Functional Characterization of the HumanRPGRProximal Promoter

Abstract

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)

RPGR: Its role in photoreceptor physiology, human disease, and future therapies

“…The RPGR gene is located on the short arm of the X chromosome (Xp21.1) ( Meindl et al., 1996; Vervoort et al., 2000 ) and expresses at least 10 alternative transcripts of which 5 are predicted to be protein coding ( Kirschner et al., 1999; Roepman et al., 2000; Neidhardt et al., 2007; Schmid et al., 2010 ). Expression of the major splice variants (see below) is at least partly driven by a TATA-less proximal promoter ( Shu et al., 2012 ), which fits with the widespread expression of RPGR in adult mammalian tissues. The promoter contains 4 transcriptional start sites which may influence expression in different tissues and within which the transcription factor SP1 was shown to activate RPGR transcription.…”

Gene Augmentation for X-Linked Retinitis Pigmentosa Caused by Mutations in RPGR

“…These results have provided invaluable information about the viral constructs currently being considered. Indeed, there was initially concern that fine-tuning of the levels of RPGR transgene expression in photoreceptors may be required via supposedly less "active" promoters (e.g., the RGPR promoter [Shu et al 2012]) than the ones currently used. Yet, the potent hRPGR immunolabeling that was observed with hIRBP and hGRK1 promoters was not limited to the connecting cilium region of transduced canine rods and cones, thus suggesting that some degree of hRPGR ex1-ORF15 overexpression is tolerable and does not induce measurable cytotoxicity.…”

Advances in gene therapy technologies to treat retinitis pigmentosa

“…This was shown to be 10% stronger then the ubiquitous cytomegalovirus promoter, and was ineffective in older animals 17. More recently, RP guanosine triphosphatase (GTPase) regulator (RPGR) promoter region was characterized, and it may be useful in future RPE targeting 35. Photoreceptors have been successfully targeted by both rhodopsin and rhodopsin-kinase promoters, with substantial activity in mice, dogs, and nonhuman primates,30,36,37 and a cone arrestin promoter has been used more recently for cone dystrophy 38,39.…”