Severe type 1 interferonopathy due to germline STAT2 gain-of-function
ABSTRACT: The transcription factor STAT2 is essential for transcriptional activation downstream of the receptors for the innate IFNs -?/? (IFNAR) and -gamma (IFNLR). STAT2 is activated by tyrosine phosphorylation, associating with STAT1 and IRF9 to form the Interferon-Stimulated Gene Factor 3 (ISGF3) to effect gene transcription. Loss-of-function variants in STAT2 increase susceptibility to viral disease. Here a transcriptome study is reported on an individual with severe early-onset neuroinflammatory disease and an elevated IFN signature. The individual had a homozygous missense variant in STAT2 and symptoms consistent with a gain-of-function effect.
Project description:The transcription factor STAT2 is essential for transcriptional activation downstream of the receptors for the innate IFNs -α/β (IFNAR) and -gamma (IFNLR). STAT2 is activated by tyrosine phosphorylation, associating with STAT1 and IRF9 to form the Interferon-Stimulated Gene Factor 3 (ISGF3) to effect gene transcription. Loss-of-function variants in STAT2 increase susceptibility to viral disease. Here a transcriptome study is reported on an individual with severe early-onset neuroinflammatory disease and an elevated IFN signature. The individual had a homozygous missense variant in STAT2 and symptoms consistent with a gain-of-function effect.
Project description:Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFN?) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFN? signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFN?-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFN?-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ?120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFN? was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFN?-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFN? signalling.
Project description:Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.
Project description:Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9-IAD alone and in a complex with STAT2-CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9-IAD and STAT2-CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells.
Project description:Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-?-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-? was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-? exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.
Project description:Interferon (IFN) ? and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFN? is reprogrammed by crosstalk with TNF. IFN? mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFN? and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFN? and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFN? and TNF.
Project description:Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.
Project description:During cellular aging, many changes in cellular functions occur. A hallmark of aged cells is secretion of inflammatory mediators, which collectively is referred to as the senescence-associated secretory phenotype (SASP). However, the mechanisms underlying such changes are unclear. Canonically, the expression of interferon (IFN)-stimulated genes (ISGs) is induced by IFNs through the formation of the tripartite transcriptional factor ISGF3, which is composed of IRF9 and the phosphorylated forms of STAT1 and STAT2. However, in this study, the constitutive expression of ISGs in human-derived senescent fibroblasts and in fibroblasts from a patient with Werner syndrome, which leads to premature aging, was mediated mainly by the unphosphorylated forms of STATs in the absence of INF production. Under homeostatic conditions, STAT1, STAT2, and IRF9 were localized to the nucleus of aged cells. Although knockdown of JAK1, a key kinase of STAT1 and STAT2, did not affect ISG expression or IFN-stimulated response element (ISRE)-mediated promoter activities in these senescent cells, knockdown of STAT1 or STAT2 decreased ISG expression and ISRE activities. These results suggest that the ISGF3 complex without clear phosphorylation is required for IFN-independent constitutive ISG transcription in senescent cells.
Project description:In response to IFN?, the IL6 gene is activated, modestly at early times by ISGF3 (IRF9 plus tyrosine-phosphorylated STATs 1 and 2), and strongly at late times by U-ISGF3 (IRF9 plus U-STATs 1 and 2, lacking tyrosine phosphorylation). A classical IFN-stimulated response element (ISRE) at -1,513 to -1,526 in the human <i>IL6</i> promoter is required. Pretreating cells with IFN? or increasing the expression of U-STAT2 and IRF9 exogenously greatly enhances IL6 expression in response to the classical NF-?B activators IL1, TNF, and LPS. U-STAT2 binds tightly to IRF9, the DNA binding subunit of ISGF3, and also to the p65 subunit of NF-?B. Therefore, as shown by ChIP analyses, U-STAT2 can bridge the ISRE and ?B elements in the <i>IL6</i> promoter. In some cancer cells, the protumorigenic activation of STAT3 will be enhanced by the increased synthesis of IL6 that is facilitated by high expression of U-STAT2 and IRF9.
Project description:TLR-stimulated cross-presentation by conventional dendritic cells (cDCs) is important in host defense and antitumor immunity. We recently reported that cDCs lacking the type I IFN signaling molecule STAT2 are impaired in cross-presenting tumor Ags to CD8(+) T cells. To investigate how STAT2 affects cross-presentation, we determined its requirements for dendritic cell activation. In this study, we report that STAT2 is essential for the activation of murine female cDCs upon TLR3, -4, -7, and -9 stimulation. In response to various TLR ligands, Stat2(-/-) cDCs displayed reduced expression of costimulatory molecules and type I IFN-stimulated genes. The cDC responses to exogenous IFN-? that we evaluated required STAT2 activation, indicating that the canonical STAT1-STAT2 heterodimers are the primary signaling transducers of type I IFNs in cDCs. Interestingly, LPS-induced production of IL-12 was STAT2 and type I IFN receptor (IFNAR) dependent, whereas LPS-induced production of TNF-? and IL-6 was STAT2 and IFNAR independent, suggesting a specific role of the IFNAR-STAT2 axis in the stimulation of proinflammatory cytokines by LPS in cDCs. In contrast, R848- and CpG-induced cytokine production was less influenced by the IFNAR-STAT2 axis. Short kinetics and IFNAR blockade studies showed that STAT2 main function is to transduce signals triggered by autocrine type I IFNs. Importantly, Stat2(-/-) cDCs were deficient in cross-presenting to CD8(+) T cells in vitro upon IFN-?, CpG, and LPS stimulation, and also in cross-priming and licensing cytotoxic T cell killers in vivo. We conclude that STAT2 plays a critical role in TLR-induced dendritic cell activation and cross-presentation, and thus is vital in host defense.