Effect Of Transcription Factor E2F1 On CD2AP Transcriptional Regulation And Its Possible Mechanism

Objective: The reduction of CD2 associated protein (CD2AP) expression was found to be a reason of glomerulosclerosis. This study was to clone E2F1 and express it in HEK-293 cells, and then to investigate the effect of transcription factor E2F1 on CD2AP transcriptional regulation and its possible mechanism.Methods: E2F1 gene was amplified by RT-PCR with the total RNA of HEK293 cells as template. The PCR fragments were cloned into pcDNA3.1 (+) vector to construct recombinant eukaryotic expression plasmids. HEK293 cells were transfected with the recombinant plasmids. Western blot was performed to evaluate the expression of E2F1 in HEK293 cells. By semi-quantitative RT-PCR and Western blot analysis we demonstrated the effect of E2F1 on the expression of CD2AP. Then we tested the effect of overexpression, RNAi of E2F1 and point mutation on human CD2AP promoter activity by Dual-Luciferase reporter assays. Lastly we performed chromatin immunoprecipitation assay to identify whether E2F1 can bind to the human CD2AP promoter.Result: In this study, we successfully constructed a eukaryotic E2F1 expression plasmid. Overexpression of E2F1 upregulated the promoter activity, mRNA and protein levels of the human CD2AP in HEK293 cells by transient transfection, luciferase activity assay, semi-quantitative RT-PCR and Western blot analysis. Knock-down of endogenous E2F1 by a shRNA strategy resulted in reduction of the CD2AP promoter activity. Chromatin Immunoprecipitation (ChIP) assay revealed that transcription factor E2F1 was capable of binding to the CD2AP promoter in vivo. Another ChIP assay indicated that overexpression of E2F1 was associated with decreasing the binding of Sp1 and increasing the binding of E2F1 to Sp1 binding sites, whereas the protein level of Sp1 remained unaffected. Lastly site-directed mutagenesis confirmed that E2F1 upregulated CD2AP promoter activity by binding to Sp1C site.Conclusin: These results indicated that E2F1 can upregulate the mRNA and protein levels of the human CD2AP in HEK293 cells, by the activation of the human CD2AP promoter via binding to the Sp1C binding site.