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ES Tasheva; Transcriptional Regulation of the Human Mimecan Gene . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3194.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mimecan is a small leucine-rich proteoglycan that may play an important role in the regulation of cellular growth as illustrated by ability of growth factors and cytokines to modulate its expression and by recent demonstration that bovine mimecan is transcriptionally activated by p53 through a conserved intronic recognition site. The purpose of this study was to clone and functionally characterize the human mimecan promoter region and identify the cis-elements and transcription factors that play a role in the regulation of mimecan expression. Methods: The 3.5kb fragment of the human mimecan gene containing the first intron and 2.5kb of the 5'-flanking region was obtained by PCR amplification of human genomic DNA. The transcription initiation sites were determined by RACE and S1 protection analyses. Luciferase reporter gene transfections, DNase1 Footprinting and EMSA were used to determine the cis-elements and transcription factors that regulate the human mimecan expression. Results: Within a 296bp upstream region required for basal gene expression there are three Inr elements, an E-box and Oct-1, MRE, and NF-kB recognition sites. USF-1, Oct-1 and MRE-binding proteins were identified as proteins that bind to these regulatory elements and modulate transcription of mimecan in both bovine corneal keratocytes and human osteosarcoma MG-63 cells. The first intron of human mimecan contains enhancer and silencer elements. Reporter gene transfections demonstrated that cooperation of upstream region and intronic enhancer elements are required for maximal gene expression in both p53-deficient and wild-type p53-expressing cells. Within the footprinted intronic enhancer region an ISRE is present. Using EMSA, IRF-1 was identified as a protein that binds to this region in MG-63 but not in U-937 cells. In vitro translated IRF-1 also was shown to bind to this ISRE. Conclusion: Our results indicate that binding of USF-1 to the E-box located 50 bp upstream of transcription initiation site is sufficient for minimal expression of the human mimecan gene in cultured corneal keratocytes. IRF-1 may play a role in the constitutive expression of human mimecan gene in MG-63 cells.
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