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D. Koleski, A.I. Jobling, M.J. Tymms; Analysis of Elf-3 Regulatory Regions in the Human TIMP-3 Promoter . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2283.
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Purpose: Accumulation of TIMP-3 in Bruchs membrane occurs in age-related macular degeneration (AMD). The absence of associated mutations in the TIMP-3 gene suggests that aberrant regulation of TIMP-3 transcription may underlie this process. Previous work has shown the human TIMP-3 promoter to be regulated by several transcription factors, including Elf-3. The present study sought to characterise the Elf-3 binding sites within the TIMP-3 promoter. Methods: The human TIMP-3 promoter was obtained by PCR. Wild type and mutant deletion constructs (ranging from 1.2kb to 200bp) were then made and linked to a luciferase reporter gene. The mutant TIMP-3 promoter constructs were generated from oligonucleotide primers containing point mutations in three specific Elf-3 binding sites (Elf-3bs). Using transient transfection assays, each construct was co-transfected with an Elf-3 expression construct into a human retinal pigment epithelial (hTERT/RPE) cell line. Regulation of TIMP-3 was assessed via changes in luciferase activity. Potential binding sites for Elf-3 were further probed using gel shift assays. Results: In hTERT/RPE cells, TIMP-3 upregulation by Elf-3 was greatest with construct -933/+276 (~8 fold), whilst the construct consisting of mutations in Elf-3bs 5 and 6 exhibited a 30% decrease in activity. A mutation in the first Elf-3bs increased activity, suggesting the upstream promoter region contained a repressor element. This is supported by data showing that removing this region increased basal level expression ~2.5-fold. Gel shift assays showed that Elf-3 binds to this region of the promoter and that mutations abolished this binding. Experiments also showed that removal of a downstream element (+69/+276) substantially decreased activity, suggesting the presence of an enhancer element. Conclusions: The present study demonstrates that the upstream region -933/-828, encompassing the first Elf-3bs, and a previously unrecognised element in the proximal promoter (+69/+276) are very important in the Elf-3 regulation of TIMP-3. This study provides insight into an alternative mechanism to explain elevated TIMP-3 expression in AMD.
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