Functional characterization of interferon regulatory factor 2 and its role in the transcription of interferon a3 in golden pompano Trachinotus ovatus (Linnaeus 1758)

Fish Shellfish Immunol. 2019 Oct:93:90-98. doi: 10.1016/j.fsi.2019.07.045. Epub 2019 Jul 18.

Abstract

Similar to mammals, fish possess interferon (IFN) regulatory factor 2 (IRF2)-dependent type I IFN responses. Nevertheless, the detailed mechanism through which IRF2 regulates type I IFNa3 remains largely unknown. In the present study, we first identified two genes from golden pompano (Trachinotus ovatus), IRF2 (ToIRF2) and IFNa3 (ToIFNa3), in the IFN/IRF-based signalling pathway. The open reading frame (ORF) sequence of ToIRF2 encoded 335 amino acids possessing four typical characteristic domains, including a conserved DNA-binding domain (DBD), an interferon association domain 2 (IAD2), a transcriptional activation domain (TAD), and a transcriptional repression domain (TRD). Furthermore, transcripts of ToIRF2 were significantly upregulated after stimulation by polyinosinic: polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS) and flagellin in immune-related tissues (blood, liver, and head-kidney). Moreover, to investigate whether ToIRF2 was a regulator of ToIFNa3, promoter analysis was performed. The results showed that the region from -896 bp to -200 bp is defined as the core promoter using progressive deletion mutations of IFNa3. Additionally, ToIRF2 overexpression led to a clear time-dependent enhancement of ToIFNa3 promoter expression in HEK293T cells. Mutation analyses indicated that the activity of the ToIFNa3 promoter significantly decreased after targeted mutation of M4/5 binding sites. Electrophoretic mobile shift assays (EMSAs) verified that IRF2 interacted with the binding site of the ToIFNa3 promoter region to regulate ToIFNa3 transcription. Last, the promoter activity of ToIFNa3-2 was more responsive to treatment with poly (I:C) than LPS and flagellin. Furthermore, overexpression of ToIRF2 in vitro obviously increased the expression of several IFN/IRF-based signalling pathway genes after poly (I:C) abduction. In conclusion, the present study provides the first evidence of the positive regulation of ToIFNa3 transcription by ToIRF2 and contributes to a better understanding of the transcriptional mechanisms of ToIRF2 in fish.

Keywords: IFNa3; IRF2; Promoter activity; Trachinotus ovatus; Transcription factors.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Female
  • Fish Diseases / immunology*
  • Fish Proteins / chemistry
  • Fish Proteins / genetics
  • Fish Proteins / immunology
  • Fishes / genetics*
  • Fishes / immunology*
  • Flagellin / pharmacology
  • Gene Expression Profiling / veterinary
  • Gene Expression Regulation / immunology*
  • Immunity, Innate / genetics*
  • Interferon Regulatory Factor-2 / chemistry
  • Interferon Regulatory Factor-2 / genetics*
  • Interferon Regulatory Factor-2 / immunology*
  • Lipopolysaccharides / pharmacology
  • Male
  • Phylogeny
  • Poly I-C / pharmacology
  • Promoter Regions, Genetic

Substances

  • Fish Proteins
  • Interferon Regulatory Factor-2
  • Lipopolysaccharides
  • Flagellin
  • Poly I-C