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D.N. Magana-Arachchi, P. Sharma, N. Fatma, D.P. Singh, T. Shinohara; Identification of Regulatory Elements in the Promoter of LEDGF . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4498.
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Purpose: LEDGF enhances survival of a wide range of cell types under stresses. It is a transcriptional factor, and it binds to the heat shock element (HSE) and stress related element (STRE) to activate stress-associated genes. LEDGF is a central regulatory factor in the stress tolerance in lens epithelial cells. In this study, we investigated regulatory elements of the LEDGF promoter to facilitate elucidation of the molecular mechanisms that influence its expression. Methods: Deletion mutant in the 5’-DNA were generated to ascertain the role of individual elements. Promoter-CAT-constructs were prepared and a transfection system was used to assay CAT value. Site-directed-mutagenesis, gel-shift assay, northern and protein blot analyses were also used. In vivo transcription system of LECs was used for the promoter activity. Results: The 5’-flanking region of the LEDGF promoter contained potential regulatory elements: AP1, HSEs, STREs, SRE, E2F1, IRF-2, IRF-1, GRE, VDR/RXR, NF-6B, SP1/GC box, TGF-ß inhibitory site (TIE), STAT, etc. Cells transfected with the construct containing VDR/RXR and TIE showed diminished CAT activity with vitamin D and TGF-ß treatment, respectively. The construct with STE and STRE showed elevated CAT expression following stress, suggesting that the ledgf gene might be auto-regulated. Cells containing the construct between -1285 and +35 showed modulation in CAT activity with TNF-α, IFN-γ and TGF-ß treatment. Conclusions: These results clearly defined that LEDGF promoter can be regulated by various factors and its regulation appears to be physiologically important and may provide new insights to molecular mechanisms regulating the ledgf gene.
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