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ST Gee, J Tombran-Tink, J-X Ma; Identification of a putative enhancer region for Pigment Epithelium-Derived Factor (PEDF) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):689.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Identify the enhancer region and transcription factor(s)(TFs) responsible for transcription of the antiangiogenic and neurotrophic factor, PEDF. Methods: The PEDF promoter region was amplified by PCR using DNA from a cosmid clone, which contained the full genomic PEDF gene, as a template. The PCR product was cloned into the pCRII vector and subcloned into the pGL3 basic vector upstream of a luciferase gene. After sequence verification, the PEDF promoter clone and a beta-galactosidase clone were co-transfected into D407, a human retinal pigment epithelium (RPE) cell line, cells using a common lipid reagent. Twenty-four hours after transfection, the cells were lysed and assayed for luciferase and beta-galactosidase activity. Upon observing luciferase activity higher than controls, 5' deletions of the PEDF promoter clone were constructed as described above and assayed in a similar manner. Furthermore, nuclear extracts were collected from D407 cells and used in gel shift analyses. Results: A PEDF promoter clone exhibited strong promoter activity in D407 cells. Four 5' deletions of this promoter sequence revealed the deletion of a 336bp region, which exhibited a 50% reduction in promoter activity, suggesting a potential enhancer in this region. Gel-shift analyses demonstrated specific binding of D407 nuclear proteins to a portion of this 336bp region. Conclusion: A 5' flanking region of the PEDF gene contains sufficient promoters and enhancers to exert a high level of promoter activity in a human RPE cell line. A putative enhancer has been identified, although further analysis will confirm its enhancer ability. Future footprinting analyses of the enhancer region will identify TFs responsible for the transcription of the PEDF mRNA. Dysregulation of PEDF appears to be involved in ophthalmic diseases such as diabetic retinopathy and retinitis pigmentosa. Identification of the enhancer and TFs responsible for PEDF transcription will aid in determining if PEDF dysregulation occurs at the transcriptional level.
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