr. Adolfo Garcia-Sastré (Icahn School of Medicine) N/A CIC-S MRC, University of Dundee ATXN1 Addgene, Cat# 48189 LentiCRISPR v2 Addgene Cat# 52961 LentiCRISPR v2-GFP Addgene Cat# 82416 pXPR-BRD003 sgATXN1L-1 Addgene Cat# 74970 pLX-304 Addgene Cat# 25890 Software and algorithms GraphPad Prism 10 GraphPad Software, Boston Fiji Schindelin et al. https://imagej.net/software/fiji/ R (version 4.0.0) The R Project for Statistical Computing https://www.r-project.org Bioconductor/diffBind package (version 3.0.15) https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf https://bioconductor.org/packages/release/bioc/html/DiffBind.html nextflow (version 20.01.0) Seqera https://www.nextflow.io bwa mem (version 0.7.15-r1140) Li H. and Durbin R. (2009) Bioinformatics, 25, 1754-1760. [PMID: 19451168]. https://github.com/lh3/bwa Ngmerge (version 0.3) Gaspar, J.M. BMC Bioinformatics 19, 536 (2018). https://doi.org/10.1186/s12859-018-2579-2 https://github.com/jsh58/NGmerge Homer (version 4.11.1) Heinz S, Benner C, Spann N, Bertolino E et al. Mol Cell 2010 May 28;38(4):576-589. PMID: 20513432 http://homer.ucsd.edu/homer/motif/ Genrich (version 0.5) Gaspar JM (2018). Genrich: Detecting sites of genomic enrichment. https://github.com/jsh58/Genrich https://github.com/jsh58/Genrich FastQC (version v0.11.5) Babraham Bioinformatics https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Open in a new tab Highlights. Conserved DNA-binding transcriptional repressor CIC prevents aberrant ISG expression The CIC-ATXN1L complex binds to CBS motifs to repress ISGs under homeostatic conditions During respiratory viral infection, degradation of CIC-ATXN1L relieves ISG repression CIC-ATXN1L acts as a novel negative regulator of cell-intrinsic antiviral responses ACKNOWLEDGEMENTS We would like to thank Dr. Adolfo Garcia-Sastré (Mount Sinai School of Medicine) for sharing numerous reagents. EMCV, Zika virus MR766 and recombinant HA proteins were obtained from BEI Resources (NIAID). Julianna Han and Olivia Vogel were partly supported by the NIH Molecular and Cellular Biology training program at The University of Chicago (T32GM007183). Julianna Han was supported by the NIH Diversity Supplement (R01AI123359-02S1). Abigail Lewis was supported by the Primary Caregiver Technical Assistance Supplement awarded to Dr. Senthamizharasi Manivasagam (PCTAS, 3R01AI165932-02S1). Drs. Balaji Manicassamy and Priya Issuree were partly supported by NIAID grant (R01AI123359) and NIGMS (1R35GM154831-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. DECLARATION OF INTERESTS The authors declare no competing interests. REFERENCES 1. Mesev EV, LeDesma RA, and Ploss A (2019). Decoding type I and III interferon signalling during viral infection. Nat Microbiol 4, 914–924. 10.1038/s41564-019-0421-x. 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NIHMS2062688-supplement-3.xlsx (2MB, xlsx) 4 Table S3: MSigDB transcription factor analysis of DEGs identified in RNA-Seq analysis of CIC KO A549 cells, related to Figure 1 and Figure S3 . NIHMS2062688-supplement-4.xlsx (15.9KB, xlsx) 5 Table S4: DE peaks identified in ATAC-Seq analysis of CIC KO A549 cells, related to Figure 3 . NIHMS2062688-supplement-5.xlsx (1.4MB, xlsx) 6 Table S5: CBS motif containing DE identified in ATAC-Seq analysis of CIC KO A549 cells, related to Figure 3 . NIHMS2062688-supplement-6.xlsx (511.1KB, xlsx) 7 Table S6: Genes identified in the integrated analysis of RNA-Seq, ATAC-Seq and CBS motif mining, related to Figure 3 . NIHMS2062688-supplement-7.xlsx (403.4KB, xlsx) 8 Table S7: Homer transcription factor analysis of 324 common genes identified in integrated analysis of mock samples, related to Figure 3 . NIHMS2062688-supplement-8.xlsx (121KB, xlsx) 9 Table S8: DEGs identified in RNA-Seq Analysis of Cic KO mouse lungs, related to Figure 7 and Figure S6 . NIHMS2062688-supplement-9.xlsx (506.1KB, xlsx) 10 Table S9: Gene ontology analysis of DEGs identified in RNA-Seq analysis of Cic KO mouse lungs, related to Figure 7 and Figure S6 . NIHMS2062688-supplement-10.xlsx (584.6KB, xlsx) Data Availability Statement RNA-seq data comparing control and CIC KO A549 cells are available at GEO ( GSE271898 ). ATAC-Seq analysis in Control and CIC KO A549 cells are available under accession number GSE271897 . Mouse lung RNA-Seq data are available at GEO ( GSE271899 ). Summary data from RNA-Seq and ATAC-Seq are included as Extended Data Tables S1-S9. There are no restrictions on data availability. 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