Clone And Identification Of The Promoter Of Human IRF-3 Gene

Objective To clone IRF-3 gene promoter and identify its core promoter region.Methods Approximately 1 kb of the 5' flanking sequence of IRF-3 was cloned from the genomic DNA of HEK 293 cells.After sequencing, the 5'flanking sequence was directly subcloned into pGL3-Basic vector and then the dual-luciferase reporter assay system was employed to identify its transcriptional activity in HEK 293 cells.In the deletion analysis of IRF-3 promoter, we prepared 5' truncations of 1 kb fragments and identified the core promoter essential for transcriptional activation.With point mutations, electrophoresis mobility shift assay (EMSA), chromatin immune precipitation (ChIP), small RNA interference and overexpression, we identified the location and features of the core promoter and its upstream regulatory region of IRF-3.Results In this study, the 5'flanking sequence of IRF-3 is consistent with the record in Genbank.In HEK 293 cells,the IRF-3 promoter exhibited high transcriptional activities.The transcriptional activity increased first and then decreased with 5' truncations.In HEK 293,-149 to -93 bp truncated fragment exhibited the maximal transcriptional activity.Sp1/3 and E2F sites were essential for maintaining the basal transcriptional activity of the human IRF-3 promoter. EMSA and ChIP assays demonstrate the occupancy of the IRF-3 promoter by Sp1,Sp3 and E2F1 in vitro or in vivo. Overexpression of Sp1 or Sp3 transactivated the human IRF-3 promoter, whereas small interfering RNA-mediated blockage of Sp1 or Sp3 gene expression inhibited markedly its activity. Overexpression of E2F1 protein reduced the transcription activity, while small interfering RNA-mediated blockage of E2F1 gene expression upregulated markedly its activity. Conclusion The -149 to -93 bp region in IRF-3 promoter was the core promoter region.Sp1,Sp3 and E2F1 played a critical role in regulating basal IRF-3 promoter activity in HEK 293 cells.