The upstream region of the Rpe65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites.
RPE65 is essential for all-trans- to 11-cis-retinoid isomerization, the hallmark reaction of the retinal pigment epithelium (RPE). Here, we identify regulatory elements in the Rpe65 gene and demonstrate their functional relevance to Rpe65 gene expression. We show that the 5' flanking region of the mouse Rpe65 gene, like the human gene, lacks a canonical TATA box and consensus GC and CAAT boxes. The mouse and human genes do share several cis-acting elements, including an octamer, a nuclear factor one (NFI) site, and two E-box sites, suggesting a conserved mode of regulation. A mouse Rpe65 promoter/beta-galactosidase transgene containing bases -655 to +52 (TR4) of the mouse 5' flanking region was sufficient to direct high RPE-specific expression in transgenic mice, whereas shorter fragments (-297 to +52 or -188 to +52) generated only background activity. Furthermore, transient transfection of analogous TR4/luciferase constructs also directed high reporter activity in the human RPE cell line D407 but weak activity in the non-RPE cell lines HeLa, HepG2, and HS27. Functional binding of potential transcription factors to the octamer sequence, AP-4, and NFI sites was demonstrated by directed mutagenesis, electrophoretic mobility shift assay, and cross-linking. Mutations of these sites abolished binding and corresponding transcriptional activity and indicated that octamer and E-box transcription factors synergistically regulate the RPE65 promoter function. Thus, we have identified the regulatory region in the Rpe65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the Rpe65 gene.
Boulanger
A
A
Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Liu
S
S
Henningsgaard
A A
AA
Yu
S
S
Redmond
T M
TM
D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005136:Eye Proteins; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes; D051840:Y-Box-Binding Protein 1; D001616:beta-Galactosidase / Q000235:genetics; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D020121:5' Untranslated Regions; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D008156:Luciferases; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; D001616:beta-Galactosidase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C070856:YBX1 protein, human; C055473:enhancer-binding protein AP-4