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 mouseRpe65 promoter/β-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 theRpe65 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 theRpe65 gene. 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 mouseRpe65 promoter/β-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 theRpe65 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 theRpe65 gene. retinal pigment epithelium nuclear factor one cellular 11-cis-retinaldehyde-binding protein polymerase chain reaction base pair(s) 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside β-galactosidase electrophoretic mobility shift assay CCAAT-box binding transcription factor/nuclear factor one helix-loop-helix All-trans- to 11-cis-isomerization of vitamin A is an obligate and tissue-specific enzymatic step in the renewal of 11-cis-retinal, the universal chromophore of rhodopsin and other visual pigment proteins, in the visual cycle (1Saari J.C. Invest. Ophthalmol. Visual Sci. 2000; 41: 337-348PubMed Google Scholar) of the retinal pigment epithelium (RPE).1 Several components, including 11-cis-retinol dehydrogenase (2Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), cellular 11-cis-retinaldehyde-binding protein (CRALBP) (3Saari J.C. Bredberg D.L. Exp. Eye Res. 1988; 46: 569-578Crossref PubMed Scopus (32) Google Scholar, 4Saari J.C. Bredberg D.L. Noy N. Biochemistry. 1994; 33: 3106-3112Crossref PubMed Scopus (80) Google Scholar) and lecithin:retinol acyltransferase (5Saari J.C. Bredberg D.L. J. Biol. Chem. 1988; 263: 8084-8090Abstract Full Text PDF PubMed Google Scholar, 6Ruiz A. Winston A. Lim Y.H. Gilbert B.A. Rando R.R. Bok D. J. Biol. Chem. 1999; 274: 3834-3841Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), all essential to the visual cycle activity, are found highly expressed, but not exclusively expressed, in the RPE. However, the retinol isomerase activity (7Bernstein P.S. Rando R.R. Biochemistry. 1986; 25: 6473-6478Crossref PubMed Scopus (54) Google Scholar, 8Deigner P.S. Law W.C. Canada F.J. Rando R.R. Science. 1989; 244: 968-971Crossref PubMed Scopus (155) Google Scholar, 9Winston A. Rando R.R. Biochemistry. 1998; 37: 2044-2050Crossref PubMed Scopus (80) Google Scholar), central to 11-cis-chromophore synthesis, is expected, mechanistically, to be highly tissue-specific. A tissue-specific component of the RPE, RPE65 (10Hamel C.P. Tsilou E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Biol. Chem. 1993; 268: 15751-15757Abstract Full Text PDF PubMed Google Scholar, 11Hamel C.P. Tsilou E. Harris E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Neurosci. Res. 1993; 34: 414-425Crossref PubMed Scopus (104) Google Scholar, 12Hamel C.P. Jenkins N.A. Gilbert D.J. Copeland N.G. Redmond T.M. Genomics. 1994; 20: 509-512Crossref PubMed Scopus (56) Google Scholar), which copurifies with 11-cis-retinol dehydrogenase (2Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), appears to play a crucial role in retinoid isomerization. Thus, in the Rpe65-deficient mouse (13Redmond T.M., Yu, S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (779) Google Scholar), rod photoreceptor function is abolished due to lack of the 11-cis-retinal chromophore. Furthermore, mutations in the human RPE65 gene cause several forms of severe early onset blindness (14Morimura H. Fishman G.A. Grover S.A. Fulton A.B. Berson E.L. Dryja T.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3088-3093Crossref PubMed Scopus (393) Google Scholar, 15Marlhens F. Bareil C. Griffoin J.M. Zrenner E. Amalric P. Eliaou C. Liu S.Y. Harris E. Redmond T.M. Arnaud B. Claustres M. Hamel C.P. Nat. Genet. 1997; 17: 139-141Crossref PubMed Scopus (507) Google Scholar, 16Marlhens F. Griffoin J.M. Bareil C. Arnaud B. Claustres M. Hamel C.P. Eur. J. Hum. Genet. 1998; 6: 527-531Crossref PubMed Scopus (56) Google Scholar, 17Gu S.M. Thompson D.A. Srikumari C.R. Lorenz B. Finckh U. Nicoletti A. Murthy K.R. Rathmann M. Kumaramanickavel G. Denton M.J. Gal A. Nat. Genet. 1997; 17: 194-197Crossref PubMed Scopus (525) Google Scholar). Clearly, RPE65 is essential to the visual cycle in general and to all-trans- to 11-cis-retinoid isomerization in particular. RPE65 is the major protein of the RPE microsomal membrane fraction. The bovine (10Hamel C.P. Tsilou E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Biol. Chem. 1993; 268: 15751-15757Abstract Full Text PDF PubMed Google Scholar), human (18Nicoletti A. Wong D.J. Kawase K. Gibson L.H. Yang-Feng T.L. Richards J.E. Thompson D.A. Hum. Mol. Genet. 1995; 4: 641-649Crossref PubMed Scopus (105) Google Scholar), dog (19Aguirre G.D. Baldwin V. Pearce-Kelling S. Narfstrom K. Ray K. Acland G.M. Mol. Vis. 1998; 4: 23PubMed Google Scholar), rat (20Manes G. Leducq R. Kucharczak J. Pages A. Schmitt-Bernard C.F. Hamel C.P. FEBS Lett. 1998; 423: 133-137Crossref PubMed Scopus (22) Google Scholar), and salamander (21Ma J. Xu L. Othersen D.K. Redmond T.M. Crouch R.K. Biochim. Biophys. Acta. 1998; 1443: 255-261Crossref PubMed Scopus (43) Google Scholar) cDNAs have been cloned, as have the human (18Nicoletti A. Wong D.J. Kawase K. Gibson L.H. Yang-Feng T.L. Richards J.E. Thompson D.A. Hum. Mol. Genet. 1995; 4: 641-649Crossref PubMed Scopus (105) Google Scholar) and mouse genes. 2S. Liu, A. Boulanger, J. Kammer, E. Harris, S. Yu, and T. M. Redmond, manuscript in preparation. 2S. Liu, A. Boulanger, J. Kammer, E. Harris, S. Yu, and T. M. Redmond, manuscript in preparation. RPE65 is specific to the vertebrate RPE and is also highly conserved at the level of protein sequence. Previous data suggest a complex transcriptional and translational regulation of RPE65. At the transcriptional level, our knowledge is limited (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar), and we lack functional evidence concerning the transcriptional elements involved in the activation of the gene and in its specific expression in the RPE. Transcription ofRpe65 appears to be developmentally regulated, with the protein first appearing at about postnatal day 4 in the rat (11Hamel C.P. Tsilou E. Harris E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Neurosci. Res. 1993; 34: 414-425Crossref PubMed Scopus (104) Google Scholar), coincident with the first appearance of the photoreceptor outer segments. Reverse transcription-polymerase chain reaction (reverse transcription-PCR) analysis of RPE65 in embryonic and newborn rat suggests a biphasic induction of RPE65 mRNA expression (20Manes G. Leducq R. Kucharczak J. Pages A. Schmitt-Bernard C.F. Hamel C.P. FEBS Lett. 1998; 423: 133-137Crossref PubMed Scopus (22) Google Scholar). At the level of translation, we have found that a 170-nucleotide region of the RPE65 3′ untranslated region acts as a translational S.Y. Redmond T.M. Biophys. 1998; PubMed Scopus Google Scholar). RPE are expression of RPE65 protein the expression of RPE65 mRNA (10Hamel C.P. Tsilou E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Biol. Chem. 1993; 268: 15751-15757Abstract Full Text PDF PubMed Google Scholar, 11Hamel C.P. Tsilou E. Harris E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Neurosci. Res. 1993; 34: 414-425Crossref PubMed Scopus (104) Google Scholar). Here, we the of the 5′ flanking region of the mouseRpe65 gene and its to the corresponding human gene We have generated transgenic constructs and show that theRpe65 region –655 to +52 reporter gene expression in the RPE. we show that also a high transcriptional activity in D407 RPE in Furthermore, by directed mutagenesis, electrophoretic mobility shift assay and we demonstrate functional binding of transcription factors to an octamer and and NFI sites and show their to the transcriptional regulation of the mouseRpe65 gene. A containing the mouse Rpe65 gene was fragments containing the 5′ region of the mouseRpe65 gene identified by to a bovine 5′ (10Hamel C.P. Tsilou E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Biol. Chem. 1993; 268: 15751-15757Abstract Full Text PDF PubMed Google Scholar). containing the 5′ region of theRpe65 gene was found to the first of mouse as as of 5′ flanking was with the of the 5′ flanking region of the human RPE65 gene, in the F. Bareil C. Griffoin J.M. Zrenner E. Amalric P. Eliaou C. Liu S.Y. Harris E. Redmond T.M. Arnaud B. Claustres M. Hamel C.P. Nat. Genet. 1997; 17: 139-141Crossref PubMed Scopus (507) Google Scholar), the and analysis transgenic mice, and containing in the mouse 5′ flanking containing sites at their 5′ The sites as and The was fragments with and J. S. Science. PubMed Scopus Google Scholar) containing an E. gene and transient transfection assay, constructs and the The the as to and fragments the of transgenic The also with the but bases at the 3′ Mutations by A of the as a and as containing mutations in or all of the elements or as a for are in The of mutations was by for and directed to to to to to to to to to binding are in are in all the (5Saari J.C. Bredberg D.L. J. Biol. Chem. 1988; 263: 8084-8090Abstract Full Text PDF PubMed Google Scholar). in a binding are in are in all the (5Saari J.C. Bredberg D.L. J. Biol. Chem. 1988; 263: 8084-8090Abstract Full Text PDF PubMed Google Scholar). constructs the of cell mouse which and their identified by of a region to all of the was by analysis with a to to reporter gene activity was the assay the the and the the RPE, and and the and for in in and in 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside The in a of in containing and in and in at a of with and for of in and to and The was and in for an of the The human retinal pigment epithelium cell line D407 was C. P.S. Bok D. J. M. Invest. Ophthalmol. Visual Sci. 1995; Google Scholar) and in high with bovine and HeLa, HepG2, and and in the as for D407 that the of bovine was and to for for in transfection of and of to the in a containing and reporter gene the reporter gene assay The of activity to activity in as a of activity. in at D407 and RPE bovine by the of Res. PubMed Scopus Google Scholar). with and of nuclear to binding 4 of and and at the assay, a of or was with the The in and by nuclear with of by or 4 of by for at to of was as a in the NFI containing with bovine and and an by with the as the as in the by in one with the in the for in a a in We have of 5′ flanking region of the transcription of the mouse Rpe65 gene with the human gene (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google and for conserved the mouse and genes 5′ flanking of the 5′ flanking region of the mouse and genes by a of was to with a region of to the 5′ of not The and of the human and mouse 5′ flanking and 5′ untranslated by conserved of the including NFI to octamer to and two E-box consensus to and to binding The is A to the human gene S. P. D.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) by Nicoletti (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar) in the 5′ flanking region is at to in the mouse Rpe65 5′ flanking is not in the mouse gene The NFI in genes a only in the human RPE65 gene (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar), but the consensus binding with the The two E-box sites are consensus helix-loop-helix protein binding sites, (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar) to octamer was also identified in by only one the consensus octamer human and mouse genes lack consensus GC and CAAT boxes. A TATA box was identified at to in the human gene (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar), as in the mouse gene, is consensus C.R. K. Mol. Biol. PubMed Scopus Google Scholar, C.R. K. 4: PubMed Scopus Google Scholar). to found in other including protein N. E. A. F. D. R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and S. P. D.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), found in the human RPE65 gene promoter (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar), is not these play in the activity of the Rpe65 gene. identify elements and transcriptional factors of Rpe65 we first the Rpe65 promoter for the in specific expression of the Rpe65 gene in the RPE. We constructs containing the of theRpe65 gene to the sequence, and we their corresponding activity in transgenic the in –188 to +52 to +52 and –655 to +52 of these constructs in the of several lines for was by the of with transgenic to of a of the The of and the of and expression of are in activity in transgenic mouse of of transgene expression in the of the and of activity. high expression in non-RPE not of of transgene expression in the of the and of activity. high expression in non-RPE not in a RPE65 been to be in the RPE of the and is not found in (11Hamel C.P. Tsilou E. Harris E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Neurosci. Res. 1993; 34: 414-425Crossref PubMed Scopus (104) Google Scholar), we expression of RPE65 gene constructs in a of We the and the the RPE, and and the and and transgenic and or for activity. We found that the constructs not and only background activity in or transgenic in all However, the an of about activity the in the but in other and The for of are in and for of are in B. for of and but was only in the of transgenic and At the level, of the a of these by the was to be in its to the RPE of transgenic whereas was in and of or was in transgenic and not of the RPE was and was by of transgenic was in not of the RPE demonstrated level of in the about of highly and with of with not for the which an expression in the RPE65 is not in (11Hamel C.P. Tsilou E. Harris E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Neurosci. Res. 1993; 34: 414-425Crossref PubMed Scopus (104) Google Scholar) or M. Redmond, the transcriptional regulation of theRpe65 gene, we for a cellular of activation of the mouse Rpe65 only of RPE65 mRNA are by in of the human RPE cell D407 not been demonstrated that these are to a human RPE65 promoter (22Nicoletti A. Kawase K. Thompson D.A. Invest. Ophthalmol. Visual Sci. 1998; 39: 637-644PubMed Google Scholar). Thus, to its activity and in the promoter to was the reporter and D407 show the cellular of the was also the non-RPE cell lines HeLa, HepG2, and HS27. of these cell lines or the which are and the RPE65 show that activity generated by was and was in all of the cell lines HeLa, HepG2, and activity was also to that by and activity generated by in D407 activity that generated by identify elements in the Rpe65 of the promoter to +52) and D407 for directed are in The the NFI R. Full Text PDF PubMed Scopus Google Scholar) at to only a promoter activity mutations of the of the two potential binding sites, and at to and to promoter activity by and mutations of the octamer at to promoter activity by of these mutations abolished promoter activity, we The of mutations in the and or octamer a promoter activity by about the promoter activity the level of a containing and octamer sites activity with the that the E-box and octamer are promoter that the NFI with an octamer the activity with the was and transcription factors binding to the potential octamer, and sites in theRpe65 promoter in and in we nuclear bovine RPE and D407 A of specific was bovine RPE or D407 nuclear with a containing the NFI of these a mobility the two nuclear and these by of a of of D407 nuclear binding to the Rpe65 promoter by The indicated and the corresponding with or bovine RPE nuclear in the 2000; or of or as are indicated by to to NFI to sequence. to to to to of bovine RPE or D407 nuclear with the A and or the and in the of a specific complex with However, transcription factors to these sites with the complex with the was only of A complex of a was with nuclear was as a all in the of an of but not in the of a of or of D407 nuclear with the in the of two The complex was by a of but not by or whereas the complex was not by and was to be one complex was the nuclear was with the complex with the mobility as the one with the D407 nuclear complex was of the with of the nuclear suggest that these sites involved in the regulation of theRpe65 and to the transcription factors involved in the binding of the D407 nuclear with the NFI and The the with NFI in the of However, a was also in the of the and the corresponding of the complex was not that other also be in the binding to the octamer be due to the of was the with the corresponding or containing bovine for A was an with the as the was as a not The lack of of as a component of the identified by with the two However, we the and a D407 nuclear in to the of the by A of and a of with of the with a of but not with the of an to for by N. R. 1988; PubMed Scopus Google Scholar). RPE65 to the only RPE-specific component of the RPE-specific all-trans- to 11-cis-retinoid isomerization as the visual the of we have theRpe65 promoter in and in we show that the –655 to +52 region of the mouseRpe65 promoter tissue-specific expression in and in and we elements that are crucial to expression. first was to identify a region of the 5′ flanking region of the Rpe65 gene that in RPE-specific expression. transgenic that the region of the Rpe65 gene to +52) RPE-specific expression in with expression in expression was also in at postnatal day 4 not with RPE65 expression (11Hamel C.P. Tsilou E. Harris E. Pfeffer B.A. Hooks J.J. Detrick B. Redmond T.M. J. Neurosci. Res. 1993; 34: 414-425Crossref PubMed Scopus (104) Google Scholar). of mouse a of the RPE-specific expression. of been in other transgenic mouse S. L. E. J. Biol. 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Thompson D.A. Invest. Ophthalmol. 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The role of NFI in the Rpe65 transcriptional regulation is not indicated that NFI to theRpe65 promoter and also that reporter activity the NFI is in with the octamer suggest a of NFI gene expression. we have that the promoter region of the mouse Rpe65 gene direct RPE-specific expression of a reporter in transgenic We have also found that and factors synergistically regulate theRpe65 gene and that mutations in these elements transcriptional activity and binding of the corresponding NFI is also involved but in a the regulation of Rpe65 gene expression a of specific gene be to these and of transcription factors binding to the transcriptional elements is are We the of the and of and and for their in the transgenic in We and for critical of the manuscript and We also for in the of for and the for the and and of of with the D407 cell

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