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• W2102870423 abstract "Vertebrate rhodopsin promoters exhibit striking sequence identities proximal to the initiation site, suggesting that conserved transcription factors regulate rhodopsin expression in these animals. We identify and characterize two transcriptional activators of the Xenopus rhodopsin gene: homologs of the mammalian Crx and Nrl transcription factors, XOtx5 and XL-Nrl (originally named XL-maf), respectively. XOtx5 stimulated transcription ∼10-fold in human 293 cells co-transfected with a plasmid containing the rhodopsin promoter (–508 to +41) upstream of luciferase, similar to the ∼6-fold stimulation with human Crx. XL-Nrl stimulated transcription ∼27-fold in mammalian 293 cells co-transfected with the rhodopsin luciferase reporter, slightly more than the ∼17-fold stimulation with Nrl. Together, the Xenopus transcription factors synergistically activated the rhodopsin promoter (∼140-fold), as well as in combination with mammalian homologs. Deletion of the Nrl-response element, TGCTGA, eliminated the synergistic activation by both mammalian and Xenopus transcription factors. Deletion of the conserved ATTA sequences (Ret-1 or BAT-1), binding sites for Crx, did not significantly decrease activation by Crx/XOtx5. However, there was increased activation by Nrl/XL-Nrl and an increased synergy when the Ret-1 site was disrupted. These results illustrate conservation of mechanisms of retinal gene expression among vertebrates. In transgenic tadpoles, XOtx5 and XL-Nrl directed premature and ectopic expression from the Xenopus rhodopsin promoter-GFP transgene. Furthermore, activation of the endogenous rhodopsin gene was also observed in some animals, showing that XOtx5 and XL-Nrl can activate the promoter in native chromatin environment. Vertebrate rhodopsin promoters exhibit striking sequence identities proximal to the initiation site, suggesting that conserved transcription factors regulate rhodopsin expression in these animals. We identify and characterize two transcriptional activators of the Xenopus rhodopsin gene: homologs of the mammalian Crx and Nrl transcription factors, XOtx5 and XL-Nrl (originally named XL-maf), respectively. XOtx5 stimulated transcription ∼10-fold in human 293 cells co-transfected with a plasmid containing the rhodopsin promoter (–508 to +41) upstream of luciferase, similar to the ∼6-fold stimulation with human Crx. XL-Nrl stimulated transcription ∼27-fold in mammalian 293 cells co-transfected with the rhodopsin luciferase reporter, slightly more than the ∼17-fold stimulation with Nrl. Together, the Xenopus transcription factors synergistically activated the rhodopsin promoter (∼140-fold), as well as in combination with mammalian homologs. Deletion of the Nrl-response element, TGCTGA, eliminated the synergistic activation by both mammalian and Xenopus transcription factors. Deletion of the conserved ATTA sequences (Ret-1 or BAT-1), binding sites for Crx, did not significantly decrease activation by Crx/XOtx5. However, there was increased activation by Nrl/XL-Nrl and an increased synergy when the Ret-1 site was disrupted. These results illustrate conservation of mechanisms of retinal gene expression among vertebrates. In transgenic tadpoles, XOtx5 and XL-Nrl directed premature and ectopic expression from the Xenopus rhodopsin promoter-GFP transgene. Furthermore, activation of the endogenous rhodopsin gene was also observed in some animals, showing that XOtx5 and XL-Nrl can activate the promoter in native chromatin environment. Photoreceptors are highly specialized cells with complex structures that permit efficient light absorption, high signal transduction amplification, rapid kinetics, and adaptation over a range of light intensities (1Dowling J.E. The Retina: An Approachable Part of the Brain. Belknap Press of Harvard University Press, Cambridge, MA1987Google Scholar). Phototransduction requires the coordinated expression of many genes, including the visual pigments that absorb light, enzymes involved in the cGMP cascade, ion channels plus multiple regulatory and structural proteins. It has been estimated using serial analysis of gene expression that ∼4% of the genes expressed in the retina encode phototransduction proteins (2Sharon D. Blackshaw S. Cepko C.L. Dryja T.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 315-320Crossref PubMed Scopus (144) Google Scholar). Many of these genes are quite conserved in vertebrates. Moreover, programs of eye development share many conserved features in the animal kingdom (3Oliver G. Gruss P. Trends Neurosci. 1997; 20: 415-421Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 4Pichaud F. Desplan C. Curr. Opin. Genet. Dev. 2002; 12: 430-434Crossref PubMed Scopus (104) Google Scholar). Even in such distantly related species as Drosophila and humans, similar transcription factors are involved in development and expression of retina-specific genes (5Kumar J.P. Moses K. Semin. Cell Dev. Biol. 2001; 12: 469-474Crossref PubMed Scopus (51) Google Scholar, 6Tahayato A. Sonneville R. Pichaud F. Wernet M.F. Papatsenko D. Beaufils P. Cook T. Desplan C. Dev. Cell. 2003; 5: 391-402Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), although the evolutionary significance is not yet settled (5Kumar J.P. Moses K. Semin. Cell Dev. Biol. 2001; 12: 469-474Crossref PubMed Scopus (51) Google Scholar, 7Cook T. Bioessays. 2003; 25: 921-925Crossref PubMed Scopus (27) Google Scholar, 8Hanson I.M. Semin. Cell Dev. Biol. 2001; 12: 475-484Crossref PubMed Scopus (90) Google Scholar). Often, proteins involved in growth and differentiation of the eye from one species can substitute for homologs in distantly related species (4Pichaud F. Desplan C. Curr. Opin. Genet. Dev. 2002; 12: 430-434Crossref PubMed Scopus (104) Google Scholar, 7Cook T. Bioessays. 2003; 25: 921-925Crossref PubMed Scopus (27) Google Scholar). This conservation also extends to the cis-acting elements in proximal promoters of retinal genes. Promoters have exhibited at least partial functionality between mammals and lower vertebrates. For example, the human β-PDE and IRBP proximal promoters were found to drive rod or photoreceptor-specific expression, respectively, in Xenopus (9Lerner L.E. Gribanova Y.E. Ji M. Knox B.E. Farber D.B. J. Biol. Chem. 2001; 276: 34999-35007Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Boatright J.H. Knox B.E. Jones K.M. Stodulkova E. Nguyen H.T. Padove S.A. Borst D.E. Nickerson J.M. FEBS Lett. 2001; 504: 27-30Crossref PubMed Scopus (15) Google Scholar). Current attention is focused upon identifying the specific sequences that are important for cell-specific expression and the transcription factors that regulate gene expression. Rhodopsin is found exclusively in rods in high abundance and its expression level is controlled primarily at the level of transcription initiation (11Treisman J.E. Morabito M.A. Barnstable C.J. Mol. Cell. Biol. 1988; 8: 1570-1579Crossref PubMed Scopus (93) Google Scholar, 12DesJardin L.E. Timmers A.M. Hauswirth W.W. J. Biol. Chem. 1993; 268: 6953-6960Abstract Full Text PDF PubMed Google Scholar). Proper rhodopsin mRNA levels are necessary for rod differentiation and photoreceptor layer maintenance (13Humphries M.M. Rancourt D. Farrar G.J. Kenna P. Hazel M. Bush R.A. Sieving P.A. Sheils D.M. McNally N. Creighton P. Erven A. Boros A. Gulya K. Capecchi M.R. Humphries P. Nat. Genet. 1997; 15: 216-219Crossref PubMed Scopus (459) Google Scholar, 14Lem J. Krasnoperova N.V. Calvert P.D. Kosaras B. Cameron D.A. Nicolo M. Makino C.L. Sidman R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 736-741Crossref PubMed Scopus (334) Google Scholar, 15Toda K. Bush R.A. Humphries P. Sieving P.A. Visual Neurosci. 1999; 16: 391-398Crossref PubMed Scopus (84) Google Scholar, 16Olsson J.E. Gordon J.W. Pawlyk B.S. Roof D. Hayes A. Molday R.S. Mukai S. Cowley G.S. Berson E.L. Dryja T.P. Neuron. 1992; 9: 815-830Abstract Full Text PDF PubMed Scopus (407) Google Scholar). Transgenic experiments have shown that rhodopsin promoters are able to direct high level expression of reporter genes to the photoreceptor layer across species: between mammals, amphibians, and fish (17Gouras P. Kjeldbye H. Zack D.J. Visual Neurosci. 1994; 11: 1227-1231Crossref PubMed Scopus (19) Google Scholar, 18Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 19Perkins B.D. Kainz P.M. O'Malley D.M. Dowling J.E. Visual Neurosci. 2002; 19: 257R-264RCrossref PubMed Scopus (38) Google Scholar, 20Zhang T. Tan Y.H. Fu J. Lui D. Ning Y. Jirik F.R. Brenner S. Venkatesh B. Gene (Amst.). 2003; 313: 189-200Crossref PubMed Scopus (13) Google Scholar), although there were some differences in early expression patterns, mosaicism, and some loss of rod-restricted expression (17Gouras P. Kjeldbye H. Zack D.J. Visual Neurosci. 1994; 11: 1227-1231Crossref PubMed Scopus (19) Google Scholar, 18Zack D.J. Bennett J. Wang Y. Davenport C. Klaunberg B. Gearhart J. Nathans J. Neuron. 1991; 6: 187-199Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 19Perkins B.D. Kainz P.M. O'Malley D.M. Dowling J.E. Visual Neurosci. 2002; 19: 257R-264RCrossref PubMed Scopus (38) Google Scholar, 20Zhang T. Tan Y.H. Fu J. Lui D. Ning Y. Jirik F.R. Brenner S. Venkatesh B. Gene (Amst.). 2003; 313: 189-200Crossref PubMed Scopus (13) Google Scholar). The conservation of transcriptional mechanisms is also apparent in the sequence similarities in the rhodopsin proximal promoter (20Zhang T. Tan Y.H. Fu J. Lui D. Ning Y. Jirik F.R. Brenner S. Venkatesh B. Gene (Amst.). 2003; 313: 189-200Crossref PubMed Scopus (13) Google Scholar, 21Batni S. Scalzetti L. Moody S.A. Knox B.E. J. Biol. Chem. 1996; 271: 3179-3186Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22Ma G.C. Wang T.M. Su C.Y. Wang Y.L. Chen S. Tsai H.J. FEBS Lett. 2001; 508: 265-271Crossref PubMed Scopus (10) Google Scholar, 23Mani S.S. Batni S. Whitaker L. Chen S. Engbretson G. Knox B.E. J. Biol. Chem. 2001; 276: 36557-36565Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Two of the most highly conserved sequences are the Ret-1 and BAT-1 elements, each of which contain a core ATTA, and are recognized by Otx family homeodomain proteins in vitro (i.e. Crx (23Mani S.S. Batni S. Whitaker L. Chen S. Engbretson G. Knox B.E. J. Biol. Chem. 2001; 276: 36557-36565Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar)). The functional role these elements play in vivo is not fully understood (23Mani S.S. Batni S. Whitaker L. Chen S. Engbretson G. Knox B.E. J. Biol. Chem. 2001; 276: 36557-36565Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Another highly conserved feature is the Nrl response element (NRE), 1The abbreviations used are: NRE, Nrl response element; RT, reverse transcriptase; XOP, Xenopus rhodopsin promoter –508/+41; GFP, green fluorescent protein. TGCTGA. The NRE is necessary for high levels of rhodopsin expression in both transfected mammalian cells and in transgenic Xenopus (20Zhang T. Tan Y.H. Fu J. Lui D. Ning Y. Jirik F.R. Brenner S. Venkatesh B. Gene (Amst.). 2003; 313: 189-200Crossref PubMed Scopus (13) Google Scholar, 23Mani S.S. Batni S. Whitaker L. Chen S. Engbretson G. Knox B.E. J. Biol. Chem. 2001; 276: 36557-36565Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 25Rehemtulla A. Warwar R. Kumar R. Ji X. Zack D.J. Swaroop A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 191-195Crossref PubMed Scopus (186) Google Scholar, 26Kumar 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: 29612-29618Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Given the functional and sequence similarities in rhodopsin promoters, it appears likely that homologous transcription factors would mediate transcriptional activation in vertebrates. Two important activators of the mammalian rhodopsin promoter are Crx and Nrl. Crx is a divergent member of the Otx5 subclass, related to the Drosophila otd (orthodenticle) protein (27Gamse J.T. Shen Y.C. Thisse C. Thisse B. Raymond P.A. Halpern M.E. Liang J.O. Nat. Genet. 2002; 30: 117-121Crossref PubMed Scopus (139) Google Scholar, 28Sauka-Spengler T. Baratte B. Shi L. Mazan S. Dev. Genes Evol. 2001; 211: 533-544Crossref PubMed Scopus (32) Google Scholar, 29Plouhinec J.L. Sauka-Spengler T. Germot A. Le Mentec C. Cabana T. Harrison G. Pieau C. Sire J.Y. Veron G. Mazan S. Mol. Biol. Evol. 2003; 20: 513-521Crossref PubMed Scopus (42) Google Scholar). Nrl, a bZIP containing transcription factor, is a member of the large Maf family (30Swaroop A. Xu J.Z. Pawar H. Jackson A. Skolnick C. Agarwal N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 266-270Crossref PubMed Scopus (269) Google Scholar, 31Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (223) Google Scholar). Nrl is expressed specifically in developing and mature rods (32Swain P.K. Hicks D. Mears A.J. Apel I.J. Smith J.E. John S.K. Hendrickson A. Milam A.H. Swaroop A. J. Biol. Chem. 2001; 276: 36824-36830Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), and has been shown to activate expression of rhodopsin both in non-retinal and retinal cell cultures (24Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 25Rehemtulla A. Warwar R. Kumar R. Ji X. Zack D.J. Swaroop A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 191-195Crossref PubMed Scopus (186) Google Scholar, 26Kumar 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: 29612-29618Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Furthermore, Nrl has been shown to bind to Crx (33Mitton K.P. Swain P.K. Chen S. Xu S. Zack D.J. Swaroop A. J. Biol. Chem. 2000; 275: 29794-29799Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and with Crx, can synergistically activate expression of the rhodopsin promoter (24Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). We show here that Xenopus XOtx5 (34Vignali R. Colombetti S. Lupo G. Zhang W. Stachel S. Harland R.M. Barsacchi G. Mech. Dev. 2000; 96: 3-13Crossref PubMed Scopus (46) Google Scholar, 35Kuroda H. Hayata T. Eisaki A. Asashima M. Dev. Growth Differ. 2000; 42: 87-93Crossref PubMed Google Scholar) and XL-maf (36Ishibashi S. Yasuda K. Mech. Dev. 2001; 101: 155-166Crossref PubMed Scopus (55) Google Scholar) are the functional Xenopus counterparts of mammalian Crx and Nrl. Phylogenic Analysis—Long maf protein sequences were retrieved from the GenBank™ data base. Their accession numbers are as follows: chicken c-maf 516681, chicken L-maf AF034570, chicken mafB 516723, chicken mafF 439705, chicken mafG 1020399, chicken mafK 439707, human Nrl 11433960, human KRML 14770835, human MafG AF059195, human MafK AF059194, human U-maf 6429133, human long c-maf AF055377, human maf 2344815, human mafF 6912489, human short c-maf AF055376, human v-maf 5453735, mouse mafF 2749779, mouse mafG 2749778, mouse mafK 976236, mouse Nrl 6679131, mouse v-maf 6754611, rat maf1 U56241, rat maf2 U56242, rat maf G-2 13646953, Xenopus L-maf AF 202059, Xenopus mafB AF202058, zebrafish c-maf 12381856, zebrafish maf AF109781, and quail MafA AAC60377. The Xenopus tropicalis L-maf was obtained from the X. tropicalis genome sequence (scaffold_1738, version 1.0 (37Institute Joint Genome Xenopus Tropicalis Database. 2004. DOE Joint Genome Institute, 2004Google Scholar)). The sequences were aligned using CLUSTALX (version 1.81) (38Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35620) Google Scholar). Phylogenetic trees were constructed using neighbor-joining (39Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar), maximum parsimony, and maximum likelihood algorithms using ProtDist and Neighbor as implemented in the PHYLIP package (40Felsenstein J. PHYLIP (Phylogeny Inference Package). Department of Genetics, University of Washington, Seattle1993Google Scholar); PAUP, version 4.0b (41Swofford D.L. PAUP*4b10 Phylogenetic Analysis Using Parsimony and Other Methods. 4.0b Ed. Sinauer Associates, Sunderland, MA1996Google Scholar); and PUZZLE, version 4.0.2, (42Strimmer K. Haesler A. Mol. Biol. Evol. 1996; 13: 964-969Crossref Scopus (2276) Google Scholar), respectively. In the neighbor-joining analysis, distances were computed with the method of Kimura (43Kimura M. J. Mol. Evol. 1980; 16: 111-120Crossref PubMed Scopus (23653) Google Scholar). The maximum parsimony tree was obtained by 10 random addition heuristic search replicates and the tree bisection-reconnection branch-swapping option. Maximum likelihood trees were constructed by the quick-add OTUs search with the JTT-f model of amino acid substitution, retaining the 2,000 top ranking trees. Bootstrap proportions were calculated by analysis of 2,000 replicates for neighbor-joining and 1000 for maximum parsimony, and 2,000 puzzles for maximum likelihood. Expression Constructs—Full-length expression constructs for pcDNA-Crx (human) and pMT3-Nrl (human) were obtained from S. Chen and A. Swaroop, respectively, and have been described previously (24Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 30Swaroop A. Xu J.Z. Pawar H. Jackson A. Skolnick C. Agarwal N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 266-270Crossref PubMed Scopus (269) Google Scholar). The pCS2-XOtx5b full-length expression construct was obtained from A. Viczian and has been described previously (34Vignali R. Colombetti S. Lupo G. Zhang W. Stachel S. Harland R.M. Barsacchi G. Mech. Dev. 2000; 96: 3-13Crossref PubMed Scopus (46) Google Scholar). The XL-maf coding region was produced by RT-PCR (see below) and the resulting PCR product was cloned into pBluescript using the engineered BamHI and EcoRI sites. This construct was then digested with SpeI and EcoRI and the XL-maf coding region was moved into the pMT3 vector that had been modified with adapters (5′-AATTAACTAGTCTGGAATTCAT-3′ top and 5′-TCGAATGAATTCCAGACTAGTT-3′ bottom). XMafB was amplified from cDNA prepared from stage 38 Xenopus embryo heads (see below) and cloned into the modified pMT vector. Promoter Constructs—The XOP-GFP reporter construct contains the Xenopus rhodopsin promoter –508/+41 (XOP), driving expression of GFP. The luciferase reporter construct, XOP-GL2, contains XOP (–503/+41) driving expression of luciferase. The XOP deletion constructs contained the full-length XOP promoter with targeted disruption of specific elements (ΔRet1, Δ–136 to –122; ΔBAT, Δ–107 to –91; ΔNRE, Δ–84 to –58), driving expression of luciferase. All reporter constructs were described previously (23Mani S.S. Batni S. Whitaker L. Chen S. Engbretson G. Knox B.E. J. Biol. Chem. 2001; 276: 36557-36565Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 293 Transfections—293 cells (human embryonic kidney cell line) were co-transfected in 24-well plates with a total of 0.7 μg of DNA and LipofectAMINE PLUS transfection reagent as per the manufacturer's protocol (Invitrogen). 0.2 μg of XOP-GL2 was transfected in each well, along with various combinations of mammalian and Xenopus expression constructs. Each transfection also contained 0.1 μg of a Renilla luciferase reporter under the control of the thymidine kinase promoter (pRL-TK, Invitrogen). Empty pMT3 vector was included in transfections when necessary to bring total DNA to 0.7 μg. Cells were transfected for 3 h in the absence of serum, and harvested 42 h post-transfection. Cell lysates were analyzed using luciferase assay reagent (Invitrogen) to assay luciferase expression. Expression levels were determined relative to each construct alone. RT-PCR for XL-maf and XMafB—RNAs from adult Xenopus retina and stage 38 Xenopus heads were reverse transcribed with gene-specific primers to produce cDNA (SuperscriptII RT polymerase, Invitrogen). XL-maf was amplified using primers as previously described (36Ishibashi S. Yasuda K. Mech. Dev. 2001; 101: 155-166Crossref PubMed Scopus (55) Google Scholar): forward, 5′-CCCGGATCCATGGCACTCGATGATCTACCC-3′ and reverse, 5′-GGGGAATTCTCACAGAAAGAGCTCAGCTCC-3′, with the addition of BamHI and EcoRI sites, respectively, to facilitate cloning. XOtx2 primers were used as a positive control (forward, 5′-AGGGAAAGGACCACTTTCAC and reverse, 5′-CCAGATGGACACAGGGGCTG). 1/10 of the RT reaction was amplified with Taq polymerase (Promega, Madison, WI) using the following PCR parameters: 1 min at 94 °C to denature, then 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C, and a final extension of 3 min at 72 °C. The PCR products were cloned into pBluescript and 14 clones were sequenced (Davis Sequencing, Davis, CA). Sequence comparisons were performed on the deduced polypeptide sequences using the Lasergene package (DNAStar, Madison, WI), after correcting sequences for nucleotide substitutions that occurred in only one clone (arising during PCR amplification). XMafB was amplified from cDNA prepared from stage 38 Xenopus embryo heads using the primers: forward, 5′-GGACTAGTCATGCGTGGAGAGTTGC and reverse, 5′-GAGAATTCCTCACATGAAGAACTCTGG. SpeI and EcoRI sites were added for cloning the insert into the modified pMT vector. PCR parameters were 1 min at 94 °C to denature, 35 cycles of 30 s at 94 °C, 30 s at 53 °C, and 45 s at 72 °C, and a final extension of 3 min at 72 °C. Clones were confirmed by sequencing (Davis Sequencing). In Situ Hybridizations—The XL-maf/pBluescriptII clone was digested with either EcoRI or SpeI and the linearized DNA was used to produce sense and antisense digoxigenin-labeled probes (Roche Diagnostics) using T3 and T7 polymerases, respectively. In situ hybridization was performed on sections of stage 48 Xenopus tadpoles and adult Xenopus eyes as described (44Shimamura K. Hirano S. McMahon A.P. Takeichi M. Development. 1994; 120: 2225-2234Crossref PubMed Google Scholar), except that the digoxigenin-labeled probe was hybridized for 2 days at 65 °C. Embryo Transfections—Embryo transfections were performed as described previously (23Mani S.S. Batni S. Whitaker L. Chen S. Engbretson G. Knox B.E. J. Biol. Chem. 2001; 276: 36557-36565Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) except that N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-triethylammonium methylsulfate was used for all transfections (Roche). 2.5 μg of XOP-GL was used per transfection and 2.5 μg of each transcription factor plasmid was used as well, with empty pMT plasmid included when necessary to bring total DNA up to 7.5 μg. The DNA to lipid ratio was 1:3. Transgenic Xenopus—Transgenic animals were produced using standard restriction enzyme-mediated integration (45Batni S. Mani S.S. Schlueter C. Ji M. Knox B.E. Methods Enzymol. 2000; 316: 50-64Crossref PubMed Google Scholar, 46Kroll K.L. Amaya E. Development. 1996; 122: 3173-3183Crossref PubMed Google Scholar), with the following modifications: pCS2+-Otx5b was linearized with SalI, XL-maf-PMT and empty pMT3 were linearized with AvrII, and XOP-GFP was linearized with XhoI (New England Biolabs, Beverly, MA). The restriction enzyme-mediated integration reaction for each combination included 0.15 units of each restriction enzyme, 400 ng of XOP-GFP reporter, 100 ng of pCS2-XOtx5b or XL-maf-PMT, and when necessary, empty pMT3 vector to bring total DNA up to 600 ng/∼104 sperm nuclei. Restriction enzyme-mediated integration reactions contained 5 μl of egg extract and frozen rather than fresh sperm were used (47Sparrow D.B. Latinkic B. Mohun T.J. Nucleic Acids Res. 2000; 28: E12Crossref PubMed Scopus (121) Google Scholar). GFP expression was followed for 5 days of development by using fluorescence microscopy of live animals. Images were produced using a SPOT CCD camera (Diagnostic Instruments, Inc., McHenry, IL) and Adobe Photoshop (Adobe, San Jose, CA). RT-PCR for GFP and Endogenous Rhodopsin—RNA was isolated from the tails of 2-day-old tadpoles positive for GFP expression for the treatments: XOP + XL-maf, XOP + XOtx5, and XOP + XL-maf + XOtx5. Five GFP positive animals from each treatment were analyzed. Three animals from the group injected with XOP + empty pMT vector were also analyzed. RNA was also prepared from one 6-day-old XOP positive tadpole (entire embryo, including the eye) as a positive control for both GFP and rhodopsin. RNA was reverse transcribed with random hexamers and SuperScriptII RT polymerase (Invitrogen), and treated with DNase (Promega) following the manufacturers' protocols. The resulting cDNA was amplified in a standard PCR with primers for GFP (forward, 5′-ATGGTGAGCAAGGGCGAGG; reverse, 5′-CCTTGAAGAAGATGGTGCGCTC) and Xenopus rhodopsin (forward, 5′-ATGAACGGAACAGAAGGTCCA; reverse, 5′-CCAGTGACCAGAGGGCC). The rhodopsin primers were designed to amplify across the first intron of the rhodopsin gene to produce a 376-bp band from cDNA. PCR parameters were as follows: GFP, 1 min at 94 °C to denature, then 36 cycles of 45 s at 94 °C, 1 min at 45 °C, and 1 min at 72 °C, and a final extension of 3 min at 72 °C; XOP, 1 min at 94 °C to denature, then 35 cycles of 45 s at 94 °C, 45 s at 55 °C, and 45 s at 72 °C, and a final extension of 3 min at 72 °C. XOtx5 Activates the Xenopus Rhodopsin Promoter—Recent studies suggest that Crx represents a divergent branch of the Otx5 family based on both phylogeny (Refs. 28Sauka-Spengler T. Baratte B. Shi L. Mazan S. Dev. Genes Evol. 2001; 211: 533-544Crossref PubMed Scopus (32) Google Scholar, 29Plouhinec J.L. Sauka-Spengler T. Germot A. Le Mentec C. Cabana T. Harrison G. Pieau C. Sire J.Y. Veron G. Mazan S. Mol. Biol. Evol. 2003; 20: 513-521Crossref PubMed Scopus (42) Google Scholar, and 48Germot A. Lecointre G. Plouhinec J.L. Le Mentec C. Girardot F. Mazan S. Mol. Biol. Evol. 2001; 18: 1668-1678Crossref PubMed Scopus (46) Google Scholar, and Supplementary Materials) and embryonic expression patterns (28Sauka-Spengler T. Baratte B. Shi L. Mazan S. Dev. Genes Evol. 2001; 211: 533-544Crossref PubMed Scopus (32) Google Scholar, 35Kuroda H. Hayata T. Eisaki A. Asashima M. Dev. Growth Differ. 2000; 42: 87-93Crossref PubMed Google Scholar, 49Viczian A.S. Vignali R. Zuber M.E. Barsacchi G. Harris W.A. Development. 2003; 130: 1281-1294Crossref PubMed Scopus (97) Google Scholar). Xenopus laevis contains two paralogous Otx5 genes (29Plouhinec J.L. Sauka-Spengler T. Germot A. Le Mentec C. Cabana T. Harrison G. Pieau C. Sire J.Y. Veron G. Mazan S. Mol. Biol. Evol. 2003; 20: 513-521Crossref PubMed Scopus (42) Google Scholar), which are expressed in photoreceptors and bipolar cells in embryonic retina (28Sauka-Spengler T. Baratte B. Shi L. Mazan S. Dev. Genes Evol. 2001; 211: 533-544Crossref PubMed Scopus (32) Google Scholar, 35Kuroda H. Hayata T. Eisaki A. Asashima M. Dev. Growth Differ. 2000; 42: 87-93Crossref PubMed Google Scholar, 49Viczian A.S. Vignali R. Zuber M.E. Barsacchi G. Harris W.A. Development. 2003; 130: 1281-1294Crossref PubMed Scopus (97) Google Scholar). To determine whether XOtx5 functions similarly to mammalian Crx, we tested the ability of XOtx5 to activate Xenopus rhodopsin. In 293 cells, the XOP-GL2 directed very weak expression of a luciferase reporter (∼3-fold over a promoter-less control, data not shown), as expected from the in vivo cell specificity. When an XOtx5 expression construct was included in the transfection, significant stimulation (∼10-fold over XOP-GL2 alone) of transcriptional activity was observed (Fig. 1, A and C). In transfections in which the XOP-GL2 reporter was increased, an even greater increase in transcriptional activity was observed (Fig. 1B). This magnitude of activation is similar to that seen in transfections with a mammalian promoter and Crx (24Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). A key feature of the Crx transcription factor is the synergistic activation of a mammalian rhodopsin promoter in the presence of Nrl. Co-transfections of 293 cells with plasmids harboring XOtx5 and human Nrl resulted in an activation of over 100-fold (Fig. 1C) (note: Nrl alone only activates the Xenopus promoter ∼17-fold, see Fig. 5A). This synergistic activation with XOtx5, although slightly lower in combination with human Nrl, is comparable with the activation of the mammalian promoter (24Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar) and Xenopus rhodopsin promoter (Fig. 1C) with Crx. These results confirm the earlier phylogenetic results and indicate that XOtx5 has the same function as mammalian Crx in transcription assays.Fig. 5Comparison of transcriptional activities with rhodopsin-targeted disruption constructs. A, the luciferase activity from 293 cells transfected with various targeted disruption constructs, human Crx, and/or human Nrl are shown relative to the activity observed by transfection of each promoter construct alone (n = 2–6). 200 ng of each construct were used, and empty pMT was included when necessary to make the total transfected DNA equ" @default.
• W2102870423 created "2016-06-24" @default.
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• W2102870423 date "2004-11-01" @default.
• W2102870423 modified "2023-09-28" @default.
• W2102870423 title "Conserved Transcriptional Activators of the Xenopus Rhodopsin Gene" @default.
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• W2102870423 doi "https://doi.org/10.1074/jbc.m406080200" @default.
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