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HGNC Approved Gene Symbol: IRF9
Cytogenetic location: 14q12 Genomic coordinates (GRCh38): 14:24,161,265-24,166,565 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 14q12 | Immunodeficiency 65, susceptibility to viral infections | 618648 | Autosomal recessive | 3 |
The IRF9 gene encodes the DNA-binding component of the interferon-stimulated gene factor-3 complex (ISGF3), which also includes STAT1 (600555) and STAT2 (600556) (Kessler et al., 1990, summary by Hernandez et al., 2018).
ISGF3 is an interferon-dependent, positive-acting transcription factor that is cytoplasmically activated, possibly through direct interaction with the interferon receptor. The attachment of interferon-alpha (IFNA; 147660) to a specific cell surface receptor activates the transcription of a limited set of genes termed the IFN-stimulated genes. In the activated genes, Levy et al. (1989) identified the IFN stimulation response element (ISRE) and a cognate transcription factor, ISGF3, whose activation paralleled the transcriptional activation pattern of the IFN-stimulation genes in cells treated with IFN-alpha. Supporting the notion that the proteins in ISGF3 are links between an occupied receptor and a limited set of genes were the observations that the proteins preexist in untreated cells, are promptly activated in an IFN-alpha-dependent fashion in the cell cytoplasm, and are subsequently translocated to the nucleus.
In its latent state, ISGF3 appears to exist as 2 independent components, ISGF3-alpha, which consists of the 84-, 91-, and 113-kD polypeptides, and ISGF3-gamma, the 48-kD protein. ISGF3-alpha and ISGF3-gamma associate only following exposure of cells to IFN-alpha. One or more of the ISGF3-alpha polypeptides serve as a target for IFN-alpha signaling, while ISGF3-gamma is a DNA-binding protein that serves as the ISRE recognition component (Kessler et al., 1990).
Fu et al. (1990) purified ISGF3, separated its component proteins, and determined their peptide sequences. Four proteins of 48 (ISGF3G), 84, 91, and 113 kD (STAT2; 600556) make up the ISGF3 complex. Using these sequences, Schindler et al. (1992) constructed degenerate oligonucleotide probes to screen for cDNA clones. They found that the 84- and 91-kD components appeared to arise from 2 differently processed RNA products derived from 1 gene (STAT1; 600555). Fu et al. (1992) proposed that the genes encoding the 113- and 91/84-kD proteins are members of a gene family whose products serve directly to receive information that a specific receptor has bound its ligand and subsequently transduces information to the nucleus.
By RT-PCR of IFN-gamma (147570)-treated HeLa cell mRNA with degenerate primers based on a partial ISGF3-gamma protein sequence, Veals et al. (1992) isolated an ISGF3-gamma cDNA. Northern blot analysis revealed that expression of the approximately 1.9-kb ISGF3-gamma mRNA in HeLa cells was induced by treatment of cells with either IFN-gamma or IFN-alpha. The predicted 393-amino acid protein contains an N-terminal region similar to the DNA-binding domains of the IRF (interferon regulatory factor) proteins IRF1 (147575), IRF2 (147576), and ICSBP (601565). Members of the IRF family accumulate in cells in response to a variety of inducers, including interferons, and bind DNA with a specificity related to, but distinct from, that of ISGF3-gamma. The authors suggested that other IRF family members may participate in signaling pathways by interacting with regulatory subunits analogous to ISGF3-alpha.
Hernandez et al. (2018) determined that the IRF9 gene contains 9 exons, with exons 2 through 9 being protein-coding.
From sequencing studies, McCusker et al. (1999) demonstrated that the gene encoding the ISGF3G protein is located close to the 2 genes for PA28 (PSME1, 600654; PSME2, 602161) on 14q11.2. Using an interspecific backcross, Suhara et al. (1996) mapped the mouse ISGF3-gamma homolog to the distal portion of mouse chromosome 14.
Blaszczyk et al. (2015) found that IFN-alpha (see IFNA1, 147660) response in human and mouse STAT1 knockout cells was diminished and correlated with diminished STAT2 phosphorylation. Overexpression of STAT2 in STAT1 knockout cells restored the IFN-alpha response, confirming this result. In STAT1 knockout cells overexpressing STAT2, STAT2 and IRF9 interacted, and the STAT2/IRF9 complex was responsible for the IFN-alpha response in the absence of STAT1. Comparative analysis of transcriptional responses in genes upregulated by both STAT2/IRF9 and ISGF3 in these cells implied functional overlap between STAT2/IRF9 and ISGF3, especially for the potential of generating an IFN-alpha-induced antiviral response. Moreover, STAT2/IRF9 was found to regulate the expression of IFN-stimulated response element (ISRE)-independent ISGs. Antiviral assays found that STAT2/IRF9 mediated an antiviral response similar to that of ISGF3 against encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV), providing further evidence for functional overlap between STAT2/IRF9 and ISGF3 in the antiviral response.
Li et al. (2017) compared the expression of genes in murine mixed glial cell cultures (MGCs) that lacked Stat1, Stat2, or Irf9 with wildtype MGCs and found that all 3 genes regulated the constitutive expression of a subset of genes that are involved in antiviral response, proteolysis, and retroviral envelope polyprotein production. The number of ISGs was significantly less in Stat1 and Stat2 knockout MGCs than in Irf9 knockout MGCs, suggesting that regulation of ISGF3-independent genes in response to IFN-alpha depends mainly on Stat1 and Stat2 signaling and to a lesser extent on Irf9 signaling. Despite functional annotation of ISGs in MGCs indicating the possibility of other signaling molecules in regulating the expression of ISGF3-independent genes, microarray results demonstrated and RNase protection assay confirmed that Stat1, Stat2, and Irf9 were the major signaling factors functionally involved in noncanonical IFN-I signaling, as only a small number of ISGs were induced when cells were deficient in all 3 signaling genes. Investigation of the interferon-regulated gene (IRG) response at different times revealed that IFN-alpha treatment induced similar response in IFN-I-signaling in mutant MGCs compared with wildtype, with prolonged kinetics due to increased time for response. Analyses of RNA from the brains of mice that lacked either Stat1 or Irf9 confirmed that IRGs were regulated by IFN-alpha in vivo.
Using quantitative RT-PCR, Wang et al. (2017) found that ISGs were expressed constitutively under homeostatic conditions in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. Knockdown of STAT1, STAT2, or IRF9 in human liver cells decreased the constitutive expression of ISG, and increased the replication of hepatitis C (HCV) and hepatitis E (HEV) viruses. Furthermore, STAT1, STAT2, and IRF9 were each necessary, but not sufficient, to drive constitutive ISG expression. Overexpression of STAT1, STAT2, and IRF9 in human liver cells revealed that these 3 factors function as the unphosphorylated ISGF3 (U-ISGF3) complex independently of activation by exogenous IFN. Analysis of the U-ISGF complex showed that it consists of IRF9 with unphosphorylated STAT1 and STAT2 in the nucleus. U-ISGF3-induced expression of ISGs was independent of IFN and upstream elements of the IFN signaling pathway.
In a 5-year-old girl, born of consanguineous Algerian parents, with immunodeficiency-65 (IMD65; 618648), Hernandez et al. (2018) identified a homozygous mutation in the IRF9 gene (147574.0001). The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. It was not found in the 1000 Genomes Project or gnomAD databases. Analysis of cells transfected with the mutation and patient-derived cells showed normal STAT1/STAT2 phosphorylation following IFN stimulation, but an inability to form a functional ISGF3 complex, resulting in loss of transcriptional activity, as evidenced by abolished luciferase production and impaired induction of certain interferon-stimulated genes (ISGs), mainly those in the type I IFN response pathway. Patient fibroblasts showed impaired antiviral immunity compared to controls after infection with influenza type A (IAV) and vesicular stomatitis virus (VSV). Transfection of patient cells with wildtype IRF9 restored expression and rescued the functional defects, consistent with the patient having IRF9 deficiency. Knockdown of IRF9 using siRNA in control fibroblasts resulted in defective control of the positive-sense RNA virus human rhinovirus (HRV). Hernandez et al. (2018) concluded that IRF9 plays an important role in type I, and likely type III, interferon responses to viral infection.
In 2 sibs, born of consanguineous parents of Portuguese origin, with IMD65, Bravo Garcia-Morato et al. (2019) identified a homozygous splice site mutation in the IRF9 gene (147574.0002). The mutation, which was found by next-generation sequencing of a customized panel and confirmed by Sanger sequencing, segregated with the disorder in the family. Patient cells and HEK293 cells transfected with the mutation had no IRF9 expression, consistent with a complete loss of function. In vitro functional expression studies showed that patient cells were unable to inhibit of RSV or HSV1 replication, and there was absence of ISG induction, including MX1 (147150), IFIT3 (604650), and ISG15 (147571), in response to IFN-alpha stimulation. STAT1 phosphorylation was normal. CRISPR/Cas9-mediated knockdown of IRF9 in control fibroblasts resulted in similar defects. Transfection of patient cells with wildtype IRF9 restored the defects.
In a 5-year-old girl, born of consanguineous Algerian parents, with immunodeficiency-65 (IMD65; 618648), Hernandez et al. (2018) identified a homozygous c.991G-A transition in the last nucleotide of exon 7 of the IRF9 gene. The mutation was predicted to result in a splice site alteration as well as possibly an asp331-to-asn (ASP331ASN, D331N) substitution. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. It was not found in the 1000 Genomes Project or gnomAD databases. Analysis of patient-derived cells showed presence of an abnormal transcript reflecting the skipping of exon 7 and an in-frame deletion; a D331N variant was not identified. Western blot analysis showed a protein with a decreased molecular weight compared to wildtype, consistent with the lack of exon 7 (142 amino acids), which forms a large portion of the domain that interacts with STAT proteins. In vitro functional expression studies showed that the mutation caused an inability to form a functional ISGF3 complex in response to stimulation, resulting in loss of transcriptional activity and impaired induction of certain interferon-stimulated genes (ISGs), mainly those in the type I IFN response pathway. Transfection of patient cells with wildtype IRF9 restored expression and rescued the functional defects. The findings were consistent with IRF9 deficiency.
In 2 sibs, born of consanguineous parents of Portuguese origin, with immunodeficiency-65 (IMD65; 618648), Bravo Garcia-Morato et al. (2019) identified a homozygous G-to-T transversion in intron 5 of the IRF9 gene (c.577+1G-T, NM_006084), resulting in a splice site alteration, the skipping of exon 5, a frameshift, and premature termination (Glu166LeufsTer80). The mutation, which was found by next-generation sequencing of a customized panel and confirmed by Sanger sequencing, segregated with the disorder in the family. Patient cells and HEK293 cells transfected with the mutation had no IRF9 expression, consistent with a complete loss of function. In vitro functional expression studies of patient cells reflected significantly impaired IRF9 function.
Blaszczyk, K., Olejnik, A., Nowicka, H., Ozgyin, L., Chen, Y.-L., Chmielewski, S., Kostyrko, K., Wesoly, J., Balint, B. L., Lee, C.-K., Bluyssen, H. A. R. STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1. Biochem. J. 466: 511-524, 2015. [PubMed: 25564224] [Full Text: https://doi.org/10.1042/BJ20140644]
Bravo Garcia-Morato, M., Calvo Apalategi, A., Bravo-Gallego, L. Y., Blazquez Moreno, A., Simon-Fuentes, M., Garmendia, J. V., Mendez Echevarria, A., del Rosal Rabes, T., Dominguez-Soto, A., Lopez-Granados, E., Reyburn, H. T., Rodriguez Pena, R. Impaired control of multiple viral infections in a family with complete IRF9 deficiency. J. Allergy Clin. Immun. 144: 309-312, 2019. [PubMed: 30826365] [Full Text: https://doi.org/10.1016/j.jaci.2019.02.019]
Fu, X.-Y., Kessler, D. S., Veals, S. A., Levy, D. E., Darnell, J. E., Jr. ISGF3, the transcriptional activator induced by interferon alpha consists of multiple interacting polypeptide chains. Proc. Nat. Acad. Sci. 87: 8555-8559, 1990. [PubMed: 2236065] [Full Text: https://doi.org/10.1073/pnas.87.21.8555]
Fu, X.-Y., Schindler, C., Improta, T., Aebersold, R., Darnell, J. E., Jr. The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family involved in signal transduction. Proc. Nat. Acad. Sci. 89: 7840-7843, 1992. [PubMed: 1502204] [Full Text: https://doi.org/10.1073/pnas.89.16.7840]
Hernandez, N., Melki, I., Jing, H., Habib, T., Huang, S. S. Y., Danielson, J., Kula, T., Drutman, S., Belkaya, S., Rattina, V., Lorenzo-Diaz, L., Boulai, A., and 22 others. Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency. J. Exp. Med. 215: 2567-2585, 2018. [PubMed: 30143481] [Full Text: https://doi.org/10.1084/jem.20180628]
Kessler, D. S., Veals, S. A., Fu, X. Y., Levy, D. E. Interferon-alpha regulates nuclear translocation and DNA-binding affinity of ISGF3, a multimeric transcriptional activator. Genes Dev. 4: 1753-1765, 1990. [PubMed: 2249773] [Full Text: https://doi.org/10.1101/gad.4.10.1753]
Levy, D. E., Kessler, D. S., Pine, R., Darnell, J. E., Jr. Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro. Genes Dev. 3: 1362-1371, 1989. [PubMed: 2606351] [Full Text: https://doi.org/10.1101/gad.3.9.1362]
Li, W., Hofer, M. J., Songkhunawej, P., Jung, S. R., Hancock, D., Denyer, G., Campbell, I. L. Type I interferon-regulated gene expression and signaling in murine mixed glial cells lacking signal transducers and activators of transcription 1 or 2 or interferon regulatory factor 9. J. Biol. Chem. 292: 5845-5859, 2017. [PubMed: 28213522] [Full Text: https://doi.org/10.1074/jbc.M116.756510]
McCusker, D., Wilson, M., Trowsdale, J. Organization of the genes encoding the human proteasome activators PA28-alpha and beta. Immunogenetics 49: 438-445, 1999. [PubMed: 10199920] [Full Text: https://doi.org/10.1007/s002510050517]
Schindler, C., Fu, X.-Y., Improta, T., Aebersold, R., Darnell, J. E., Jr. Proteins of transcription factor ISGF-3: one gene encodes the 91- and 84-kDa ISGF-3 proteins that are activated by interferon alpha. Proc. Nat. Acad. Sci. 89: 7836-7839, 1992. [PubMed: 1502203] [Full Text: https://doi.org/10.1073/pnas.89.16.7836]
Suhara, W., Yoneyama, M., Yonekawa, H., Fujita, T. Structure of mouse interferon stimulated gene factor 3 gamma (ISGF3 gamma/p48) cDNA and chromosomal localization of the gene. J. Biochem. 119: 231-234, 1996. [PubMed: 8882710] [Full Text: https://doi.org/10.1093/oxfordjournals.jbchem.a021227]
Veals, S. A., Schindler, C., Leonard, D., Fu, X.-Y., Aebersold, R., Darnell, J. E., Jr., Levy, D. E. Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and myb families of DNA-binding proteins. Molec. Cell. Biol. 12: 3315-3324, 1992. [PubMed: 1630447] [Full Text: https://doi.org/10.1128/mcb.12.8.3315-3324.1992]
Wang, W., Yin, Y., Xu, L., Su, J., Huang, F., Wang, Y., Boor, P. P. C., Chen, K., Wang, W., Cao, W., Zhou, X., Liu, P., van der Laan, L. J. W., Kwekkeboom, J., Peppelenbosch, M. P., Pan, Q. Unphosphorylated ISGF3 drives constitutive expression of interferon-stimulated genes to protect against viral infections. Sci. Signal. 10: eaah4248, 2017. Note: Electronic Article. [PubMed: 28442624] [Full Text: https://doi.org/10.1126/scisignal.aah4248]