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N.W. Keiser, A.M. Maguire, J. Bennett; Expression and Regulation of the Choroideremia Gene, Rep-1 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1537.
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Purpose: Choroideremia is a rare X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP-1), which is involved in intracellular vesicle trafficking. To attempt to better understand the molecular basis behind the manifestation of choideremia, we investigated both the location of REP-1 expression in the retina and the regulation of the gene by analyzing regions of the REP-1 promoter. Methods: To determine the location of REP-1 transcripts, we performed in situ hybridization on cryosections of mouse and monkey retina. The locations of the REP-1 and REP-2 proteins in the mouse retina were determined by immunohistochemistry. A 2.8 kb region of the human REP-1 promoter was cloned by PCR, and nested deletions were made starting from the 5' end of the fragment, allowing for the activity of each promoter region to be determined by luciferase assay. A highly active region of the promoter was cloned into an AAV2/5 vector expressing EGFP, which was injected subretinally into mice, allowing for the activity of this promoter fragment to be determined in vivo. A region of the REP-1 promoter containing a tandem GC-box was analyzed by electrophoretic mobility shift assay (EMSA) and supershift assay. Results: REP-1 mRNA transcripts localized to the photoreceptor inner segments, RPE, and choroid in the monkey and mouse. The REP-1 protein was found in the photoreceptor outer segments, the RPE, and the choroid; the related protein REP-2 localized to the outer segments only. The 2.8-kb upstream region of the human REP-1 gene provided both promoter and enhancer activity in 293 and ARPE-19 cells in vitro, and a highly active region of this promoter was sufficient to provide EGFP expression in vivo. Analysis of a tandem GC-box in this region revealed that it likely binds the Sp1 transcription factor. Conclusions: REP-1 mRNA and protein localizes to distinct cell types in the retina, and the REP-1 protein localization pattern differs from that of REP-2. These proteins have overlapping function, so this difference in expression patterns may play a role in the manifestation of choroideremia. The upstream region of the REP-1 gene provides promoter and enhancer activity in vitro and in vivo, most likely through general transcription factors such as Sp1.
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