title: Functional characterization of the human RPGR proximal promoter.
abstract: OBJECTIVE
  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 5'-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 5'-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 (-952 to +28) 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. β-galactosidase expression was predominantly found in mouse retina, brain, and kidney. In the retina, the photoreceptor cell layer showed the strongest β-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.
authors.affiliation: Department of Life Sciences, Glasgow Caledonian University, Glasgow, United Kingdom. xinhua.shu@gcu.ac.uk
authors.forename: Xinhua
authors.initials: X
authors.lastname: Shu
authors.forename: Julie R
authors.initials: JR
authors.lastname: Simpson
authors.forename: Alan W
authors.initials: AW
authors.lastname: Hart
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zeng
authors.forename: Sarita Rani
authors.initials: SR
authors.lastname: Patnaik
authors.forename: Philippe
authors.initials: P
authors.lastname: Gautier
authors.forename: Emma
authors.initials: E
authors.lastname: Murdoch
authors.forename: Brian
authors.initials: B
authors.lastname: Tulloch
authors.forename: Alan F
authors.initials: AF
authors.lastname: Wright
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D005136:Eye Proteins; D020662:Guanine Nucleotide Exchange Factors; C099929:RPGR protein, human; D014157:Transcription Factors