The Functional Characteristics Of The New Promoter In The Second Intron Of Interferon Regulatory Factor 3

Objective Interferon regulatory factor 3 is a member of IRF family. It is the key transcription factor to regulate type I interferon (IFNαandβ) gene expression. It was involved in resistance to viral infections, including SARS virus and hepatitis C virus infection, intracellular bacterial infection. To construct the plasmid of the new variant(int2V2) promoter in the second intron of human interferon regulatory factor 3 (IRF-3). The transcriptional regulation mechanisms and the influence of different viruses analogues to the new variant promoter were studied. The study provides a further insight into the transcriptional regulation mechanisms of different variants of IRF-3.Methods A 800bp fragment in the second intron upstream of the new transcription start site was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase reporter plasmid. Using TFSEARCH ver.1.3 software analysied the new variant promoter and predicted transcription factor binding sites. According to transcription factors and transcription start site, a series of deletion mutants was constructed by PCR. Transfection of AD-293 cells and Hela cells with the promoter - driven luciferase construct was performed to induce luciferase gene expression and calculate the relative luciferase activity unit (RLU). The regulatory function of the promoter by a transcription factor was tested by the mothods of point mutation and overexpression. The promoter activity and mRNA levels of the new variant were studied by cotransfecting different viruses analogues (polyI: C, polydA: dT).Results It was successful to construct the plasmid of IRF-3 gene in the second intron promoter. Promoter activity analysis showed that compared with normal pGL3-Basic plasmid, the new promoter activity was increased. The fragment with promoter activity was located upstream of the new transcription start sites between -317 bp to -110 bp. Bioinformatics analysis indicated that the region include CREB transcription factor binding site. Mutation of this CREB binding site reduced the promoter activity. Overexpression of CREB increased the transcription activity. Both promoter activity and mRNA level of the new variant were elevated after virus analogues stimulated HEK293 cells.Conclusion The 5'flank region of the second transcription start site in the second intron of IRF-3 has promoter activity. A region between -317～- 110bp has important regulatory elements. Transcription factor CREB may be involved in their transcriptional regulation. Transcription factors CREB can regulate the promoter activity. Virus analogues can affect the activity of new promoter and its expression.