Abstract
Interferon (IFN) regulatory factor 1 (IRF1), a transcription factor with a novel helix–turn–helix DNA-binding domain, plays a crucial role in innate immunity by regulating the type I IFN signaling pathway. However, the regulatory mechanism through which IRF1 regulates type I IFN in fish is not yet elucidated. In the present study, IRF1 was characterized from golden pompano, Trachinotus ovatus (designated ToIRF1), and its immune function was identified to elucidate the transcriptional regulatory mechanism of ToIFNa3. The full-length complementary DNA (cDNA) of IRF1 is 1763 bp, including a 900-bp open reading frame (ORF) encoding a 299-amino-acid polypeptide. The putative protein sequence has 42.7–71.7% identity to fish IRF1 and possesses a representative conserved domain (a DNA-binding domain (DBD) at the N-terminus). The genomic DNA sequence of ToIRF1 consists of eight exons and seven introns. Moreover, ToIRF1 is constitutively expressed in all examined tissues, with higher levels being observed in immune-relevant tissues (whole blood, gill, and skin). Additionally, Cryptocaryon irritans challenge in vivo increases ToIRF1 expression in the skin as determined by Western blotting (WB); however, protein levels of ToIRF1 in the gill did not change significantly. The subcellular localization indicates that ToIRF1 is localized in the nucleus and cytoplasm with or without polyinosinic/polycytidylic acid (poly (I:C)) induction. Furthermore, overexpression of ToIRF1 or ToIFNa3 shows that ToIRF1 can notably activate ToIFNa3 and interferon signaling molecule expression. Promoter sequence analysis finds that several interferon stimulating response element (ISRE) binding sites are present in the promoter of ToIFNa3. Additionally, truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that ToIRF1 M5 ISRE binding sites are functionally important for ToIFNa3 transcription. These results may help to illuminate the roles of teleost IRF1 in the transcriptional mechanisms of type I IFN in the immune process.
Highlights
Interferon (IFN) regulatory factors (IRFs) are important transcriptional modulators of bacteria, parasite, virus, and IFN-induced signaling pathways in the process of response to virus infection, immune response, cell growth, and apoptosis [1,2,3,4,5]
Truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that ToIRF1 M5 interferon stimulating response element (ISRE) binding sites are functionally important for ToIFNa3 transcription
These results may help to illuminate the roles of teleost IRF1 in the transcriptional mechanisms of type I IFN in the immune process
Summary
Interferon (IFN) regulatory factors (IRFs) are important transcriptional modulators of bacteria-, parasite-, virus-, and IFN-induced signaling pathways in the process of response to virus infection, immune response, cell growth, and apoptosis [1,2,3,4,5]. Some research showed that IRF1 can be upregulated after challenge with different types of bacteria, viruses, or polyinosinic/polycytidylic acid (poly (I: C)) in fish [37,38,39,40,41], suggesting that IRF1 plays a role in host antiviral and antibacterial responses These findings raise the question of whether IRF1 plays a similar role in antiparasitic diseases in T. ovatus. The role of the IRF1 M5 binding site in the ToIFNα3 promoter was explored using an electrophoretic mobility shift assay (EMSA) These results may help to characterize the regulation associated with type I IFN-associated signaling in marine fish
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