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

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.

Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA.

PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5' UTR (36 bp), a 3' UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.

Chimeric murine interferon regulatory factor-2 (IRF-2) binds to IRF-E (IRF binding element), VREß (virus response element) but not to VREα1.

Interferon regulatory factor-2 (IRF-2) is a multifunctional transcription factor having gene activation, repression and synergistic effect in conjunction with IRF-1. IRF-2 is also involved in type I IFN signalling by repressing INFß gene. So far, the molecular mechanism of its DNA binding activity remains elusive. We have carried out molecular sub-cloning, expression and electrophoretically mobility shift assay study of chimeric murine IRF-2. Here, we report expression of chimeric murine IRF-2 as GST-IRF-2 fusion protein in Escherichia coli/BL21 cells and demonstrated DNA binding activity by gel retardation technique using radio (32) P-labelled IRF-E motif (GAAAGT)4 , virus response element (VRE) of human INFß and IFNα1 gene. We observed five different masses DNA/GST-IRF-2 complexes (1-5) with IRF-E motif, three different masses DNA/GST-IRF-2 complexes (1-3) with VREß , but we could not observe any complex of DNA/GST-IRF-2 with VREα1 . The specific binding on IRF-E motif was confirmed by carrying out 100-X fold cold competition with (32) P-labelled IRF-E motif. In contrast to specific binding on VREß , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG-induced extract. As far as binding on VREα1 is concerned, we could not observe any complex in negative control as well as in IPTG-inducible clones extract. Chimeric IRF-2 binds with IRF-E motif and VREß but not with VREα1. This study is first of its kind and paves the way to understand the differential DNA binding and molecular mechanism of DNA binding activity of the IRF-2 molecule, which is crucial for its function(s).

Association between SNPs in interferon regulatory factor 2 (IRF-2) gene and resistance to Aeromonas hydrophila in freshwater mussel Hyriopsis cumingii.

Interferon regulatory factor 2 (IRF-2) is a multi-functional transcription factor in the IRF family exhibiting both transcriptional activating and repressing activities. In this study, an IRF-2 gene (HcIRF-2) from Hyriopsis cumingii was identified and characterized. The cDNA sequence consisted of 2688 bp, encoding a 329 amino acid-protein. The amino acid sequence had a highly conserved N-terminal DBD structure, containing characteristic repeats of six tryptophan residues. The 5'-flanking region contained several transcription regulation elements such as AP1, CdxA, HSF, NIT2 and HNF-3b. Nine SNPs were obtained through direct sequencing of HcIRF-2 from resistant and susceptible stock. Only +2365T/C SNP was significantly associated with resistance/susceptibility of H. cumingii to Aeromonas hydrophila both in genotype (P = 0.021) and allele (P = 0.006) analysis. The SNPs +2248T/C and +2365T/C were in high linkage disequilibrium, and haplotype analysis revealed that haplotype TT frequency in the resistant group was significantly higher than in the susceptible group. The mortality in +2248CC genotype individuals was significantly higher than in CT and TT genotype individuals. These results indicated that haplotype TT and genotype +2248CT and +2248GT individuals were resistant to A. hydrophila, which could make them potential markers in selective breeding of H. cumingii.

Molecular characterization of interferon regulatory factor 2 (IRF-2) homolog in pearl oyster Pinctada fucata.

Interferon regulatory factors (IRFs) control many facets of the innate and adaptive immune responses, regulate the development of the immune system itself and involve in reproduction and morphogenesis. In the present study, the IRF-2 homology gene, PfIRF-2 from pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfIRF-2 encodes a putative protein of 350 amino acids, and contains a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. Comparison and phylogenetic analysis revealed that PfIRF-2 shared a relatively higher identity with other mollusk but relatively lower identity with vertebrate IRF-2, and was clustered with IRF-1 subfamily composed of IRF-2 and IRF-1. Furthermore, gene expression analysis revealed that PfIRF-2 involved in the immune response to LPS and poly(I:C) stimulation. Immunofluorescence assay showed that the expressed PfIRF-2 was translocated into the nucleus and dual-luciferase reporter assays indicated that PfIRF-2 could involved and activate interferon signaling or NF-κB signal pathway in HEK293 cells. The study of PfIRF-2 may help better understand the innate immune in mollusk.

Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus.

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappaB and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C.

Replacement of the C-terminal tetrapeptide (314 PAPV 317 to 314 SSSM 317) in interferon regulatory factor-2 alters its N-terminal DNA-binding activity.

Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein-protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (314PAPV317) of mouse IRF-2 to be different (314SSSM317) from human IRF-2. Recombinant GST-IRF-2 with 314PAPV317 (wild type) and 314SSSM317 (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with 32P-(GAAAGT) 4 by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA-protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA-protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and influence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.

Interferon regulatory factor-2 in orange-spotted grouper (Epinephelus coioides): gene, inductive expression pattern and subcellular localization.

Interferon regulatory factor 2 (IRF-2) is a multifunctional transcription factor which exhibits both transcriptional activating and repressing activities in the IRF family. In this study, we report an IRF-2 gene isolated from orange-spotted grouper (Epinephelus coioides). The 1854bp full-length cDNA sequence of EcIRF-2 has been cloned, encoding a putative peptide of 336 amino acids which is highly consistent with the feature of IRF family members. The genomic fragment of EcIRF-2 contains nine exons and eight introns, spanning over approximate 8.8kb. The expression of EcIRF-2 gene was detected in various tissues of healthy orange-spotted grouper and in four tissues after being challenged with poly I:C or LPS. EcIRF-2 gene is ubiquitously expressed in various healthy fish tissues and is up-regulated in vivo in response to poly I:C or LPS. Subcellular localization analysis of EcIRF-2 suggests it is an intranuclear protein in the fish cells. We believe this research is the first report of fish IRF-2 protein localization. The results in this research establish the base for further study of function mechanism of IRF family members in orange-spotted grouper.