Interplay Between Purα And Egr-1in The Transcriptional Regulation Of Amyloid Precursor Protein Gene Expression

Objective: Amyloid precursor protein（APP）is a single transmembrane protein,and itis the precursor of A-β when APP is abnormally metabolized and it also played an importantrole in the genesis of Alzheimer disease. Our previously published data demonstrated that thePurα negatively regulated the expression of APP gene but Egr-1could up-regulated the APPgene expression. Purα has five functional DNA binding domains,so far it is not very clearwhich domain played the putative function on the regulation of APP gene expression and howit interacted with Eg-1protein in the APP gene expression. This work focused on thestructural study of the Purα proteins with the different deletions of Purα protein in order toverify the functional domain of the Purα proteins and confirm the regulation mechanisms bywhich both Purα and Egr-1interplay in the regulation of APP gene expression.Method:(1) Primers were designed according to the Purα and Egr-1gene sequence.We constructed pCDNA3-Purα mutants (deletions) and pCDNA3-Egr-1plasmid andpGEX4T1-Purα mutants and pGEX4T1-Egr-1plasmids.(2)We transfected the Purα mutantsand APP promoter into U87cell to verify the function of different deletions on APP geneexpression and to confirm the main functional domain of Purα protein by luciferase assay.(3)The pull-down assay and Co-Immunoprecipitation to detect the physical interaction of Purαwith Egr-1protein.Results:(1) Double endonuclease digestions and sequencing proved that recombinantplasmid pCDNA3-Purα mutants, pCDNA3-Egr-1plasmid, pGEX4T1-Purα mutants and pGEX4T1-Egr-1plasmid were successfully constructed;(2) Luciferase assay confirmed thatPurα full-length APP has largest down regulative effect on the APP gene expression, the otherdeletions had different down regulative effects on APP gene expression;(3) Pull-down assayand Cp-Immunoprecipitation did not find the physical interaction between the Purα and Egr-1proteins.Conclusion: The effects of Purα on the regulation of APP gene expression is dependenton the structural intact and all the five domains worked together to play the largest regulativeeffect, Purα protein and Egr-1proteins did not have physical interactions. The functionalinteraction could be a competitive binding to the APP promoter since both binding sites onAPP promoter were overlapped, this results confirmed our previous hypothesis that there isdisplacement mechanism for those two protein in the regulation of APP gene expression.