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R. E. Carpio, S. Schimpf, E. Zrenner, B. Wissinger; Characterization of the Human CNGA3 Gene Promoter. Invest. Ophthalmol. Vis. Sci. 2009;50(13):480.
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The cone photoreceptor cyclic nucleotide-gated (CNG) channel is a heterotetramer composed of two alpha and two beta subunits and plays a fundamental role in phototransduction. Achromatopsia is an inherited autosomal recessive retinal disorder characterized by a lack of cone function and frequently caused by mutations in the CNGA3. We previously identified a minimal promoter region of the CNGA3 gene located upstream of exon 1, therefore examination of transcription factors binding to the CNGA3 promoter may provide insights into the mechanisms of cone specific gene expression and reveal important sequences that should be included in mutation screening studies.
5’ RACE experiments were performed with human and mouse retinal mRNA. Interspecies sequence comparison and predictions of putative transcription factor binding sites were done to identify potential regulatory candidates. Luciferase reporter gene constructs containing the putative human CNGA3 promoter were transfected into HEK 293, retinoblastoma Y79, WERI-RB-1 cells and 661W cells and promoter activity assayed by luminometry. In addition, deletions via mutagenesis of the putative transcription factor binding sites identified potential regulatory regions. Ex-vivo mouse retina electroporation served to identify retina expression of the minimal promoter.
5’ RACE analysis revealed the presence of two transcription start sites in CNGA3 gene in human retinal tissue. Reporter gene assays identified retina transcription factor specificity within the promoter located upstream of exon 1 that yielded significant reporter activity compared with other non retinal transcription factors. The highest CNGA3 expression was observed upon transfection of the cone photoreceptor derived 661W mouse cell line.
The results presented here verify the location of the basic CNGA3 gene promoter immediately upstream of exon1 and identify potential transcription factors binding to this promoter with most significant activity in the cone photoreceptor, derived, mouse 661W cell line.
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