Preliminary Analysis Of The Transcription Reg- Ulation And Function Of Irf11 In Japanese Eel, Anguilla Japonica

Interferon regulatory factors?IRFs?constitute a family of transcription factors that play es-sential roles in regulation of antiviral immune response,antitumor immunity,cell differentiation and apoptosis,by binding to the promoter region of interferons and interferon-stimulated genes,thereby regulating their transcription.To data,a total of eleven IRFs,termed IRF1-11,have been identified in teleost.Of which,IRF11 was found only in teleost.In this study,we have character-ized IRF11in Japanese eel,Anguilla japonica,and conducted a functional analysis of this gene.The results are as follows:The determined cDNA sequence of eel IRF11?AjIRF11?is 1321 bp in length,encoding a281 amino acid protein with a calculated molecular mass of 31.97 kD.Sequence analysis revealed that AjIRF11 belongs to IRF1 subgroup,with six regularly spaced tryptophan residues present in the DNA binding domain?DBD?.Synteny analysis result shows that AjIRF11 is franked by CHS1and kif1 binding protein?KIF1BP?on the 5'and 3'side respectively,and the syntenic block re-tained in spotted gar?Lepisosteus oculatus?and coelacanth?Latimeria chalumnae?.While in Acanthomorphata species,IRF11 is sandwiched between interleukin 4 receptor,tandem duplicate1?IL4R.1?and nucleolar pre-rRNA processing protein?NIP7?at the 5'and 3'side respectively,indicating that IRF11 loci might have undergone lineage-specific genomic rearrangements during the evolution of teleost fish.Expression analysis revealed that AjIRF11 was predominantly ex-pressed in gill,and the increased expression of AjIRF11 was observed in gill at 8 hpi after chal-lenged with Edwardsiella tarda,being 3.6-fold higher than control,and in intestine and gill at 72hpi,being 1.4-fold and 4.04-fold higher than control respectively.No significant change can be detected following poly I:C stimulation.The promoter region of AjIRF11 was cloned and analyzed,which revealed the presence of several binding site for SP1 and NF-?B,and no existence of ISRE or GAS.The luciferase reporter construct encompassing the promoter of AjIRF11 was generated,as well as expression vectors of AjIRFs,STATs and several key effectors of the RLR or TLR signal pathways.Dual luciferase assay showed that the overexpression of these genes did not affect the luciferase activity of AjIRF11 promoter reporter.Immunofluorescence data shows that AjIRF11 is constitutively pre-sented in the nucleus,which may driven by two nuclear localization signals?NLS?,NLS1(⁷⁵KTW-KANFR⁸22 and ⁹⁵HDKSIKK¹⁰¹)and NLS2(¹¹⁹KRRK¹²²),predominantly by NLS1.Functional studies demonstrate that overexpression of AjIRF11 significant increased AjIFNs and AjMxs pro-moter activity and the expression level of fish antiviral genes,including IFNa,Mx1,PKR,Viperin,ISG15.Taken together,IRF11 was cloned and characterized from Japanese eel,and its expression changes following poly I:C stimulation and Edwardsiella tarda infection were investigated.More-over,its cellular distribution and regulation on antiviral genes were also characterized.Overall,our results laid the foundation for further research of the innate immunity function of teleost IRF11.