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Originally published In Press as doi:10.1074/jbc.M406080200 on August 30, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49010-49018, November 19, 2004
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Conserved Transcriptional Activators of the Xenopus Rhodopsin Gene*{boxs}

S. Leigh Whitaker and Barry E. Knox{ddagger}

From the Departments of Biochemistry & Molecular Biology and Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210

Received for publication, June 1, 2004 , and in revised form, July 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1). 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 (2). Many of these genes are quite conserved in vertebrates. Moreover, programs of eye development share many conserved features in the animal kingdom (3, 4). Even in such distantly related species as Drosophila and humans, similar transcription factors are involved in development and expression of retina-specific genes (5, 6), although the evolutionary significance is not yet settled (5, 7, 8). Often, proteins involved in growth and differentiation of the eye from one species can substitute for homologs in distantly related species (4, 7). 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 {beta}-PDE and IRBP proximal promoters were found to drive rod or photoreceptor-specific expression, respectively, in Xenopus (9, 10). 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 (11, 12). Proper rhodopsin mRNA levels are necessary for rod differentiation and photoreceptor layer maintenance (13–16). 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 (17–20), although there were some differences in early expression patterns, mosaicism, and some loss of rod-restricted expression (17–20). The conservation of transcriptional mechanisms is also apparent in the sequence similarities in the rhodopsin proximal promoter (20–23). 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 (23, 24)). The functional role these elements play in vivo is not fully understood (23). Another highly conserved feature is the Nrl response element (NRE),1 TGCTGA. The NRE is necessary for high levels of rhodopsin expression in both transfected mammalian cells and in transgenic Xenopus (20, 23, 25, 26). 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 (27–29). Nrl, a bZIP containing transcription factor, is a member of the large Maf family (30, 31). Nrl is expressed specifically in developing and mature rods (32), and has been shown to activate expression of rhodopsin both in non-retinal and retinal cell cultures (24–26). Furthermore, Nrl has been shown to bind to Crx (33), and with Crx, can synergistically activate expression of the rhodopsin promoter (24). We show here that Xenopus XOtx5 (34, 35) and XL-maf (36) are the functional Xenopus counterparts of mammalian Crx and Nrl.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Phylogenic Analysis—Long maf protein sequences were retrieved from the GenBankTM data base. Their accession numbers are as follows: chicken c-maf 516681, chicken L-maf AF034570 [GenBank] , chicken mafB 516723, chicken mafF 439705, chicken mafG 1020399, chicken mafK 439707, human Nrl 11433960, human KRML 14770835, human MafG AF059195 [GenBank] , human MafK AF059194 [GenBank] , human U-maf 6429133, human long c-maf AF055377 [GenBank] , human maf 2344815, human mafF 6912489, human short c-maf AF055376 [GenBank] , human v-maf 5453735, mouse mafF 2749779, mouse mafG 2749778, mouse mafK 976236, mouse Nrl 6679131, mouse v-maf 6754611, rat maf1 U56241 [GenBank] , rat maf2 U56242 [GenBank] , rat maf G-2 13646953, Xenopus L-maf AF 202059, Xenopus mafB AF202058 [GenBank] , zebrafish c-maf 12381856, zebrafish maf AF109781 [GenBank] , and quail MafA AAC60377 [GenBank] The Xenopus tropicalis L-maf was obtained from the X. tropicalis genome sequence (scaffold_1738, version 1.0 (37)). The sequences were aligned using CLUSTALX (version 1.81) (38). Phylogenetic trees were constructed using neighbor-joining (39), maximum parsimony, and maximum likelihood algorithms using ProtDist and Neighbor as implemented in the PHYLIP package (40); PAUP, version 4.0b (41); and PUZZLE, version 4.0.2, (42), respectively. In the neighbor-joining analysis, distances were computed with the method of Kimura (43). 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 (24, 30). The pCS2-XOtx5b full-length expression construct was obtained from A. Viczian and has been described previously (34). 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 ({Delta}Ret1, {Delta}–136 to –122; {Delta}BAT, {Delta}–107 to –91; {Delta}NRE, {Delta}–84 to –58), driving expression of luciferase. All reporter constructs were described previously (23).

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 (36): 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 (44), except that the digoxigenin-labeled probe was hybridized for 2 days at 65 °C.

Embryo Transfections—Embryo transfections were performed as described previously (23) 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 (45, 46), 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 (47). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
XOtx5 Activates the Xenopus Rhodopsin Promoter—Recent studies suggest that Crx represents a divergent branch of the Otx5 family based on both phylogeny (Refs. 28, 29, and 48, and Supplementary Materials) and embryonic expression patterns (28, 35, 49). Xenopus laevis contains two paralogous Otx5 genes (29), which are expressed in photoreceptors and bipolar cells in embryonic retina (28, 35, 49). 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 (24). 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 (24) 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.



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  		FIG. 1.
XOtx5 and XL-Nrl activate the Xenopus rhodopsin promoter. A, Xenopus transcription factors XOtx5 and XL-maf activate the XOP promoter in a dose-dependent manner. 293 cells were transiently co-transfected with 200 ng of XOP-GL2 and increasing amounts of expression constructs, showing a dose-dependent activation of the promoter with saturation at higher concentrations of transcription factor. Error bars represent S.D. Data are presented as relative light units (RLU). B, 293 cells were transiently co-transfected with 200 ng of expression constructs and increasing amounts of XOP-GL2. Addition of increasing amounts of promoter results in increased activity indicating that the transcriptional machinery of the 293 cells is not saturated at the concentrations used in C. Error bars represent S.D. C, XOP-GL2 was transfected into 293 cells with XOtx5 or Crx both individually and in combination with Nrl. 200 ng of each construct was used in each transfection. The luciferase activities are shown relative to the activity observed by the XOP promoter alone. Data are presented as mean ± S.E. D, same as A, with XL-Nrl instead of XOtx5.

 



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  		FIG. 5.
Comparison 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 equivalent. Fold activities are presented as mean ± S.E. B, same as A, using the Xenopus transcription factors XOtx5 and XL-Nrl (n = 3–6). Deletion of the NRE stimulated activity by Xotx5 (p = 0.028), decreased activity stimulated by Nrl or XL-Nrl (p = 0.026, 0.029), and decreased activation stimulated by the co-transfected transcription factors (Crx/Nrl p = 0.029, XOtx5/XL-Nrl p = 0.002). Deletion of the Ret-1 element increased activity stimulated by Nrl or XL-Nrl (p = 0.004, 0.002), and increased activation stimulated by the co-transfected transcription factors (Crx/Nrl p = 0.019, XOtx5/XL-Nrl p = 0.005). Deletion of the BAT-1 element increased activity stimulated by Nrl or XL-Nrl (p = <0.001 for both), but did not significantly change the activity stimulated by co-transfection of the transcription factors (Crx/Nrl p = 0.668, Otx5/XL-Nrl p = 0.099).

 
XL-Nrl Activates the Xenopus Rhodopsin Promoter—We aligned 23 large maf amino acid sequences and used this alignment for phylogenetic analysis. There were 485 amino acids, 157 characters were constant, 65 variable characters were parsimony uninformative, and 264 were parsimony informative. Phylogenetic trees were constructed using neighbor-joining, maximum parsimony, and maximum likelihood. The most parsimonious tree had the following parameters: length = 989 consistency index = 0.8534 and retention index = 0.8861. The maximum likelihood tree length was ln L = –6333.09. Both X. laevis and tropicalis L-maf were found with mammalian Nrl in all three tree reconstructions supported by high bootstrap values (99–100%), demonstrating that they form a distinct subgroup of large maf proteins, separate from chicken L-maf (Fig. 2 and Supplementary Materials). In addition, the Xenopus proteins did not contain the region of histidine repeats found in the other large mafs (Ref. 31 and Supplementary Materials). These results suggested that XL-maf could be the amphibian counterpart to mammalian Nrl. In fact, previous work showed that XL-maf is expressed in the differentiating retina (as well as prominent expression in the developing lens) at early stages, whereas later stages of development were not examined (36).



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  		FIG. 2.
Phylogenetic relationships of large vertebrate Maf genes. Representative phylogenetic tree calculated using maximum parsimony. No root was assumed but for display purposes the tree is shown with mammalian MafB roots. Species abbreviations are as follows: Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Xl, X. laevis; Xt, X. tropicalis; Gg, Gallus gallus; Dn, Danio rerio; Cj, Coturix japonica. Numbers indicate bootstrap values supporting each node. XL-Nrl clusters with mammalian Nrl in all trees (brackets). The scale bar represents the number of steps. Accession numbers are as described under "Experimental Procedures."

 
We investigated XL-maf expression in the mature retina. RT-PCR with XL-maf-specific primers revealed that XL-maf is expressed in stage 38 Xenopus heads as previously reported. Additionally, when RNA was prepared from adult Xenopus retinas and subjected to RT-PCR, the XL-maf-specific primers amplified a band corresponding to the expected size of the XL-maf coding region (data not shown). We sequenced 14 individual clones isolated from the RT-PCR products and found that all were nearly identical to the published XL-maf sequence (36), further confirming the expression of this gene in the adult retina (Supplemental Materials). Translation of nucleotide sequence and alignment of predicted amino acid sequences revealed that all 14 clones and the X. tropicalis sequence had a Gly at position 247 instead of an Arg as previously reported (36). Although the carboxyl termini of the Xenopus and mammalian Nrl/XL-maf are not the same size, alignments also place a Gly at the equivalent position (226) in mammalian Nrl. There were 13 additional positions that exhibited polymorphorisms in more than one clone and they formed four separate groups when aligned (Supplemental Materials). This most likely is a result of gene duplications and the resulting pseudotetraploid Xenopus genome (50).

Whole mount in situ hybridizations on stage 34 Xenopus embryos using a full-length digoxigenin-labeled RNA probe revealed prominent lens expression, confirming the early lens expression previously described (36) (data not shown). To determine the XL-maf expression pattern in young tadpole and adult retina, we performed in situ hybridization on sections. XL-maf RNA was found only in the photoreceptor layer (Fig. 3, A and C), and no signal was detected with the sense probe (Fig. 3, B and D). The RNA was clearly present in rods, however, we could not exclude expression in some cones. These results together with the phylogenetic analysis suggest that XL-maf is the Xenopus homolog of Nrl. We therefore suggest renaming XL-maf to XL-Nrl (Xenopus lens and neural retinal leucine zipper) to emphasize both the unique early lens expression (36) and the conservation with Nrl. We will hereafter refer to XL-maf as XL-Nrl.



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  		FIG. 3.
XL-Nrl is expressed in the photoreceptor layer of immature and adult Xenopus tadpoles. In situ hybridizations were performed on stage 48 (A and B) and adult (C and D) Xenopus sections. Full-length antisense (A and C) and sense (B and D) digoxigenin-labeled RNA probes to XL-Nrl were used. BM purple was used to detect an alkaline peroxidase-conjugated digoxigenin antibody. Blue purple staining can be seen in the outer nuclear layer of the photoreceptors. The light blue staining on the adult sections is 4,6-diamidino-2-phenylindole to visualize nuclei. Bright field (BM purple) and dark field (4,6-diamidino-2-phenylindole) images were merged in Adobe Photoshop. OS, outer segment; RPE, retina pigment epithelia; GC, ganglion cell layer; white arrowhead, outer nuclear layer; open arrowhead, inner nuclear layer. Scale bars, 50 µm (A and C); 100 µm (B and D).

 
We tested the ability of XL-Nrl to activate rhodopsin transcription, an important function of mammalian Nrl. In transfected 293 cells, we found significant stimulation of Xenopus opsin promoter transcription by XL-Nrl (~27-fold over XOP-luciferase alone) (Fig. 1, A and D). Up to ~40-fold increase in promoter activation with a given amount of XL-Nrl plasmid was observed after increasing the amount of XOP-luciferase reporter (Fig. 1B). Co-transfections including either XL-Nrl/XOtx5 or XL-Nrl/human Crx exhibited a remarkably similar synergistic activation of ~150-fold (Fig. 1D).

Specificity of Synergy—Because Otx family proteins have very similar homeodomains (24), we investigated the specificity of synergy by performing transfections with another Otx protein, XOtx2. In transfected 293 cells XOtx2 was able to activate the rhodopsin promoter to similar levels as XOtx5 (Fig. 4A). XOtx2 was also able to synergistically activate the Xenopus rhodopsin promoter together with human Nrl. These results suggest that XOtx2, which shares ~75% overall identity with XOtx5, has enough structural similarity to interact with Nrl and produce the synergistic activation. On the other hand, XMafB, a large maf protein with ~47% identity to XL-Nrl, was able to activate the rhodopsin promoter similarly to XL-Nrl when transfected alone, but showed reduced activation in combination with human Crx (Fig. 4B).



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  		FIG. 4.
XOtx2 and MafB are able to activate the rhodopsin promoter. A, 293 cells were transfected with XOP-GL2 either alone or with various combinations of XOtx5, XOtx2, and human Nrl. The luciferase activities from treatments are shown relative to the activity observed by transfection with XOP-GL2 alone. Data are presented as mean ± S.E. B, XOP-GL2 was transfected either alone or with various combinations of XL-Nrl, XMafB, and human Crx (200 ng of each plasmid used). The luciferase activities are shown relative to the activity observed by transfection with the Xenopus rhodopsin promoter alone. Data are presented as mean ± S.E.

 
cis-Elements in the Xenopus Rhodopsin Promoter—We investigated whether putative cis-elements in the rhodopsin promoter were targets for XOtx5 or XL-Nrl. We focused on three elements in the Xenopus promoter: NRE, which is necessary for high level expression in cultured cells and tadpoles; BAT-1, a highly conserved CRX binding site; and Ret-1, a binding site for a number of paired-like homeodomain transcription factors including Crx (24, 51, 52). In transfected 293 cells disruption of BAT-1 or Ret-1 individually in the opsin promoter did not have a significant effect on transcriptional activation by either Crx or XOtx5 (Fig. 5, A and B). However, activation by Nrl and XL-Nrl was dramatically affected. Disruption of either Crx binding site resulted in increased activation by Nrl/XL-Nrl, Ret-1 ~2-fold, and BAT-1 ~4-fold over the intact promoter. Deletion of the Ret-1 element also produced an increase in the synergistic activation as well, approximately doubling the activation compared with the intact XOP promoter. Deletion of the NRE reduced, but did not completely eliminate activation by Nrl and XL-Nrl. Although activation was observed with the combined transcription factors, synergy was effectively abolished with the NRE deletion construct. These results identify a crucial role for the NRE in mediating synergistic activation of the rhodopsin promoter by Crx/XOtx5 and Nrl/XL-Nrl and highlight the complex role of ATTA containing cis-elements in transcriptional regulation.

Activation of the XOP Promoter in Transfected Xenopus Embryos—The previous experiments demonstrate that these transcription factors are able to activate rhodopsin expression in a human embryonic kidney cell line. To determine whether Nrl and Crx are able to activate the rhodopsin promoter in embryos, we performed transfections of Xenopus embryonic heads and trunks. In previous experiments (23) we have shown that the XOP promoter is active in heads, but has very little activity in trunks (Fig. 6A, inset). Heads and trunks were transfected with the XOP-luciferase reporter construct and either Crx, Nrl, or both. We found that activity in heads was only minimally stimulated with the addition of the transcription factors (1.3-fold with Nrl, 2-fold with Crx, and 2.4-fold with both) (Fig. 6A). In trunks, although activity with addition of transcription factors was quite variable, we did see stimulation. Nrl stimulated activity 3-fold, whereas Crx stimulated activity 10-fold. Activation did increase with addition of both transcription factors (22-fold), but did not exhibit the large synergistic effects observed in 293 cell transfections (Fig. 5A). The variability of reporter expression is likely a reflection of transformation efficiency and the likelihood of individual cells being transfected with both transcription factors. It is possible that the 22-fold stimulation observed in these experiments would translate into a larger effect if transfection efficiency was higher. Nevertheless, these results indicate that these transcription factors are able to activate rhodopsin expression from plasmid DNA in Xenopus embryos.



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  		FIG. 6.
Activation of the rhodopsin promoter in transient transfections of heads and trunks of Xenopus embryos. A, the luciferase activity from embryo heads transfected with the XOP-GL2 and human Crx, human Nrl, or both are shown relative to the activity observed from the XOP-GL2 (n = 2–4). Fold activities are presented as mean ± S.E. Inset, representative results of a transfection experiment comparing luciferase activity from the XOP promoter in heads and trunks. Data are presented as mean ± S.D. RLU is relative light units. B, the luciferase activity from embryo trunks transfected with the wild type rhodopsin construct and human Crx, human Nrl, or both are shown relative to the activity observed from the XOP promoter alone (n = 2–3). Fold activities are presented as mean ± S.E.

 
XL-Nrl and XOtx5 Activate XOP in Transgenic Animals—To examine whether these transcription factors were able to activate expression in a chromatin environment, transgenic animals were made and analyzed. We have previously shown that the proximal XOP reporter can direct eye and pineal specific expression of GFP in transgenic animals at stage ~40 (53), when initiation of endogenous rhodopsin expression begins. In a small fraction of animals transgenic with wild type XOP, GFP expression can be seen in areas outside of the eye. These embryos are usually deformed and represent expression seen because of integration site.

In contrast, when transgenic animals were produced using either XL-Nrl and XOtx5 in addition to the XOP GFP reporter construct, we observed a dramatically different spatial and temporal expression pattern of the reporter (Table I). Expression in many animals began at the early neurulation stages. Subsequently, we observed 5–10 times more animals expressing GFP earlier and outside of the eye than we did in the controls (XOP-GFP and empty pMT vector), indicating that XL-Nrl and XOtx5 are able to stimulate expression from the integrated XOP promoter (Fig. 7, A–D). Similar results were obtained with the both Nrl and Crx (Table I and Supplemental Materials). There was a slight increase in the percentage of early GFP expressing animals when both Nrl and Crx were used together, compared with either construct alone (Table I). However, GFP expression levels did not appear to be synergistically elevated as found in 293 transfections (Supplemental Materials).


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  		TABLE I
Activation of Xenopus opsin promoter in transgenic animals ectopically expressing Crx/XOtx5, Nrl/XL-Nrl or both

 



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  		FIG. 7.
XOtx5 and XL-Nrl activate a rhodopsin promoter in transgenic animals. Images of representative transgenic tadpoles generated using XOP-GFP and either XL-Nrl (A and B), XOtx5 (C and D), or an empty pMT expression vector (E and F). Panels A, C, and E show expression at 2 days of development and panels B, D, and F show expression at 5 days of development. Fluorescent images show wide-spread expression throughout the body with addition of both XL-Nrl and XOtx5.

 
To determine whether the early presence of these transcription factors is able to activate endogenous rhodopsin, bodies of 2-day-old GFP positive transgenic embryos (made with XL-Nrl, XOtx5, or both) were subjected to RT-PCR with primers for Xenopus rhodopsin. Control GFP primers produced two GFP-specific bands varying in size by only a few base pairs (Fig. 8, top panel), and one nonspecific amplification product that was also observed in animals that had been made with pMT only (data not shown). Out of 15 transgenic animals, two showed a band corresponding to the expected rhodopsin product (Fig. 8, bottom panel). pMT controls did not show any ectopic rhodopsin expression. These results indicate that the presence of XL-Nrl, at least, is able to activate low levels of the endogenous rhodopsin gene in some animals.



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  		FIG. 8.
XL-Nrl can activate endogenous rhodopsin promoter in transgenic animals. RNA from transgenic embryos produced as described in the legend to Fig. 7 was subjected to RT-PCR using primers to amplify GFP (top row) or the endogenous rhodopsin gene (bottom row). The middle panel shows the samples prepared without reverse transcription. The rhodopsin primers encompass an intron, further ensuring that genomic DNA is not contributing to the band observed. Endogenous rhodopsin expression is seen in two out of 15 animals analyzed. The positive controls shown are from cDNA prepared from the head of a 5-day-old tadpole positive for XOP-GFP (GFP expressed only in the eye).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using 293 transfections and transgenic Xenopus embryos, we have shown that transcription factors XOtx5 and XL-Nrl are homologs of mammalian Crx and Nrl, and are able to synergistically activate the Xenopus rhodopsin promoter, either with each other or with their mammalian counterparts. Previous transgenic experiments have shown that mammalian retinal promoters can function in lower vertebrates (i.e. IRBP-(10) and {beta}-PDE-(9)). Moreover, zebrafish Crx (which groups phylogenetically with Fugu Otx5 and as a paralog of zebrafish Otx5) is able to activate a mammalian rhodopsin promoter in the presence of Nrl (albeit to a lesser extent than Crx) (54). All of these experiments indicate that the regulation of retina-specific genes share some conserved features between vertebrates. Taken together with phylogenetic studies and analysis of expression patterns, these experiments provide convincing evidence that Otx5 is involved in rhodopsin regulation in a manner similar to mammalian Crx (28, 29, 34, 48, 49). Experiments in this report show this conservation functionally for the first time using a non-mammalian promoter.

Crx proteins in mammals are highly divergent members of the vertebrate Otx5 homeobox gene class (28, 29, 48). It has been argued that a gene duplication event occurred early in the evolutionary history of these genes allowing for a relaxation of evolutionary constraints in the regulatory and coding regions (29). Several phylogenetic studies of the Otx gene families support this conclusion and another suggests that XOtx5 can bias neural retinal cells to a photoreceptor fate in Xenopus retinas (49). The importance of Crx during photoreceptor development is clear from knock-out experiments. However, the role of Crx in the maintenance of retina-specific gene expression in the mature retina is less clear. Crx mutations are often associated with degenerative disease with variable onset (55–59). However, it is also well established that over and underexpression of rhodopsin can cause photoreceptor degeneration (13, 14, 16). It is difficult to assess whether the pathologic phenotype of Crx mutations is due directly to changes in rhodopsin expression, or because of disturbances in the transcription of other genes critical to photoreceptor processes. The identification of the amphibian homolog of Crx will allow for experiments addressing these questions in the Xenopus model system.

Despite their many similarities, Crx and XOtx5 exhibit differences in their temporal and spatial expression patterns. XOtx5 is first detected in the dorsal lip, and subsequently in the anterior neuroectoderm (34, 35). During neurulation the expression pattern is dynamic, first disappearing from the presumptive eye field at stage 13 and later reappearing in the developing eye and pineal gland at stage 24 (34). Expression in the eye is restricted to the photoreceptor layer, the bipolar cell layer, and the RPE by stage 41 (28, 49). The spatial restriction and later onset of expression suggests that Crx has evolved a specialized role in retinal differentiation (29). The ability of Crx and XOtx5 to act synergistically with Nrl, and the similarity of their expression patterns in the retina, support a common evolutionary past. The functions of Otx1, Otx2, and even Drosophila otd are, for the large part, equivalent in vivo, with specificity of action determined by the expression pattern rather than any particular functional differences (60–63). In our transfection studies we show that such may be the case with XOtx2, XOtx5, and Crx. Although functional differences between XOtx5 and XOtx2 have been described (49), the mechanism controlling these differences has not yet been elucidated. It is possible that interactions with other cell-specific factors modify the functions of XOtx2 and XOtx5, or perhaps other mechanisms of control such as post-translational modifications and/or control of subcellular localization (64, 65).

We have also identified a homolog of the photoreceptor-specific basic leucine zipper protein Nrl, XL-maf, which we suggest be renamed to XL-Nrl. Like Nrl, XL-Nrl is a member of the Maf family of proteins (30), which are involved in regulation of gene expression in many diverse tissues (31). Although the rhodopsin promoters of non-mammalian species contain NRE-like elements (20, 22, 23), this is the first time a homolog of Nrl has been characterized in lower vertebrates.

Our analysis of XL-Nrl shows that it shares many of the protein domain features common to large Maf proteins. However, like Nrl, it is missing a region of histidine repeats that all other large Maf proteins share. Furthermore, the phylogenetic analysis clearly places XL-Nrl in a clade with mammalian Nrl proteins, rather than with chicken L-maf. Although previous research identified XL-Nrl (XL-maf) transcripts expressed predominantly in the lens (36), the in situ analysis of the adult retina revealed that the expression pattern of XL-Nrl, like XOtx5, undergoes dynamic changes during development, with strong photoreceptor expression exhibited in the mature retina. This, along with the fact that XL-Nrl is able to activate the rhodopsin promoter both alone and synergistically with either XOtx5 or Crx, support the conclusion that XL-Nrl is the amphibian homolog of Nrl. Furthermore, the observation that another large maf protein, MafB, was able to activate the rhodopsin promoter but produced a reduced activation with Crx, suggests that the structure of XL-Nrl may be specialized for interaction with an XOtx5/Crx-like protein.

293 transfection analysis allowed us to both dissect the effects of each transcription factor in the absence of individual elements and to compare the mammalian and Xenopus transcription factors. Deletion of the NRE resulted in a dramatic reduction in activity stimulated by these transcription factors, highlighting the importance of the NRE for the synergistic activation by Crx/Otx5 and Nrl/XL-Nrl. However, some residual activity was observed in the 293 transfections with the {Delta}NRE construct, both with Nrl/XL-Nrl alone and in combination with Crx/Otx5. These results corroborate our previous observation that deletion of the NRE dramatically reduces, but does not completely abolish rod-specific expression in transgenic animals (23). Other transfection experiments have not revealed this residual activity, perhaps because of the sensitivity allowed by the experimental conditions (23, 26). The reason for this residual activity is unclear, but it is possible that there is another weak recognition site for Nrl/XL-Nrl on the rhodopsin promoter. Alternatively, it is possible that other transcription factors present in 293 cells are able to interact with Nrl and recruit its activity to the promoter. In support of this, it was recently reported that Crx and Nrl are able to stimulate reporter activity from a construct containing multimers of the Ret-1 site even though Nrl was not able to bind to this site, suggesting that Nrl through binding to Crx can enhance its activity without actually binding directly to DNA (51). Many homeobox containing genes (such as members of the Hox, Pax, Emx, and Six families) are active during kidney development (66), and it is likely that some of these factors are present in 293 cells. Regardless, these results demonstrate the importance of the NRE for high levels of expression mediated by Crx/Otx5 and Nrl/XL-Nrl.

The observation that Crx transactivation of the Xenopus rhodopsin promoter was not significantly reduced when either Ret-1 or BAT-1 were deleted could be explained in several different ways. One simple model is that Crx/Otx5 binding sites are redundant, as has been previously suggested (23). However, redundancy of Crx/Otx5 binding sites cannot explain the increase in activity stimulated by Nrl/XL-Nrl when these sites are deleted. Furthermore, the synergistic activity of Nrl/XL-Nrl and Crx/Otx5 increased slightly when BAT-1 was deleted and increased dramatically when Ret-1 was deleted. These results suggest a role for these sites in negative regulation of the rhodopsin gene. We have previously shown that in Xenopus head transfections deletion of the Ret-1 element caused a similar increase in activity demonstrating that this negative regulation is not exclusive to 293 cells (23). Because both Ret-1 and BAT-1 are in close proximity to the NRE, binding of another protein to these sites could alter either the ability of Nrl/XL-Nrl to bind to the promoter or its ability to interact with co-activators. Indeed, several other paired-like transcription factors bind to Ret-1 and/or BAT-1, including Rx, Qrx, and the bipolar specific Chx10 (51). This potential negative regulation could be important both in rod cells for maintaining proper levels of rhodopsin expression, and in other retinal cells such as bipolar cells and cones (which express Crx (67, 68)) for preventing low levels of rhodopsin expression. Whereas these results do not rule out the importance of Crx/Otx5 binding to various sites on the promoter, including Ret-1 and BAT-1, they do support a critical role for protein-protein interactions between Nrl/XL-Nrl and Crx/XOtx5 primarily mediated through the NRE.

Transgenic Xenopus experiments with XOtx5 and XL-Nrl provided a chance to examine the regulation of the rhodopsin promoter in a chromatin environment. Under the low DNA concentrations used in the restriction enzyme-mediated integration, only transgenic animals harboring integrated XOP reporter express GFP at the stages examined. Only a fraction (~20–50%) of the resulting embryos express GFP, indicating that they have the linearized plasmid incorporated into their genome (46, 53).

When XOtx5 and XL-Nrl were included in the transgenic experiments we observed widespread GFP expression that mimicked the expected expression patterns from the viral promoters used to drive expression of the transcription factors. These transcription factors are able to activate rhodopsin promoter integrated into chromatin, lending further support to the results from the transfection experiments, in which expression was driven from plasmid DNA. No bias in the GFP expression pattern was observed in any particular tissue type. This indicates that any co-activators (i.e. p300 (69), BAF1 (67)) or additional transcription factors required by XOtx5/XL-Nrl are widely expressed or have close homologs. There were no apparent differences in intensity of GFP expression between those injected with one transcription factor and those injected with both, which agrees with the results obtained from the transient transfections of embryo heads and trunks. It is difficult to determine whether the GFP expression in the trunks of these animals arises through synergistic activation involving the exogenously provided transcription factor and endogenous coactivators, or arises from saturation of the promoter transgene by dramatic overexpression from the viral promoter.

The onset of GFP expression was variable but significantly earlier in animals that were co-transgenic for either exogenously added transcription factor. The first detectable expression occurred in the early neurul stages, in contrast to the XOP transgene alone, which is not detected until stage 41 (Ref. 23 and data not shown). The ectopic transgene expression lagged behind the onset of general transcription at the mid-blastula transition. Onset of transgene expression has been observed close to the mid-blastula transition, with other promoters (46). The delay observed here may reflect the time required to accumulate sufficient levels of exogenously added transcription factors, although this seems unlikely because the strong viral promoters are able to drive expression of protein to sufficient levels by stage 10 (46). Alternatively, it is possible that time is required to accumulate GFP protein, perhaps because of a very low transcription rate involving the transgenes. Another possibility is that developmental changes in transcription impact the timing of transgene activation. In any case both XOtx5 and XL-Nrl were able to drive expression from the transgenic promoter.

Even when Crx and Nrl are overexpressed in 293 cells they do not stimulate transcription from the human endogenous rhodopsin gene in cell transfections (24). In contrast, we found that these transcription factors ectopically activated the endogenous gene in several embryos. However, the level of rhodopsin expression was low in comparison with that of GFP transgene. It is not clear whether this expression arises from very low expression in a wide variety of cell types, or if expression was confined to particular cells or tissues. Nonetheless, these results reinforce the conclusion that XL-Nrl and XOtx5 activate rhodopsin. In contrast to the kidney cells, the developing embryonic genome may have some plasticity with regards to activation. The low frequency of this activation may indicate that the transcriptional activation is sensitive to the timing and levels of transcription factor present in the embryo. Future work will be directed toward exploring the possible epigenetic control mechanisms present on the rhodopsin promoter.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants EY-11256 and EY-12975 (to B. E. K.), an unrestricted grant to the Research to Prevent Blindness to SUNY Upstate Medical University Department of Ophthalmology, and the Lions of Central New York. 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. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S4. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St. Syracuse, NY 13210. Tel.: 315-464-8719; Fax: 315-464-8750; E-mail: knoxb{at}upstate.edu.

1 The abbreviations used are: NRE, Nrl response element; RT, reverse transcriptase; XOP, Xenopus rhodopsin promoter –508/+41; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank A. Viczian, A. Swaroop, and S. Chen for kindly providing the XOtx5b, Nrl, and Crx plasmids, respectively. We also thank S. Babu for assisting in the making of transgenic animals, and K. Babu and A. Dukipati for assistance in performing the 293 cell transfections, and S. Babu, M. Zuber, A. Viczian, and S. Mani for the helpful comments and discussions regarding this work.



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