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• W2020149574 abstract "The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photo-receptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5′-up-stream 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 RPE-specific 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. The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photo-receptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5′-up-stream 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 RPE-specific 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. The retinal pigment epithelium (RPE) 1The 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-β-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. 1The 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-β-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. 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 (1Bok D. J. Cell Sci. Suppl. 1993; 17: 189-195Google Scholar). 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 (2Mullen R.J. LaVail M.M. Science. 1976; 192: 799-801Google Scholar, 3D'Cruz P.M. Yasumura D. Weir J. Matthes M.T. Abderrahim H. LaVail M.M. Vollrath D. Hum. Mol. Genet. 2000; 9: 645-651Google Scholar). 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 (4Klein R. Klein B.E. Linton K.L. Ophthalmology. 1992; 99: 933-943Google Scholar, 5Smith W. Assink J. Klein R. Mitchell P. Klaver C.C. Klein B.E. Hofman A. Jensen S. Wang J.J. de Jong P.T. Ophthalmology. 2001; 108: 697-704Google Scholar).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 (6Redmond 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: 344-351Google Scholar, 7Gu 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: 194-197Google Scholar, 8Marlhens 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: 139-141Google Scholar, 9Burstedt M.S. Sandgren O. Holmgren G. Forsman-Semb K. Investig. Ophthalmol. Vis. Sci. 1999; 40: 995-1000Google Scholar, 10Maw M.A. Kennedy B. Knight A. Bridges R. Roth K.E. Mani E.J. Mukkadan J.K. Nancarrow D. Crabb J.W. Denton M.J. Nat. Genet. 1997; 17: 198-200Google Scholar, 11Morimura H. Berson E.L. Dryja T.P. Investig. Ophthalmol. Vis. Sci. 1999; 40: 1000-1004Google Scholar, 12Morimura H. Saindelle-Ribeaudeau F. Berson E.L. Dryja T.P. Nat. Genet. 1999; 23: 393-394Google Scholar, 13Felbor U. Suvanto E.A. Forsius H.R. Eriksson A.W. Weber B.H. Am. J. Hum. Genet. 1997; 60: 57-62Google Scholar, 14Petrukhin K. Koisti M.J. Bakall B. Li W. Xie G. Marknell T. Sandgren O. Forsman K. Holmgren G. Andreasson S. Vujic M. Bergen A.A. McGarty-Dugan V. Figueroa D. Austin C.P. Metzker M.L. Caskey C.T. Wadelius C. Nat. Genet. 1998; 19: 241-247Google Scholar, 15Marquardt A. Stohr H. Passmore L.A. Kramer F. Rivera A. Weber B.H. Hum. Mol. Genet. 1998; 7: 1517-1525Google Scholar). 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 (16Weingeist T.A. Kobrin J.L. Watzke R.C. Arch. Ophthalmol. 1982; 100: 1108-1114Google Scholar, 17Frangieh G.T. Green W.R. Fine S.L. Arch. Ophthalmol. 1982; 100: 1115-1121Google Scholar, 18Stone E.M. Nichols B.E. Streb L.M. Kimura A.E. Sheffield V.C. Nat. Genet. 1992; 1: 246-250Google Scholar). 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 (19Sun H. Tsunenari T. Yau K.W. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4008-4013Google Scholar, 20Tsunenari T. Sun H. Williams J. Cahill H. Smallwood P. Yau K.W. Nathans J. J. Biol. Chem. 2003; 278: 41114-41125Google Scholar).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 (21Boulanger A. Liu S. Henningsgaard A.A. Yu S. Redmond T.M. J. Biol. Chem. 2000; 275: 31274-31282Google Scholar, 22Boulanger A. Redmond T.M. Curr. Eye Res. 2002; 24: 368-375Google Scholar), which is specifically expressed in the RPE and cone photoreceptors (23Znoiko S.L. Crouch R.K. Moiseyev G. Ma J.X. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1604-1609Google Scholar). 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 (21Boulanger A. Liu S. Henningsgaard A.A. Yu S. Redmond T.M. J. Biol. Chem. 2000; 275: 31274-31282Google Scholar). The human RPE65 5′-upstream region has also been studied using sequence analyses, cell transfection assays, and DNase I footprinting (24Nicoletti A. Kawase K. Thompson D.A. Investig. Ophthalmol. Vis. Sci. 1998; 39: 637-644Google Scholar). Analysis of the 5′-up-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 (25Kennedy B.N. Goldflam S. Chang M.A. Campochiaro P. Davis A.A. Zack D.J. Crabb J.W. J. Biol. Chem. 1998; 273: 5591-5598Google Scholar). 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 type-specific expression and developmental regulation in melanocytes and the RPE (26Beermann F. Schmid E. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2809-2813Google Scholar). 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 (27Goding C.R. Genes Dev. 2000; 14: 1712-1728Google Scholar, 28Yasumoto K. Yokoyama K. Takahashi K. Tomita Y. Shibahara S. J. Biol. Chem. 1997; 272: 503-509Google Scholar). Consistent with this binding, MITF is sufficient to direct pigment cell-specific transcription of TYR (28Yasumoto K. Yokoyama K. Takahashi K. Tomita Y. Shibahara S. J. Biol. Chem. 1997; 272: 503-509Google Scholar, 29Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Mol. Cell. Biol. 1994; 14: 8058-8070Google Scholar). 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 (27Goding C.R. Genes Dev. 2000; 14: 1712-1728Google Scholar, 30Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Google Scholar, 31Hughes M.J. Lingrel J.B. Krakowsky J.M. Anderson K.P. J. Biol. Chem. 1993; 268: 20687-20690Google Scholar, 32Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Google Scholar, 33Steingrimsson E. Tessarollo L. Pathak B. Hou L. Arnheiter H. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4477-4482Google Scholar, 34Widlund H.R. Fisher D.E. Oncogene. 2003; 22: 3035-3041Google Scholar). Mice with mutations in Mitf (Mitfmi/Mitfmi) 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 (35Packer S.O. J. Exp. Zool. 1967; 165: 21-46Google Scholar, 36Hero I. Farjah M. Scholtz C.L. Investig. Ophthalmol. Vis. Sci. 1991; 32: 2622-2635Google Scholar, 37Nakayama A. Nguyen M.T. Chen C.C. Opdecamp K. Hodgkinson C.A. Arnheiter H. Mech. Dev. 1998; 70: 155-166Google Scholar, 38Nguyen M. Arnheiter H. Development. 2000; 127: 3581-3591Google Scholar).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 PROCEDURESCloning, 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 32P-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 (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 2.60-2.121Google Scholar). 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™ at National Center for Biotechnology Information (accession number NT_033903 for human and NT_039687 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) (40Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Google Scholar).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) (41Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar).Primer extension was performed according to standard procedures (42Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K.S. Current Protocols in Molecular Biology. John Wiley and Sons, Hoboken, NJ1994: 4.8.1-4.8.5Google Scholar) using 10 μg 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 for 1 h.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′-CATTCTTTCCATAGCCCACA-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′-TCCTTTTCAGATAAGGGCAC-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′-AGGCTGTGCTAGCCGTTGCT-3′; -104 to +38 bp, 5′-AAGGACTCCTTTGTGGAGGT-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′-GGGCAGTGCCAGCCTCTAAG-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′-AAGGACTCCTTTGTGGAGGTCCTGGCTTAGGGAGTCAAGTGACGGCGGCTCAGCACTCACGTGGGCAGTGCCAGCCTCTA-3′) or -71 to -22 bp (5′-GTCAAGTGACGGCGGCTCAGCACTCACGTGGGCAGTGCCAGCCTCTAAGA-3′) and contained mutation m1, m2, or m1m2 were annealed to a common reverse oligonucleotide that corresponded to +38 to -41 bp (5′-GGTCTGGCGACTAGGCTGGTGGGACTCCCTGGGACTCTGTGGCCAGTGCCCCTGCCCACTCTTAGAGGCTGGCACTGCC-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.Fig. 4Effect of mutation in E-box elements on VMD2 promoter activity. A, sequence of the VMD2 proximal promoter containing two E-box elements. The transcription start site (TS, numbered +1) is indicated by an angled arrow. Two E-box sites, E-box 1 and E-box 2, are highlighted in boldface. Mutations in E-box 1 and E-box 2 are indicated under the boldfaced consensus sequence and designated as m1 and m2, respectively. A probe for EMSA that contains E-box 1 (Probe -55/-34) is underlined. B, effect of mutation in E-box elements on VMD2 promoter activity. Luciferase constructs containing mutations in E-box elements in the context of both the -104 to +38 and -71 to +38 bp fragments as well as the constructs containing wild-type fragments and pGL2-Basic vector were transiently transfected into D407 cells. Relative luciferase activities were calculated and presented as described in Fig. 3B. The constructs containing mutations of both E-box 1 and E-box 2 are designated as m1m2.View Large Image Figure ViewerDownload (PPT)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′-AGAAAGCTTGAACAAGTGTGCTCCGTCTCTTC-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 (43Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Google Scholar). 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 32P-labeled 3.1-kb lacZ fragment according to standard procedures for Southern blotting (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 2.60-2.121Google Scholar). Primers for PCR were forward 5′-ACATCAGCCGCTACAGTCAA-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-β-d-galactopyranoside (X-gal), eyes were then cryoprotected in 25% sucrose in PBS at 4 °C for 12-18 h, mounted in OCT medium (Sakura Finetek, Torrance, CA), and cut at 10 μm on a cryostat. Sections were stained in 1 mg/ml X-gal, 5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6·3H2O, 1 mm MgCl2 in PBS at 37 °C for 12-48 h (43Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Google Scholar). 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 24-48 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 μg 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 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′-CACTGCAAACGGGGAAATGG-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 μg 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 (44Davis A.A. Bernstein P.S. Bok D. Turner J. Nachtigal M. Hunt R.C. Investig. Ophthalmol. Vis. Sci. 1995; 36: 955-964Google Scholar), ARPE19 (45Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. Exp. Eye Res. 1996; 62: 155-169Google Scholar), and telomerase-immortalized hTERT-RPE1 (46Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Google Scholar) (Clontech, Palo Alto, CA), and a human neuroepithelioma cell line, SK-N-MC (47Biedler J.L. Spengler B.A. J. Natl. Cancer Inst. 1976; 57: 683-695Google Scholar), were selected. Each cell line was cultured in the medium suggested in the references. Cells were transfected at 40-48 h after plating into 60-mm dishes at 70-80% confluency using LipofectAMINE PLUS reagent (Invitrogen). Plasmid DNA for each dish included 9 μg of a luciferase construct and 1 μg of pCMV-lacZ as an internal control for transfection efficiency. For co-transfection studies, 0, 0.5, or 2.5 μg of a human MITF-M expression vector were added to each DNA mixture, and the total amount of expression vector was adjusted to 2.5 μg by adding pcDNA3.1 vector when necessary. Transfections were performed six independent times in duplicate each time. Cell lysates were prepared at 48-60 h after transfection using 300 μl of Reporter Lysis Buffer (Promega). Luciferase and β-galactosidase activities were measured as described previously (48Kumar R. Chen S. Scheurer D. Wang Q.L. Duh E. Sung C.H. Rehemtulla A. Swaroop A. Adler R. Zack D.J. J. Biol. Chem. 1996; 271: 29612-29618Google Scholar) except that Softmax Pro 2.6.1 program with SpectraMax Plus 96-well plate reader (Molecular Devices, Sunnyvale, CA) was used for β-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. (49Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). EMSA was performed according to standard methods as described previously (50Nie Z. Chen S. Kumar R. Zack D.J. J. Biol. Chem. 1996; 271: 2667-2675Google Scholar). 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 [α-32P]dCTP and Klenow fragment. Labeled probe (20,000-40,000 cpm) was incubated with 3-5 μg of nuclear extracts in binding solution (25 mm HEPES, pH 7.6, 60 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 5% glycerol) containing either 0.1, 0.5, or 2.5 μg of poly(dI-dC) on ice for 30 min and analyzed on a 5% polyacrylamide gel. For supershift assays with Probe 24, nuclear extracts were incubated with 2 μl of each antibody at room temperature for 1 h before addition of the probe. Anti-CCAAT/enhancer-binding protein (CEBP) antibodies used (Santa Cruz Biotechnology, Santa Cruz, CA) were anti-CEBPβ (sc-746, which reacts with CEBPβ, -α, -δ, and -ϵ), anti-CEBPα (sc-9314), anti-CEBPβ (sc-150), and anti-CEBPδ (sc-151). Anti-acetyl-histone H3 antibody (Upstate Biotechnology, Charlottesville, VA) was used as control.Two oligonucleotide probes were made corresponding to -55 to -34 bp (Fig. 4A, underlined), one with wild-type sequence and the other with mutation of E-box 1 (CACGTG to ACCGTA). For cold oligomer competition experiments, 1-, 10-, and 100-fold molar excess of unlabeled annealed oligonucleotides, wild-type or E-box 1 mutation, were added to binding reactions containing 0.5 μg of poly(dI-dC), and the mixtures were kept on ice for 10 min before adding 32P-labeled wild-type probe.RESULTSCloning and Sequence Comparison of the 5′-Upstream Region of Human and Murine Vmd2—Genomic clones corresponding to human and murine Vmd2 were obtained as described under “Experimental Procedures.” The transcription start site for VMD2 was determined as 111 bp upstream from the initiation ATG by 5′-rapid amplification of cDNA ends using human RPE RNA (data not shown). This result was confirmed by primer extension using two independent primers (Fig. 1A).The 5′-upstream regions of human and murine Vmd2 were sequenced 2.3 and 5.7 kb from the transcription start site, respectively. Within 2.3 kb of the 5′-flanking region of human VMD2, two repetitive sequences are present: Alu at -2391 to -2113 bp and L1 at -1471 to -1280 bp. In murine Vmd2, the B1 repeat is located at -396 to -267 bp (assuming the transcription start site based on homology to the human gene). Sequence alignment of these 5′-flanking regions of the two species demonstrated many blocks of conserved sequences particularly in three regions: -1579 to -1523, -664 to -530, and -244 bp to the 5′-untranslated region (data not shown).The VMD2 5′-upstream region contains several consensus transcription factor binding sites, including three E-box, E-box 1 (-47 to -42 bp), E-box 2 (-69 to -64 bp), and E-box 3 (-847 to -842 bp); NF1/CTF1 (-782 to -769 bp); two octamer (-420 to -413 and -591 to -584 bp); CEBP (-565 to -556 bp); and PCE-1-like (-174 to -168 bp) binding sites (Fig. 1B). Four Crx/Otx-like homeodomain protein binding sites are also present at -81 to -76, -127 to -122, -1088 to -1083, and -1553 to -1548 bp. Although many blocks of conserved sequences are seen in the 5′-upstream region between the two species, some of the above mentioned consensus sequences are not found in murine Vmd2. For instance, among three E-box sites found in VMD2, only E-box 2 is conserved in the murine gene, and neither the NF1/CTF1, CEBP, nor one octamer (-420 to -413 bp) binding site is well conserved. In contrast, a PCE-1-like sequence is identified in both species, and it differs by only one nucleotide from the consensus sequence CAATTAG. Interestingly all three E-box sites, of which a consensus is CANNTG, are flanked by 5′ ANT residues, creating ANTCANNTG, similar to the M-box in which a core CATGTG motif is flanked by specific 5′ re" @default.
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• W2020149574 date "2004-04-01" @default.
• W2020149574 modified "2023-10-17" @default.
• W2020149574 title "Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation" @default.
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• W2020149574 doi "https://doi.org/10.1074/jbc.m309881200" @default.
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