Mechanism Research On Human IRF-5 Gene Transcriptional Regulation

Objective:To analyse the different expression of interferon regulatory factor 5 (IRF-5), IFN-α and Spl in childhood-onset systemic lupus erythematosus (SLE) and healthy controls. To clone and analyse the promoter of human IRF-5 gene and to find the core promoter areas. To identify the key transcription factor which regulates IRF-5. To detect the impacts of histone deacetylase inhibitors (HDACi) and histone acetyltransferase (HAT) on IRF-5 transcription, and define the related mechanism.Methods:Peripheral blood mononuclear cells were extracted from 12 children with childhood-onset SLE and healthy controls. The different expression of IRF-5, IFN-a and Sp1 was detected by real time fluorescence quantitative PCR. The core promoter area was identified using a series of 5’deletion promoter plasmids in luciferase reporter assays. Prediction of potential transcription factor binding sites in IRF-5 core promoter was using TFSEARCH online software and JASPAR database. Transcription factors regulating IRF-5 gene were identified by mutational analysis, RNA interference, overexpression experiments, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecitation assay (ChIP). The IRF-5 promoter activity, mRNA and protein expression levels were observed by using two kinds of HDACi-trichostatin A (TSA) and valproic acid (VPA) as well as two kinds of HAT-P300 and PCAF. The production of IFN-a and TNF-α in supernatant fluid in TSA and VPA treated THP-1 cells was measured by ELISA. Meanwhile, the DNA binding of Spl, Pol II, HDAC3, P300 and PCAF to IRF-5 promoter was detected by ChIP-qPCR.Results:1. The expression of IRF-5, IFN-a and Spl increased 55%,61% and 85% respectively in childhood-onset SLE compared with healthy controls. Moreover, the expression of Spl and IFN-α was positive correlated with IRF-5 (P<0.05).2. Luciferase assays of series 5’truncated reporter plasmids revealed that the-179bp truncated plasmids still exhibited high luciferase activity. Further 5’deletion from-179bp to -40bp, this construct promoter activity declined significantly in all three cell lines, these results indicated that the core promoter area of IRF-5 was located within the region-179/+62 relative to the TSS.3. Bioinformatics identified that the core promoter contained three Spl binding sites. Mutational analysis, RNA interference, overexpression experiments, EMSA, ChIP and real time quantitative PCR assays revealed that Spl could be combined with IRF-5 promoter and regulate its expression.4. TSA and VPA reduced IRF-5 expression and inhibited the production of IFN-α and TNF-α in supernatant fluid in induced THP-1 cells. P300 also reduced IRF-5 expression. However, PCAF had no effect on IRF-5 expression. ChIP-qPCR assays revealed TSA and VPA inhibited DNA binding of Spl, Pol Ⅱ, HDAC3 and P300 to the core promoter regions of IRF-5 gene and did not inhibit DNA binding of PCAF to the core promoter region.Conclusions:1. The expression of IRF-5, IFN-a and Spl was higher in childhood-onset SLE compared with healthy controls. The expression of Spl and IFN-α was positive correlated with IRF-5. This revealed that IRF-5 might be involved in the pathogenesis of childhood-onset SLE.2. The core promoter of IRF-5 gene was located between-179bp and+62bp, and Sp1 might be involved in IRF-5 transcriptional regulation. This would provide the target for treating IRF-5 related diseases.3. HDACi-TSA and VPA could reduce IRF-5 expression and inhibit the production of IFN-α and TNF-α. HAT-P300 could also reduce IRF-5 expression but PCAF had no effect on IRF-5 expression. This study would provide theoretical foundation for HDACi in the treatment of autoimmune diseases such as SLE.