Biochemistry and Molecular Biology Functional Characterization of the Human RPGR Proximal Promoter Xinhua Shu,*,1,2 Julie R. Simpson,2 Alan W. Hart,2 Zhihong Zeng,3 Sarita Rani Patnaik,1 Philippe Gautier,2 Emma Murdoch,2 Brian Tulloch,2 and Alan F. Wright*,2 PURPOSE. Mutations in the retinitis pigmentosa (RP) GTPase promoter will facilitate understanding of the functional role regulator (RPGR) gene account for more than 70% of X-linked of RPGR in the retina and gene therapy of X-linked RP. (Invest RP cases. This study aims to characterize the proximal Ophthalmol Vis Sci. 2012;53:3951–3958) DOI:10.1167/ promoter region of the human RPGR gene. iovs.11-8811 0 METHODS. The 5 -flanking region (5 kb) of human RPGR was cloned and sequenced. A potential transcription start site and etinitis pigmentosa (RP) is a genetically heterogeneous transcription factor binding motifs were identified by bio- Rgroup of retinal degenerations that affect 1 in 4000 in the informatic analysis. Constructs containing the putative human general population.1,2 Most cases are inherited in an autosomal RPGR promoter region upstream of a luciferase reporter gene dominant, autosomal recessive, X-linked, or mitochondrial were generated and analyzed by transient transfection and manner, but oligogenic inheritance has been established in a luciferase assays. Transgenic mouse lines carrying a 3-kb small proportion of families.3 X-linked RP (XLRP) is one of the human RPGR promoter sequence fused to lacZ were generated most consistently severe forms of RP, with a reported average and RPGR proximal promoter activity was analyzed by X-gal age at onset of 7.2 6 1.7 years.4 XLRP affects 10% to 20% of all staining. RP patients5 and has been genetically mapped to six loci: RP2, 0 RESULTS. Bioinformatic analyses of the human RPGR 5 -flanking RP3, RP6, RP23, RP24, RP34 (http://www.sph.uth.tmc.edu/ region uncovered potential transcription factor binding sites retnet/). The RPGR gene is mutated in the RP3 form of XLRP, and a CpG island. Transient transfection assays with RPGR which accounts for 70% to 80% of affected families.5–8 promoter/luciferase reporter constructs revealed a 980-bp The human RPGR gene is located in chromosomal region fragment (À952 to þ28) that produced higher levels of Xp21.1 and spans 172 kilobases.8 There are multiple alterna- luciferase activity. Mutagenesis identified a putative Sp1 tively spliced transcripts, all of which encode an amino (N)- binding site that was critical for regulating transcriptional terminal RCC1-like (RCCL) domain, which is structurally activity. We generated transgenic mice in which a lacZ reporter similar to the RCC1 protein, a guanine nucleotide exchange gene was controlled by the 3-kb upstream region of RPGR. b- factor for the small guanosine triphosphate–binding protein, 9 galactosidase expression was predominantly found in mouse Ran. The RPGR gene that was initially identified contained 19 ex1–19 6,7 retina, brain, and kidney. In the retina, the photoreceptor cell exons (RPGR ), encoding a predicted 90-kDa protein. layer showed the strongest b-galactosidase staining. Exons 2 to 11 encode the RCCL domain, whereas exons 12 to 19 encode a carboxyl (C)-terminal domain rich in acidic CONCLUSIONS. Our study defined the human RPGR proximal residues and ending in an isoprenylation anchorage signal.6,10 promoter region in which a 3-kb fragment contained sufficient Mutations found in RPGRex1–19 account for only 15% to 20% of regulatory elements to control RPGR expression in mouse XLRP patients and subsequent studies revealed many more retina and other tissues. Characterization of the RPGR disease-causing mutations within one or more transcripts containing an alternatively spliced C-terminal exon called ORF15 (RPGRORF15).8 Exon ORF15 encodes a repetitive From the 1Department of Life Sciences, Glasgow Caledonian glycine and glutamic acid–rich domain of unknown function University, Glasgow, United Kingdom; 2MRC Human Genetics Unit, and contains a conserved basic C-terminal domain. The ORF15 Institute of Genetics and Molecular Medicine, Edinburgh, United exon harbors a high frequency of microdeletions, frameshift, Kingdom; and 3Genome Damage and Stability Centre, University of and premature stop mutations.8 In total, 296 RPGR mutations Sussex, Brighton, United Kingdom. have been identified to date, which can give rise to both Supported by RP Fighting Blindness and the Medical Research central and peripheral retinal dystrophies, including X-linked Council(UK)(AFW),theRoyalSocietyofLondon,TENOVUS Scotland,NationalEyeResearchCentre,VisualResearchTrust,the forms of RP (95% of subjects); human cone-rod, cone, and W.H. Ross Foundation, the Rosetrees Trust, the Carnegie Trust for macular dystrophies (3% of subjects); or syndromal forms of the Universities Scotland, and the Nuffield Foundation (XS). XLRP with hearing loss and primary ciliary dyskinesia (2% of Submitted for publication October 17, 2011; revised April 25, subjects).11,12 2012; accepted April 25, 2012. RPGR interacts with a number of photoreceptor and ciliary Disclosure: X. Shu,None;J.R. Simpson,None;A.W. Hart, proteins. The RCCL domain was shown to interact with the None; Z. Zeng,None;S.R. Patnaik,None;P. Gautier,None;E. delta subunit of the rod cyclic GMP phosphodiesterase Murdoch,None;B. Tulloch,None;A.F. Wright,None (PDE6D), a highly conserved protein capable of binding *Each of the following is a corresponding author: Alan F. Wright, several prenylated proteins, including Rab13, Ras, Rap, and MRC Human Genetics Unit, Institute of Genetics and Molecular 13 Medicine, Edinburgh EH4 2XU, United Kingdom; Rho6. The RCCL domain also interacts with the RPGR- [email protected]. interacting protein 1 (RPGRIP1), which, like RPGR, is localized 14–16 Xinhua Shu, Department of Life Sciences, Glasgow Caledonian to the photoreceptor connecting cilium. Mutations in University, 70 Cowcaddens Road, Glasgow G4 0BA, United RPGRIP1 cause a severe early-onset form of retinal degener- Kingdom; [email protected]. ation, Leber’s congenital amaurosis.17,18 The exact function of Investigative Ophthalmology & Visual Science, June 2012, Vol. 53, No. 7 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. 3951 Downloaded from iovs.arvojournals.org on 09/28/2021 3952 Shu et al. IOVS, June 2012, Vol. 53, No. 7 RPGRIP1 is unknown, but it is necessary for photoreceptor disc formation and morphogenesis.19 The disease course in the Rpgrip1 knockout (KO) mouse was more severe than that seen in mice lacking RPGR,19,20 which is consistent with the differential severity of these two disorders in humans. RPGR was absent from the connecting cilia in Rpgrip1 KO mice, suggesting that Rpgrip1 is necessary for the proper localization of RPGR.19 The C-terminal domain of the RPGRORF15 isoform was found to interact with nucleophosmin,21 a chaperone involved in many cellular processes, including centrosome duplication, folding of denatured proteins and histone chaper- oning. Co-immunoprecipitation studies showed that RPGR interacts with several basal body/axonemal proteins (CEP290/ NPHP6, NPHP5, IFT88, gamma-tubulin, 14-3-3 epsilon, RPGRIP1L) and microtubule transport proteins (kinesin II– related proteins KIF3A and KAP3, dynein heavy and interme- diate chains, dynactin subunits p150-Glued, and p50-dynami- tin), supporting a role for RPGR in microtubular organization and transport between photoreceptor inner and outer segments.22–24 RPGR is widely expressed and shows a complex expression pattern. At the mRNA level, RPGR transcripts were detected in different tissues, including brain, eye, kidney, lung, and testis in several different species.25–29 At the protein level, RPGR has 0 been detected in retina, trachea, brain, and testis. In human, FIGURE 1. (A) Schematic representation of the 5 region of the human mouse, and bovine retina, RPGR mainly localizes to photore- RPGR gene investigated using reporter constructs generated in the ceptor connecting cilia,29 but expression has also been pGL-3 vector. (B) The corresponding activities of the luciferase reported in outer segments in some species.30 RPGR is reporter gene in RPE1 and HEK 293T cell lines. The promoter-less expressed in the transitional zone of motile cilia and within plasmid pGL3-Basic was used as a negative control and the pGL3- 29,31 Control plasmid containing an SV40 promoter and enhancer was used human and monkey cochlea. Overexpression of mouse as a positive control. The Renilla luciferase plasmid was used as an RPGR results in male infertility because of defects in flagellar internal control for the normalization of transfection efficiency. The formation.32 The severity of the flagellar defect is correlated activities of the reporter gene are expressed as -fold change relative to with increased RPGR copy number, suggesting that RPGR the activity of pGL3-Basic (an activity value of 1.0). expression is tightly controlled. Our study aimed to increase our understanding of the regulation of human RPGR expres- primer, as shown in Supplementary Table S1 (see Supplementary sion and to provide appropriate expression in therapeutic Material, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs. strategies for treating patients with RPGR mutations. 11-8811/-/DCSupplemental). The five PCR fragments: 5098 bp (À5070 to þ28 bp), 3093 bp (À3065 to þ28 bp), 2005 bp (À1977 to þ28 bp), 1508 bp (À1480 to þ28 bp), and 980 bp (À952 to þ28 bp) MATERIALS AND METHODS were ligated into the pGEM-T Easy vector (Promega, Southampton, Bioinformatic Analysis of Human RPGR Promoter UK), sequenced, and then subcloned into the pGL3 basic vector (Fig. 2A). An additional 222-bp (À268 to À47 bp) fragment containing two A 5098 nucleotide bp sequence upstream from human RPGR exon 1 putative Sp1 binding sites was cloned into the pGEM-T Easy vector was analyzed using Gene2promoter software (Genomatix, Munich, using RPGR222bp For and RPGR222bp Rev primers (see Supplemen- Germany), which provides access to promoter sequences of all genes tary Table S1, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.