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M. Akimoto, L. Whitaker, J.F. Friedman, H. Khanna, E. Filippova, A.J. Mears, D.L. Turner, B.E. Knox, A. Swaroop; Distal conserved region is required for the rod photoreceptor specific expression of Nrl in both mouse and frog . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2251.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: NRL is required for the expression of rod–specific genes and is essential for rod photoreceptor differentiation. The goal of this study is to identify cis–regulatory elements and trans–acting factors that control the rod–specific expression of NRL in developing retina. Methods: We studied the regulation of the Nrl gene using transgenic mice with three different lengths of 5’ –flanking promoter sequences of mouse Nrl tagged to the GFP reporter gene. Similar constructs with human NRL promoter sequences were used for studies in transgenic Xenopus. DNaseI footprinting was carried out for all conserved sequences within the Nrl–5’–upstream region. Gelshift and supershift assays were performed using bovine retinal nuclear extracts. Co–transfection of candidate regulators, myocyte enhancer factor (Mef2C) and bHLH proteins, were performed in cultured cells using luciferase reporter assays. Results: Four highly conserved regions (I–IV) were identified between the 5’–upstream regions of the human and mouse Nrl genes. The distal conserved region IV contains Mef2 and E–box–like sites. Two different deletion mutant promoters of this region result in ectopic expression of GFP in both mouse and frog. Supershift assay revealed specific binding of Mef2C to the cis–element. Mef2C and Mash1 cooperatively transactivate Nrl expression, whereas regulation by NeuroD is mediated by the proximal conserved region I. Conclusions: Distal conserved region of the Nrl promoter is required for rod –specific expression. Mef2C and bHLH proteins play an important role in Nrl expression in early rod progenitors.
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