| Interferon Regulatory Factor(IRF) forms a family of transcription factor involved in transcriptional regulation of type I Interferon(IFN) and IFN-stimulated genes(ISG) in cells. IRF plays a significant role in host defense against virus infection, immune response and cell growth regulation. IRF-2 is a multifunctional transcription factor and plays a pivotal role in regulation of apoptosis and cell cycle control. The available information shows that IRF-2 works as a negative regulatory factor in the transcription of IFN and ISG genes. IRF-2 can also actively induce certain genes such as other cytokines and chemokines. Therefore, it simultaneously plays the different roles in cell-mediated immune responses. For instance, IRF-2 expects as a transcriptional repressor of interferon system genes, but also acts as a transcriptional activator of certain genes, such as those interleukin 7(IL-7), class II transactivator(CIITA) and Gig2.To explore the molecular mechanism of fish IRF-2 regulating the transcription of fish IFN and ISGs, much attention were payed to the functional analysis of CiIRF-2. IRF-2 of grass carp had been recorded. Amino acid sequence analysis disclosed that CiIRF-2 shares remarkable homology to the known IRF-2 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with zebra fish IRF-2. CiIRF-2 was widely expressed at low level in all detected grass carp tissues and significantly up-regulated except in brain following poly I:C 6-12 h post stimulation. It turned out that CiIRF-2 plays a significant role in host defense against virus infection.CiIFN promoter region contains four putative IRF-E that located at the position from-67 to-78,-107 to-119,-456 to-468 and-646 to-658 respectively. The potential ISRE motif sequence was located at-48 to-58 in CiPKZ promoter and-39 to-51 in CiPKR promoter, respectively. For purpose of analyzing the interaction of CiIRF-2 with Ci IFN, CiPKR and CiPKZ promoter sequences. We constructed the prokaryotic expression plasmid including the full ORF and nDBD domain of CiIRF-2, and the expressed protein was cleansed by affinity chromatography with Ni-NTA His-Bind Resin. In vitro, gel mobility shift assays were applied to analyze the interaction of CiIRF-2 protein with promoters of CiIFN, CiPKR and CiPKZ respectively. Afterwards, recombinant plasmids of pGL3-Ci IFN, pGL3-CiPKR and pGL3-CiPKZ were constructed and transiently co-transfected with pcDNA3.1-CiIRF-2 or pcDNA3.1-CiIRF-1 respectively into C.idella kidney(CIK) cells. Dual-luciferase reporter assays demonstrated that CiIRF-2 down-regulates the transcription activity of CiIFN, CiPKR and CiPKZ genes in CIK cells. CIK cells were transiently co-transfected with pGL3-PKZ, pcDNA3.1-CiIRF-2 or pcDNA3.1-CiIRF-2-nDBD has no significant difference with control group. The results revealed that CiIRF-2 bound to these promoters with high affinity by means of its DBD.To further understand that the molecular mechanisms of CiIRF-2 down-regulating the transcription level of CiIFN, CiPKR and Ci PKZ, expression plasmids(pcDNA3.1-IRF-2 and pcDNA3.1-IRF-1) were transiently co-transfected with pGL3-IFN, pGL3-PKZ or pGL3-CiPKZ into CIK cells, respectively. Results of the study revealed that Ci IRF-2 plays an antagonistic role to CiIRF-1 in transcriptional regulation of IFN and ISG genes. CIK cells were transiently co-transfected with pGL3-IFN, pcDNA3.1-Ci IRF-3 or pcDNA3.1-CiIRF-7 and pcDNA3.1-CiIRF-2 using FuGENE®6 in 24-well plates, respectively, pcDNA3.1-Basic as the control vector. Meanwhile, the experimental results indicate that Ci IRF-2 inhibits the transcriptional level of IFN, acting as an antagonist to positive regulatory factors IRF-3 or IRF-7. |
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