PURPOSE. Mutations in the retinitis pigmentosa (RP) GTPase regulator (RPGR) gene account for more than 70% of X-linked RP cases. This study aims to characterize the proximal promoter region of the human RPGR gene. METHODS. The 50 -flanking region (5 kb) of human RPGR was cloned and sequenced. A potential transcription start site and transcription factor binding motifs were identified by bioinformatic analysis. Constructs containing the putative human RPGR promoter region upstream of a luciferase reporter gene were generated and analyzed by transient transfection and luciferase assays. Transgenic mouse lines carrying a 3-kb human RPGR promoter sequence fused to lacZ were generated and RPGR proximal promoter activity was analyzed by X-gal staining. RESULTS. Bioinformatic analyses of the human RPGR 50 -flanking region uncovered potential transcription factor binding sites and a CpG island. Transient transfection assays with RPGR promoter/luciferase reporter constructs revealed a 980-bp fragment (À952 to þ28) that produced higher levels of luciferase activity. Mutagenesis identified a putative Sp1 binding site that was critical for regulating transcriptional activity. We generated transgenic mice in which a lacZ reporter gene was controlled by the 3-kb upstream region of RPGR. bgalactosidase expression was predominantly found in mouse retina, brain, and kidney. In the retina, the photoreceptor cell layer showed the strongest b-galactosidase staining.CONCLUSIONS. Our study defined the human RPGR proximal promoter region in which a 3-kb fragment contained sufficient regulatory elements to control RPGR expression in mouse retina and other tissues. Characterization of the RPGR promoter will facilitate understanding of the functional role of RPGR in the retina and gene therapy of X-linked RP. (Invest Ophthalmol Vis Sci. 2012;53:3951-3958)
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