Characterization of Receptor Protein Tyrosine Phosphatase Epsilon (PTPRE) Gene Promoter
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BACKGROUND: Receptor protein tyrosine phosphatase epsilon (PTPRE) is a receptor bound phosphatase that has been shown to be downregulated in Wilms' tumors compared to normal tissue, and could potentially be a target for future therapy. Our objective is to identify and characterize the promoter of the PTPRE gene and define the critical role of Wt1 transcription factor (commonly downregulated in Wilm's tumor) in PTPRE gene expression and in Wilms' tumor progression. METHODS: Our first step involved cloning and sequence analysis of the upstream region of the human PTPRE gene followed by PCR primer design and PCR amplification. The amplified fragment was then cloned into a promoterless reporter vector (pGl3 Basic) and transfected in Hek293 cells. Promoter DNA was used for deletion analysis where multiple PCRs were performed using a single forward primer and multiple reverse primers with nucleotides sequentially deleted from the 3' end. The different size PCR products were then cloned into pGL3 Basic vector DNA, transfected into HEK cells and had reporter assay (luciferase assay) performed to calculate fold change in PTPRE expression over promoterless vector. After the critical transcription factor binding motif was identified, PCR was performed to amplify full length promoter lacking 76 bases. The role of the deleted nucleotides was confirmed via luciferase assay. The sequence of deleted nucleotides was then analyzed for the presence of transcription factor binding motifs. Next, a predicted Wt1 transcription factor binding motif was mutated using site directed mutagenesis. Mutated fragment was cloned into pGl3Basic vector. Both wild type and mutated vector were transfected and luciferase assay was performed to confirm role of Wt1 binding motif for PTPRE promoter activity. Chromatin immunoprecipitation assay was then performed for further evidence. Promoter activity was also compared in two cells lines having differential expression of Wt1. Western blot and semi-quantitative PCR are used to confirm the expression levels of Wt1. RESULTS: Promoter deletion analysis confirmed that the Wt1 binding motif present at -16 position is critical for PTPRE expression and mutation of this site results in 95% loss in promoter activity in Hek293 cells. PTPRE promoter activity was shown to be high in Hek293 cells and low in Hela cells (high and low WT1 expression respectively), suggesting WT1 driving promoter activity. ChiP using WT1 antibody confirmed WT1 binding of the critical transcription factor binding motif. CONCLUSION: These results shed light on why PTPRE expression is lower in Wilms' tumors and reveals potential future targeted therapy.