Sp1, IRF1and IRF2Regulate The Expression Of Apolipoprotein L1

Objective The aim of this project was to further investigate the transcriptionalregulation mechanism that Sp1, IRF1and IRF2regulate the expression ofapolipoprotein L1.Methods Interferon regulation factor2was cloned into pFLAG-CMV-2, whichcalled pFLAG-IRF2, the authenticity of the construct was confirmed by sequencing.Other plasimics were obtained from biotechnology company, which were respectlynamed pFLAG-Sp1, pFLAG-Sp3, pFLAG-KLF4, pFLAG-KLF6, pFLAG-IRF1,pFLAG-IRF3, pFLAG-IRF9. After the high concentration of all those expressionplasmics were obtained, cells were cotransfected with promoter and those expressionplasmids.48h post-transfection, cell extracts were prepared and analyzed for therelative luciferase activity by a dual-light reporter gene assay. Cotransfection withSp1, IRF1or IRF2expressing plasmids lead to remarkable increase of luciferaceactivity. So Sp1, IRF1or IRF2may play an important role in the transcriptionalactivity of apolipoproteinL1. These results were further comfirmed throughelectrophoretic mobility shift assay. However the supershift band of IRF1didn’tappear in the first time of EMSA, due to the low expression of IRF1in cells. SinceIFN-γis the major activator of IRF1gene expression, cells were treated with500ng/ml IFN-γfor48h. Nuclear protein was extracted and the increase of IRF1gene expression was confirmed by western blot. EMSA was performed again for IRFsite, the supershift band of IRF1appeared successfully.Results1. IRF-2expression plasmid was successfully constructed.2. ApolipoproteinL1promoter activities can be enhanced by overexpression of Sp1, IRF1and IRF2.3.IFN-γcan enhance the expression of IRF1, and have no influence on IRF2.Conclusions1. ApoL1gene is regulated by Sp1, IRF1and IRF2transcription factors.2. IFN-γcan enhance the expression of IRF1, and have no influence on IRF2.