IRF9 (interferon regulatory factor 9, ISGF3-gamma, p48) is the DNA-binding subunit of the interferon-stimulated gene factor 3 (ISGF3) transcription factor. After type I interferon (IFN-alpha/beta) activates the JAK kinases TYK2 and JAK1, IRF9 associates through its C-terminal IRF-association domain with tyrosine-phosphorylated STAT2 and STAT1, and the ISGF3 complex accumulates in the nucleus, where the N-terminal tryptophan-pentad DNA-binding domain of IRF9 recognizes the interferon-stimulated response element (ISRE) and drives RNA polymerase II transcription of interferon-stimulated genes, establishing an antiviral state. The donkey protein belongs to the IRF9 subfamily of the interferon regulatory factor family, conserves the DNA-binding domain, including all five pentad tryptophans, and the IRF-association domain, and carries a 33-residue C-terminal extension relative to human IRF9.
Existing Annotations Review
GO Term
Evidence
Action
Reason
GO:0000976 transcription cis-regulatory region binding
IEA GO_REF:0000120
TOO GENERAL
Summary: IRF9 binds the ISRE in promoters of interferon-stimulated genes; true, but entailed by the more specific RNA polymerase II cis-regulatory sequence-specific DNA binding term accepted in this review.
Reason: The claim is correct. GO:0000978 (RNA polymerase II cis-regulatory region sequence-specific DNA binding), accepted below on the basis of the conserved IRF DNA-binding domain and human IRF9 ISRE-binding data, is an is_a descendant of this term (verified in OLS), so this row adds no information.
98 of 108 residues identical in donkey A0A9L0I7X9.
GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding
IEA GO_REF:0000118
ACCEPT
Summary: IRF9 recognizes ISRE sequences in RNA polymerase II promoters of interferon-stimulated genes.
Reason: The TreeGrafter transfer places the donkey protein in PANTHER subfamily PTHR11949:SF26 (IRF9), the same subfamily as human Q00978 and mouse Q61179, so the subfamily-level DNA-binding function applies. Human ISGF3-gamma binds DNA sequence-specifically in vitro, and IRF9-deficient human cells lose ISRE binding. The donkey DNA-binding domain is 91% identical to human with all five pentad tryptophans conserved.
ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells
Summary: IRF9 is a sequence-specific RNA polymerase II transcription factor acting within ISGF3.
Reason: Core molecular function, transferred within PANTHER subfamily PTHR11949:SF26. IRF9 supplies the sequence-specific DNA recognition of ISGF3; loss of IRF9 abolishes ISRE-driven reporter transcription in human cells, and the donkey protein retains the DNA-binding and IRF-association domains.
U2A cells transfected with IRF9-Ξex7 exhibited abolished luciferase transcription when luciferase production was driven from an IFN-stimulated response element (ISRE) promoter
Summary: True but uninformative; IRF9's immune role is specifically type I interferon signalling.
Reason: The family-level PAINT term is correct for IRF9, but the specific, well-established process is type I interferon-mediated signalling, in which IRF9 is the DNA-binding component of ISGF3 that carries the signal to interferon-stimulated gene promoters. IRF9-deficient human cells fail to form ISGF3 in response to IFN-alpha2b. This replacement is the transducing step IRF9 itself performs, not a downstream organismal phenotype.
Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-Ξ±2b.
Summary: True but entailed by the sequence-specific RNA polymerase II cis-regulatory DNA-binding annotation.
Reason: IRF9 binds DNA, but RNA polymerase II cis-regulatory region sequence-specific DNA binding, accepted in this review, is a descendant of this term (verified in OLS) and already entails it.
Supporting Evidence:
file:EQUAS/IRF9/IRF9-bioinformatics/dbd_check.md
98 of 108 residues identical in donkey A0A9L0I7X9.
Summary: Correct, but the parent of the RNA polymerase II-specific transcription factor activity accepted in this review.
Reason: The claim is true. GO:0000981 (DNA-binding transcription factor activity, RNA polymerase II-specific), accepted above as the core function, is an is_a descendant of this term (verified in OLS), so this row is entailed.
Summary: ISGF3 containing IRF9 acts in the nucleus; IRF9 shuttles and accumulates there upon interferon stimulation.
Reason: Nuclear localization of human IRF9 is shown by fractionation, IRF9 is regulated by nucleocytoplasmic redistribution, and IRF9 acts in the nucleus as the DNA-binding subunit of ISGF3. The nucleus is where the core activity is executed.
Summary: IRF9 regulates transcription; it does not perform it.
Reason: RNA polymerase II executes transcription; a sequence-specific transcription factor is properly described by its DNA-binding transcription factor activity and by positive regulation of transcription by RNA polymerase II, both accepted here. The UniRule keyword mapping to transcription itself overstates IRF9's role.
Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form.
GO:0006355 regulation of DNA-templated transcription
IEA GO_REF:0000120
TOO GENERAL
Summary: Correct; IRF9 regulates transcription of interferon-stimulated genes, but this is entailed by positive regulation of transcription by RNA polymerase II.
Reason: The claim is true but uninformative here. GO:0045944 (positive regulation of transcription by RNA polymerase II), accepted below, is a descendant of this term (verified in OLS).
U2A cells transfected with IRF9-Ξex7 exhibited abolished luciferase transcription when luciferase production was driven from an IFN-stimulated response element (ISRE) promoter
GO:0006357 regulation of transcription by RNA polymerase II
IEA GO_REF:0000118
TOO GENERAL
Summary: Correct; IRF9 regulates RNA polymerase II transcription of interferon-stimulated genes, but this is entailed by the positive-regulation child accepted in this review.
Reason: The PAINT node assertion is sound for the IRF9 subfamily, but IRF9 within ISGF3 is an activator, and GO:0045944 (positive regulation of transcription by RNA polymerase II), accepted below, is a descendant of this term (verified in OLS).
Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form.
GO:0045944 positive regulation of transcription by RNA polymerase II
IEA GO_REF:0000117
ACCEPT
Summary: ISGF3 activates RNA polymerase II transcription of interferon-stimulated genes.
Reason: Activation of interferon-stimulated genes through the ISRE is the output of IRF9 activity. The ARBA source is not itself evidence, but human IRF9 is required for ISRE-driven transcription, and the donkey protein sits in the same PANTHER subfamily (PTHR11949:SF26) with conserved DNA-binding and IRF-association domains.
U2A cells transfected with IRF9-Ξex7 exhibited abolished luciferase transcription when luciferase production was driven from an IFN-stimulated response element (ISRE) promoter
ISS file:EQUAS/IRF9/IRF9-bioinformatics/human_Q00978-uniprot.txt
NEW
Summary: IRF9 is the DNA-binding subunit of ISGF3 with STAT1 and STAT2.
Reason: The donkey GOA record has no complex annotation. ISGF3 membership is the defining context of IRF9 activity; IRF9 is itself a structural subunit of the complex, not a substrate or regulator of it. Human IRF9-deficient cells cannot form ISGF3, the donkey protein is in the IRF9 PANTHER subfamily, and it retains the IRF-association domain (Pfam IRF-3) that binds STAT2.
Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-Ξ±2b.
ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma.
file:EQUAS/IRF9/IRF9-uniprot.txt
PTHR11949:SF26; INTERFERON REGULATORY FACTOR 9
Core Functions
Sequence-specific DNA-binding subunit of ISGF3 that binds ISRE elements and, with STAT1/STAT2, activates RNA polymerase II transcription of interferon-stimulated genes in response to type I interferon.
Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-Ξ±2b.
file:EQUAS/IRF9/IRF9-uniprot.txt
PTHR11949:SF26; INTERFERON REGULATORY FACTOR 9
file:EQUAS/IRF9/IRF9-bioinformatics/dbd_check.md
98 of 108 residues identical in donkey A0A9L0I7X9.
Q: Is the 33-residue C-terminal extension of donkey IRF9 encoded by an expressed exon, and does it affect STAT2 binding by the adjacent IRF-association domain?
Suggested Experiments
Experiment: Interferon-alpha stimulation of donkey fibroblasts with ISRE-reporter and STAT2 co-immunoprecipitation assays, comparing endogenous IRF9 with a construct lacking the C-terminal extension.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Assessment of 3 ProtNLM2 GO predictions for donkey IRF9 (A0A9L0I7X9), the DNA-binding subunit of the ISGF3 transcription factor. The donkey protein sits in PANTHER subfamily PTHR11949:SF26 (IRF9) with human Q00978 and conserves the IRF DNA-binding domain (98 of 108 residues identical, all five pentad tryptophans conserved). All three predictions are biologically correct and already present as exact terms in the donkey GOA record. Positive regulation of transcription by RNA polymerase II matches the accepted core process (CNN). Transcription cis-regulatory region binding and DNA-binding transcription factor activity are true but less specific than the RNA polymerase II cis-regulatory sequence-specific DNA binding and RNA polymerase II-specific transcription factor terms accepted for this protein in the gene review (LSP). ProtNLM2 emitted no non-GO function-text or subcellular-location output for this accession in the frozen snapshot.
Review score: 2 = concordant with evidence; 1 = uncertain; 0 = discordant with evidence. This is an assessment score, not a model probability.
GO:0045944 positive regulation of transcription by RNA polymerase IIGO_BP
CNN β Correct but not novel Review score: 2/2
Prediction method: ProtNLM2 Β· Version: UniProt API snapshot 2026-10-14
Review rationale: ISGF3, of which IRF9 is the DNA-binding subunit, activates RNA polymerase II transcription of interferon-stimulated genes, and human IRF9 is required for ISRE-driven reporter transcription. The donkey protein is in the same PANTHER subfamily (PTHR11949:SF26) with a conserved DNA-binding domain, which justifies the transfer. The exact term is already in the donkey GOA record and is accepted as the core process in the gene review, so the prediction is correct but not novel.
Supporting Evidence:
PMID:1630447: "Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form."
PMID:30143481: "U2A cells transfected with IRF9-Ξex7 exhibited abolished luciferase transcription when luciferase production was driven from an IFN-stimulated response element (ISRE) promoter"
GO:0000976 transcription cis-regulatory region bindingGO_MF
LSP β Less precise than existing annotation Review score: 2/2
Prediction method: ProtNLM2 Β· Version: UniProt API snapshot 2026-10-14
Review rationale: IRF9 binds the interferon-stimulated response element through its tryptophan-pentad DNA-binding domain, which is conserved in the donkey protein; human ISGF3-gamma binds DNA sequence-specifically. The term is correct and already in the donkey GOA record, but the gene review accepts the more specific descendant GO:0000978 (RNA polymerase II cis-regulatory region sequence-specific DNA binding; is_a ancestry checked in OLS4) on the basis of the PTHR11949:SF26 subfamily transfer and human data, and marks this term TOO_GENERAL. The prediction is therefore less precise than an existing supported annotation.
Supporting Evidence:
PMID:1630447: "ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells"
LSP β Less precise than existing annotation Review score: 2/2
Prediction method: ProtNLM2 Β· Version: UniProt API snapshot 2026-10-14
Review rationale: IRF9 supplies the sequence-specific DNA recognition of ISGF3 and acts as an RNA polymerase II transcription factor. The term is correct and already in the donkey GOA record, but the gene review accepts the more specific descendant GO:0000981 (DNA-binding transcription factor activity, RNA polymerase II-specific; is_a ancestry checked in OLS4) as the core function and marks this term TOO_GENERAL. The prediction is therefore less precise than an existing supported annotation.