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

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.

Toll-Like Receptor 5 of Golden Pompano Trachinotus ovatus (Linnaeus 1758): Characterization, Promoter Activity and Functional Analysis

“…Previous studies have demonstrated that poly(I:C), LPS, and flagellin can cause defense responses against pathogens in pompano [ 37 , 38 , 39 , 40 , 41 ]. Many studies have found that these PAMPs can induce the expression of TLR5M or TLR5S genes in immune-related tissues (liver, intestine, blood, kidney, and skin) [ 17 , 20 , 21 , 36 , 42 , 43 ].…”

IFN regulatory factor 3 of golden pompano and its NLS domain are involved in antibacterial innate immunity and regulate the expression of type I interferon (IFNa3)

“…IFNa3 is a member of type I IFNs in T. ovatus . Previous study demonstrated that IRF1, IRF2, IRF5, and IRF7 positively regulate IFNa3 expression in T. ovatus ( 41 , 42 ). The purpose of this study was to assess the antibacterial immune effects of IRF3 in T. ovatus (TroIRF3) and its role in the regulation of IFNa3.…”