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Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation*

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 18, Issue of April 30, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Analysis of the
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 18, Issue of April 30, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation* Received for publication, September 5, 2003, and in revised form, January 30, 2004 Published, JBC Papers in Press, February 24, 2004, DOI /jbc.M Noriko Esumi, a,b,c Yuji Oshima, a,b,d Yuanyuan Li, a,b,e Peter A. Campochiaro, a,b,f,g and Donald J. Zack a,b,f,h,i,j From a The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology at the Wilmer Eye Institute, the Departments of b Ophthalmology, f Neuroscience, and h Molecular Biology and Genetics, and i The McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5 -upstream region of human VMD2, a gene that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Transgenic mouse studies with VMD2 promoter/lacz constructs demonstrated that a 253 to 38 bp fragment is sufficient to direct RPEspecific expression in the eye. Transient transfection assays using the D407 human RPE cell line with VMD2 promoter/luciferase reporter constructs identified two positive regulatory regions, 585 to 541 bp for high level expression and 56 to 42 bp for low level expression. Mutation of a canonical E-box located in the 56 to 42 bp region greatly diminished luciferase expression in D407 cells and abolished the bands shifted with bovine RPE nuclear extract in electrophoretic mobility shift assays. Independently a candidate approach was used to select microphthalmia-associated transcription factor (MITF) for testing because it is expressed in the RPE and associated with RPE abnormalities when mutated. MITF-M significantly increased luciferase expression in D407 cells in an E-box-dependent manner. These studies define the VMD2 promoter region sufficient to drive RPE-specific expression in the eye, identify positive regulatory regions in vitro, and suggest that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression. * This study was supported in part by grants from the National Eye Institute, Foundation Fighting Blindness, Steinbach Foundation, Macula Vision Foundation, and by a generous gift from Robert and Clarice Smith. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. c To whom correspondence should be addressed: The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology at the Wilmer Eye Inst., The Johns Hopkins University School of Medicine, 832 Maumenee Bldg., 600 N. Wolfe St., Baltimore, MD Tel.: ; Fax: ; d Present address: Dept. of Ophthalmology, Faculty of Medicine, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan. e Present address: Laboratory of Neurogenetics, Kennedy Krieger Inst., The Johns Hopkins University School of Medicine, Baltimore, MD g The George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience. j The Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology and recipient of a Research to Prevent Blindness Senior Investigator Award The retinal pigment epithelium (RPE) 1 is a monolayer of cuboidal cells located between the photoreceptors and choroid of the eye. It has many specialized functions that support and nourish photoreceptors, including important roles in retinoid metabolism (visual cycle), phagocytosis of shed photoreceptor outer segments, maintenance of the blood-retina barrier, movement of ions and water, and synthesis and transport of substances that constitute the interphotoreceptor matrix (1). The importance of the RPE in maintaining retinal photoreceptors is highlighted by the Royal College of Surgeons rat that exhibits a markedly reduced capacity for phagocytosis of outer segments by the RPE that results in the degeneration and loss of photoreceptor cells (2, 3). In addition, in humans, RPE dysfunction has been implicated in the pathogenesis of age-related macular degeneration, which is the leading cause of irreversible blindness in elderly people in western countries (4, 5). Mutations in several genes that are specifically or preferentially expressed in the RPE, such as RPE65, RLBP1, RGR, TIMP3, and VMD2, are associated with inherited human retinal dystrophies (6 15). Mutations in VMD2 result in Best disease (vitelliform macular dystrophy (VMD)), an autosomal dominant, juvenile onset macular dystrophy characterized by a striking accumulation of lipofuscin-like material within and beneath the RPE (16 18). VMD2 encodes a multispan transmembrane protein, bestrophin, that is expressed preferentially in the RPE and functions as an oligomeric chloride channel that is thought to be responsible for the characteristic abnormality observed in the electrooculogram in patients with Best disease (19, 20). Despite the key role of the RPE in vision and the importance of the genes that are specifically or preferentially expressed in the RPE, relatively few studies have focused on the regulation of RPE gene expression. Perhaps the best studied model of RPE gene regulation to date has been the RPE65 gene (21, 22), which is specifically expressed in the RPE and cone photoreceptors (23). A combination of transgenic and cell culture experiments have identified a murine promoter region sufficient for RPE-specific expression and suggested that octamer and E-box binding transcription factors may play important regulatory roles (21). The human RPE65 5 -upstream region has also been studied using sequence analyses, cell transfection assays, and DNase I footprinting (24). Analysis of the 5 -up- 1 The abbreviations used are: RPE, retinal pigment epithelium; VMD, vitelliform macular dystrophy; PCE-1, photoreceptor consensus element-1; Tyr, tyrosinase; MITF, microphthalmia-associated transcription factor; RT, reverse transcription; CMV, cytomegalovirus; PBS, phosphate-buffered saline; X-gal, 5-bromo-4-chloro-3-indolyl- -D-galactopyranoside; EMSA, electrophoretic mobility shift assay; NF1/CTF1, nuclear factor 1/CCAAT-box-binding transcription factor 1; CEBP, CCAAT/enhancer-binding protein; Hh, Hedgehog. This paper is available on line at Analysis of VMD2 Promoter stream region of the human cellular 11-cis-retinaldehyde-binding protein gene (RLBP1), which is preferentially expressed in RPE and Muller cells, suggested that a photoreceptor consensus element-1 (PCE-1, CAATTAG) located in the proximal promoter region might act as a positive regulator of RPE gene expression (25). This is interesting because PCE-1 was generally considered a photoreceptor element, and a sequence similar to PCE-1 is also present within the proximal promoter region of both human and murine Rpe65; however, its biological relevance has not yet been experimentally tested. Promoter regions have also been analyzed for genes that are expressed in the RPE but are not RPE-specific. For example, transgenic studies demonstrated that 270 bp of the murine tyrosinase gene (Tyr) upstream region is sufficient to direct cell typespecific expression and developmental regulation in melanocytes and the RPE (26). At the biochemical level, the human TYR promoter can be bound in vitro by microphthalmia-associated transcription factor (MITF) with binding mediated by an M-box, which contains a core CATGTG E-box motif (27, 28). Consistent with this binding, MITF is sufficient to direct pigment cell-specific transcription of TYR (28, 29). MITF is a member of the basic helix-loop-helix leucine zipper family of transcription factors and expressed in several cell lineages including melanocytes, RPE, osteoclasts, and mast cells (27, 30 34). Mice with mutations in Mitf (Mitf mi /Mitf mi ) show loss of pigmentation, microphthalmia, and early onset of deafness. The RPE in these mice forms a multilayered structure resembling the neural retina, suggesting a critical role of MITF in RPE development (35 38). To gain further insights into the molecular mechanisms mediating RPE-specific gene regulation, we analyzed VMD2 as a model system. Using a combination of transgenic and transient transfection approaches, we found that a fragment as small as 253 to 38 bp is sufficient to direct RPE-specific expression in the eye, and two regions, 585 to 541 bp and 56 to 42 bp, are important for high level and low level expression in vitro, respectively. We also present evidence that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression. EXPERIMENTAL PROCEDURES Cloning, Sequencing, and Comparison of the 5 -Upstream Region of Human and Murine Vmd2 A P1 human genomic library (Genome Systems, St. Louis, MO) was screened with a 32 P-labeled bovine Vmd2 cdna clone according to the company s instructions. P1 plasmid DNAs were purified from purchased Escherichia coli clones and analyzed by Southern hybridization to confirm that the clones contained the 5 flanking region of VMD2. A 129/SvJ mouse genomic library in Lambda FIX II vector (Stratagene, La Jolla, CA), which was a kind gift from Dr. Se-Jin Lee (The Johns Hopkins University), was screened with the same bovine Vmd2 cdna probe according to standard methods (39). Both P1 and phage DNAs were directly sequenced using the Thermo Sequenase cycle sequencing kit (USB Corp., Cleveland, OH) with 2.5% dimethyl sulfoxide added in the reactions. Later the 5 -flanking sequences of both human and murine Vmd2 were also obtained from the GenBank TM at National Center for Biotechnology Information (accession number NT_ for human and NT_ for mouse). Computer programs, GeneWorks 2.5 (Oxford Molecular Group, Beaverton, OR) and Vector NTI version 7 (Invitrogen) were used for sequence alignment. To look for the presence of known transcription factor binding sites, MatInspector professional version 6.0 was used with the database of Matrix Family Library version 3.0 (Genomatix, Munich, Germany) (40). Primer Extension Human donor eyes were obtained from eye banks through the National Disease Research Interchange. Eyes were dissected equatorially, the retina was removed, RPE cells were collected by gentle scraping, and total RNA was extracted using TRIzol reagent (Invitrogen) (41). Primer extension was performed according to standard procedures (42) using 10 g of human RPE total RNA as template with two primers complementary to VMD2 mrna sequence. The locations of Primer A (5 -TAAGTGATGGTCATGGCCAGGCAGTGG-3 ) and Primer B (5 - AGGTGGGGTTCCAGGTGGGTCCGATGATCC-3 ) are shown in Fig. 1B. The end-labeled primers were annealed to RNA at 65 C for 90 min and extended using Thermoscript reverse transcriptase (Invitrogen) at 65 C for1h. Plasmid Construction Four VMD2 promoter/lacz reporter constructs were made for transgenic mouse studies. First, a 7.2-kb BamHI/ SphI fragment containing 3.5 kb 5 -upstream of VMD2 was purified from human P1 DNA and subcloned into pbluescript vector (Stratagene). Then two genomic fragments were generated by digestion with BspHI/XcmI for Construct 1 ( 2948 to 38 bp) and EcoRI/XcmI for Construct 2 ( 585 to 38 bp). Two shorter fragments were generated by PCR using the following primers and confirmed by sequencing: forward primer for Construct 3 ( 424 to 38 bp), 5 -CATTCTTTCCAT- AGCCCACA-3 ; forward primer for Construct 4 ( 253 to 38 bp), 5 -CTCTGGATTTTAGGGCCATG-3 ; reverse primer for both fragments, 5 -GGTCTGGCGACTAGGCTGGT-3. These four fragments were blunt ligated into SmaI site upstream of lacz gene in placf vector (a kind gift of Dr. Jacques Peschon, Immunex Corp., Seattle, WA). Fourteen VMD2 promoter/luciferase reporter constructs were made for transient transfection assays, including the four fragments described above. Additional 10 fragments were generated by PCR using the primers below and confirmed by sequencing. All fragments were blunt ligated into SmaI site of pgl2-basic vector, which contains luciferase gene (Promega, Madison, WI). Primers were as follows: forward primer for fragment 540 to 38 bp, 5 -TCCTTTTCAGATA- AGGGCAC-3 ; 500 to 38 bp, 5 -AAACCTACCCGGCGTCACCA-3 ; 460 to 38 bp, 5 -GACCAGAAACCAGGACTGTT-3 ; 204 to 38 bp, 5 -CCTGGTCTCAGCCCAACACC-3 ; 154 to 38 bp, 5 -AGGCTGT- GCTAGCCGTTGCT-3 ; 104 to 38 bp, 5 -AAGGACTCCTTTGTG- GAGGT-3 ; 86 to 38 bp, 5 -GTCCTGGCTTAGGGAGTCAA-3 ; 71 to 38 bp, 5 -GTCAAGTGACGGCGGCTCAG-3 ; 56 to 38 bp, 5 - CTCAGCACTCACGTGGGCAG-3 ; and 41 to 38 bp, 5 -GGGCAGT- GCCAGCCTCTAAG-3. Reverse primer for all fragments was the same as that used for transgenic constructs. Mutated VMD2 promoter/luciferase constructs were made using synthetic oligonucleotides containing a mutation (CANNTG to ACNNTA) in E-box 1 ( 47 to 42 bp, designated m1), E-box 2 ( 69 to 64 bp, m2), or both (m1m2) in the context of both the 104 to 38 and 71 to 38 bp fragments (Fig. 4A). Forward oligonucleotides that corresponded to 104 to 25 bp (5 -AAGGACTCCTTTGTGGAGGTCCTGGCTTAGGG- AGTCAAGTGACGGCGGCTCAGCACTCACGTGGGCAGTGCCAGCC- TCTA-3 ) or 71 to 22 bp (5 -GTCAAGTGACGGCGGCTCAGCACT- CACGTGGGCAGTGCCAGCCTCTAAGA-3 ) and contained mutation m1, m2, or m1m2 were annealed to a common reverse oligonucleotide that corresponded to 38 to 41 bp (5 -GGTCTGGCGACTAGGCTGG- TGGGACTCCCTGGGACTCTGTGGCCAGTGCCCCTGCCCACTCTT- AGAGGCTGGCACTGCC-3 ). The annealed oligonucleotides were extended to both ends by Klenow fragment, blunt ligated into SmaI site of pgl2-basic vector, and verified by sequencing. To construct an expression vector, a human MITF-M cdna was generated by reverse transcription (RT)-PCR using human RPE total RNA as template with a forward primer containing an EcoRI site (5 -ACTGAATTCATTGTTATGCTGGAAATGCTAGA-3 ) and a reverse primer containing a HindIII site (5 -AGAAAGCTTGAACAAGTGT- GCTCCGTCTCTTC-3 ). The cdna fragment was then inserted into EcoRI/HindIII sites downstream of CMV promoter in pcdna3.1( )/ Myc-His B vector (Invitrogen). Generation of Transgenic Mice The transgenic constructs were microinjected into mouse one-cell embryos (B6/SJL F2 hybrid) at the Transgenic Mouse Core Facility of The Johns Hopkins University School of Medicine as described previously (43). Mouse pups were screened to determine positive transgenic founders by both Southern blot analysis and PCR of tail DNAs. Ten micrograms of mouse tail DNAs were digested with BamHI and hybridized with a 32 P-labeled 3.1-kb lacz fragment according to standard procedures for Southern blotting (39). Primers for PCR were forward 5 -ACATCAGCCGCTA- CAGTCAA-3 and reverse 5 -GCGAGATGCTCTTGAAGTCT-3. Transgenic founders were mated with wild-type BALB/cJ mice (The Jackson Laboratory, Bar Harbor, ME) to generate progeny with albino background. Histology, X-Gal Staining, Flat Mount, and RT-PCR Analysis for Transgenic Mice Mice were euthanized, eyes were enucleated, and the whole eyes were fixed at 4 C for 1 h in 2%paraformaldehyde and 0.25% glutaraldehyde in phosphate-buffered saline (PBS) for eye sections or in 0.5% glutaraldehyde in PBS for RPE/choroid flat mounts. For histochemical staining with 5-bromo-4-chloro-3-indolyl- -D-galactopyranoside (X-gal), eyes were then cryoprotected in 25% sucrose in PBS at 4 C 19066 Analysis of VMD2 Promoter FIG. 1. The 5 -upstream region of human VMD2. A, transcription start site of the VMD2 determined by primer extension. The extension products obtained using 10 g of total RNA extracted from human RPE cells with two primers complementary to VMD2 mrna sequence were analyzed next to a DNA sequence ladder. The locations of Primer A and Primer B are shown in B. B, nucleotide sequence of the 5 -upstream region of human VMD2. The transcription start site determined by primer extension is numbered 1, and the numbers on the left are the nucleotide positions relative to it. Intron 1 is shown by dotted line, and its sequence (1343 bp) is not presented. E- box, NF1/CTF1, octamer, CEBP, PCE-1- like, and Crx/Otx consensus binding sites are underlined and labeled. Three EMSA probes that cover the 585 to 541 bp region (Probes 23, 24, and 25) and the probe that contains E-box 1 (Probe 55/ 34) are indicated by dotted underlines. The locations and directions of Primer A and Primer B used for primer extension are shown by arrows (5 3 3 ). The initiation ATG is also underlined. for h, mounted in OCT medium (Sakura Finetek, Torrance, CA), and cut at 10 m on a cryostat. Sections were stained in 1 mg/ml X-gal, 5mM K 3 Fe(CN) 6,5mM K 4 Fe(CN) 6 3H 2 O,1mM MgCl 2 in PBS at 37 C for h (43). Sections of non-transgenic mice were also stained by hematoxylin and eosin. For RPE/choroid flat mounts, fixed whole eyes were washed in PBS and stained in the X-gal solution at 37 C for h. The stained eyes were cut at the equatorial zone, the anterior portion was removed, the retina was removed, and the RPE/choroid eye cup was cut from the periphery by a pair of microsurgical scissors. To check the expression of lacz reporter in other tissues, RT-PCR was performed using 1 g of total RNA extracted by TRIzol from liver, brain, kidney, spleen, testis, and eye of each founder. First strand cdna was synthesized with oligo(dt) primer and Superscript II reverse transcriptase (Invitrogen), and PCR was performed using 35 cycles with the Analysis of VMD2 Promoter same primer set as used for the tail DNA screening. As a control for RNA and RT reaction, a cdna fragment of ribosomal protein S16 was also amplified using forward primer 5 -CACTGCAAACGGGGAAATG- G-3 and reverse primer 5 -TGAGATGGACTGTCGGATGG-3. Cell Cultures and Transient Transfections Nine human cell cultures were screened for the expression of VMD2 by RT-PCR using 1 g of total RNA, and a 269-bp VMD2 fragment was amplified using forward primer 5 -CATAGACACCAAAGACAAAAGC-3 and reverse primer 5 -GTGCTTCATCCCTGTTTTCC-3. As a control, S16 was used as described above. Based on these results, three human RPE cell lines, D407 (44), ARPE19 (45), and telomerase-immortalized htert-rpe1 (46) (Clontech, Palo Alto, CA), and a human neuroepithelioma cell line, SK-N-MC (47), were selected. Each cell line was cultured in the medium suggested in the references. Cells were transfected at h after plating into 60-mm dishes at 70 80% confluency using LipofectAMINE PLUS reagent (Invitrogen). Plasmid DNA for each dish included 9 gof a luciferase construct and 1 g of pcmv-lacz as an internal control for transfection efficiency. For co-transfection studies, 0, 0.5, or 2.5 g ofa human MITF-M expression vector were added to each DNA mixture, and the total amount of expression vector was adjusted to 2.5 g by adding pcdna3.1 vector when necessary. Transfections were performed six independent times in duplicate each time. Cell lysates were prepared at h after transfection using 300 l of Reporter Lysis Buffer (Promega). Luciferase and -galactosidase activities were measured as described previously (48) except that Softmax Pro program with SpectraMax Plus 96-well plate reader (Molecular Devices, Sunnyvale, CA) was used for -galactosidase assay. Electrophoretic Mobility Shift Assays (EMSAs) RPE cells from 203 bovine eyes yielded 4.4 mg of nuclear extract by the method of Dignam et al. (49). EMSA was performed according to standard methods as described previously (50). For scanning EMSA, a total of 26 oligonucleotide probes, each of which was 30 bp long and overlapped the adjacent probes by 10 bp at each end, were designed in the VMD2 5 -upstream region from 50 to 280 bp (Probes 1 16) and from 400 to 610 bp (Probes 17 26). Oligonucleotide pairs complementary to each other were annealed and labeled by fill-in reaction with [ - 32 P]dCTP and Klenow fragment. Labe
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