Expression regulation of zebrafish interferon regulatory factor 9 by promoter analysis

We previously showed that a fish interferon (IFN) regulatory factor 9 (IRF9) homologue, crucian carp Carassius auratus IRF9, displays constitutively nuclear localization and involvement in fish IFN-dependent JAK-STAT signaling; however, little is known about the expression regulation of fish IRF9. Here, we characterized the expression of zebrafish IRF9 by promoter analysis. Zebrafish IRF9 gene promoter contained several putative transcription factor binding sites, including one ISRE (IFN-stimulated response element), one GAS (IFN gamma activation sequence) and three GATEs (IFN gamma activated transcriptional element, GATE1/2/3). Further sequence analyses revealed that GAS and GATE motifs existed in all promoters of IRF9 from mammals and fishes. Luciferase assays confirmed that zebrafish IRF9 promoter could be activated by zebrafish IFN phi s and zebrafish IFN gamma 2, as well as transcription factors IRF3, IRF7, and combination of IRF9 and STAT2. Treatment of recombinant crucian carp IFN protein or overexpression of zebrafish IFN gamma 2 both led to significant increase in crucian carp IRF9 mRNA and protein in cultured fish cells. Comparison of IFN-stimulated promoter activity revealed much more significant induction of zebrafish IRF9 by zebrafish IFN gamma 2 than by zebrafish IFN phi s. Mutation analyses showed that the putative GAS and GATE3 contributed to zebrafish IFN gamma 2-triggered IRF9 expression, whereas the putative ISRE and the other two GATEs were not functional for induction of zebrafish IRF9. These results together indicated that the expression property of IRF9 might be conserved from fish to mammals and that some not yet identified mechanisms could exist in IRF9 gene transcription regulation in response to IFNs. (C) 2013 Elsevier Ltd. All rights reserved.