Construction And Identification Of The Promoter Of Murine IRF-3Gene

Objective: To isolate and identify murine Interferon regulatory factor3（mIRF-3）gene promoter and characterize its core promoter region．Methods: Promoter sequence nearby the transcription initiation site about1479bp(-1290~+189bp) of mIRF-3was amplified by PCR with mouse genomic DNA fromNIH3T3cells as a template. After sequencing, the PCR product was directlysubcloned into pGL3-Basic vector and then the dual-luciferase reporter assay systemwas employed to identify its transcriptional activity in NIH3T3cells．Using a seriesof5’deletion promoter plasmids in luciferase reporter assays, we identify the corepromoter essential for mIRF-3transcriptional activation. With point mutations, RNAinterference experiments and sequential chromatin immunoprecipitation (ChIP) assay,we identified the location and features of the core promoter and its upstreamregulatory region of mIRF-3.Results: The successful construction of the plasmid pmIRF-3was verified by DNAsequencing and restriction endonuelease analysis. The mIRF-3promoter exhibitedhigh transcriptional activities in NIH3T3cells. Transient transfection of a series of5’deletion of the the mIRF-3promoter plasmids and luciferase assay illustrated themaximal transcriptional activity of mIRF-3promoter was located within the region-301/-196relative to the TSS. Egr2and YY1sites were essential for maintaining thebasal transcriptional activity of the human IRF-3promoter. Mutational analysis, RNAinterference experiments demonstrated that transcription factors Egr2or YY1geneexpression inhibited markedly its activity. ChIP assays demonstrated the occupancyof the mIRF-3promoter by YY1in vitro. Conclusion: MIRF-3gene core promoter was located within the region-301/-196bprelative to the TSS. Egr2and YY1played a critical role in regulating basal mIRF-3promoter activity in NIH3T3cells.