Functional Analysis of IRF1 Reveals its Role in the Activation of the Type I IFN Pathway in Golden Pompano, (Linnaeus 1758).

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, (designated ToIRF1), and its immune function was identified to elucidate the transcriptional regulatory mechanism of . The full-length complementary DNA (cDNA) of 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 consists of eight exons and seven introns. Moreover, is constitutively expressed in all examined tissues, with higher levels being observed in immune-relevant tissues (whole blood, gill, and skin). Additionally, 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 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 can notably activate and interferon signaling molecule expression. Promoter sequence analysis finds that several interferon stimulating response element (ISRE) binding sites are present in the promoter of . Additionally, truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that M5 ISRE binding sites are functionally important for transcription. These results may help to illuminate the roles of teleost in the transcriptional mechanisms of type I IFN in the immune process.