Immediate Upstream Sequence of Arrestin Directs Rod-specific Expression in Xenopus*

Shobana S. ManiDagger §, Joseph C. Besharse, and Barry E. Knoxparallel **

From the Dagger  Department of Biochemistry and Molecular Biology and parallel  Center for Vision Research, Department of Ophthalmology, SUNY Health Science Center at Syracuse, New York 13210 and  Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arrestins are a family of proteins that modulate G protein-coupled receptor responses with distinct arrestin genes expressed in rods and cones. To understand the regulatory mechanisms controlling rod-specific expression, the abundant Xenopus rod arrestin cDNA and a partial genomic clone, containing the immediate upstream region and amino terminus of the polypeptide, have been characterized. The deduced polypeptide has ~69% identity to other vertebrate rod arrestins. Southern blot analysis and polymerase chain reaction of intronic sequences demonstrated multiple alleles for rod arrestin. DNase I footprinting with retinal proteins revealed four major DNA binding sites in the proximal promoter, coinciding with consensus sequences reported in mammalian promoters. Purified bovine Crx homeodomain and mouse Nrl proteins protected a number of these sites. A dual approach of transient embryo transfections and transgenesis was used to locate transcriptional control sequences essential for rod-specific expression in Xenopus. Constructs containing -1287/+113 of 5' upstream sequence with or without intron 1 directed high level expression, specifically in rods. A construct containing only -287/+113 directed expression of green fluorescent protein solely in rod cells. These results suggest that the Crx and Nrl binding sites in the proximal promoter are the primary cis-acting sequences regulating arrestin gene expression in rods.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arrestins (1, 2) are a large family of proteins found in both vertebrates and invertebrates, including distinct classes of visual arrestin, rod (3-9), cone (6, 10, 11) and invertebrate photoreceptors (12-14), and beta -arrestins, which are involved in regulating responses to hormones (15, 16). Visual arrestin (48 kDa) is expressed in photoreceptor cells (17) and the pineal gland (18, 19). Light-activated rhodopsin is phosphorylated by rhodopsin kinase enabling the binding of arrestin (20, 21). This association prevents additional interaction between rhodopsin and transducin, effectively terminating the response. The regulatory role of arrestin has been highlighted by arrestin knock-out mice, which exhibit prolonged rod photoresponses (22). Arrestin also appears to be critical for recycling of the light-exposed rhodopsin in Drosophila (23, 24). Moreover, a frameshift mutation in the human arrestin gene causes Oguchi's disease, which is characterized by slowed dark adaptation and reduced sensitivity (25).

The expression pattern of visual arrestins is restricted to a few cell types. For example, mouse rod arrestin is found predominantly in the retinal photoreceptor layer and pinealocytes (18), whereas rat cone arrestin was found in cone photoreceptors and pinealocytes (10). In Xenopus, arrestin expression has been detected in photoreceptors and pinealocytes (26). The onset of rod arrestin gene transcription in mouse occurs before rod outer segment formation (27), whereas in the bovine retina, arrestin expression is concurrent with other genes involved in phototransduction (28). Transcription of arrestin also appears to be regulated by diurnal or circadian rhythms in certain species (29, 30, 31). However, the mechanisms that control arrestin expression are not understood.

The regulation of mammalian rod arrestin gene expression has been studied using transgenic mice (18, 32, 33) and by transient transfections in heterologous primary chicken retinal cultures (34). A 1.3-kbp1 fragment encompassing the 5'-proximal and a portion of the untranslated region of the mouse arrestin gene was found to direct reporter expression in photoreceptor cells, pineal, lens, and brain (18). Further analysis showed that shorter fragments could direct expression in the retina and lens to variable levels (32). Using sequence comparisons and biochemical assays, several cis-acting elements in the proximal promoter of mammalian rod arrestin genes have been suggested to play a role in regulating expression (32, 35). In vitro, these elements in the arrestin gene can serve as binding sites for homeodomain transcription factors, Rx splice variant (36) and Crx (35). Transfection experiments in HEK293 cells demonstrated an increase in the transcription of arrestin reporter constructs in the presence of Crx (35). In addition to these sites, there may be other cis-acting elements in the arrestin promoter (32, 37).

Xenopus is particularly well suited for studying retinal development and gene regulation; rapid histogenesis (38, 39), relative abundance of rods (55%) and cones (45%) (40, 41), efficient methods for gene delivery (42, 43) and transgenesis (44, 45) provide unique experimental strategies to address questions of cell-specific gene expression in vertebrates. We have cloned genomic fragments that contain 5' regulatory regions of the Xenopus rod arrestin gene and used transient transfections and transgenic approaches to study promoter function. Our studies show that the immediate 5' upstream sequence of arrestin is sufficient for directing rod specific expression in vivo and suggest that transcription is regulated by Crx- and Nrl-like factors in lower vertebrates.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Clones-- An oligo(dT)-primed cDNA library (43) was screened using a 1.6-kbp EcoRI fragment of bovine rod arrestin, BSC 70 (5). Positive plaques were purified and characterized by restriction and sequence analysis (46, Sequenase Ver 2.0; U. S. Biochemical Corp). Using antisense primers: 5'-ggatccaccaagaacaactc-3' and P2, 5'-ttgcccaggtacacgct-3', RACE PCR (47) was carried out on retinal poly(A)+ RNA. Sequences from two independent RACE clones were determined on both strands.

Genomic Clones-- A Xenopus genomic library (a gift of E. deRobertis, UCLA) was screened using a radiolabeled 748-bp SmaI-KpnI cDNA fragment (Probe 1, Fig. 1). Positive plaques were isolated, purified, and restriction-mapped according to standard protocols (48). BamHI subclones were characterized, and 1.3 kbp of upstream and 3.5 kbp of intron I sequence were obtained on both strands using an automated ABI stretch DNA sequencer (DNA services, Cornell University, Ithaca, NY).

DNA Sequence Analysis-- DNA sequences were assembled and analyzed using the Genetics Computer Group Wisconsin Package (GCG9, Madison, WI). Multiple sequence alignments were performed on both DNA and amino acid sequence files using the GCG program, PILEUP. The resulting alignments were used to run PAUP*4.0 (Phylogeny Analysis Using Parsimony (49)) implemented in GCG. A PAUP heuristic search with bootstrap option (100 bootstrap samples) was performed to find a majority rule consensus unrooted tree.

RNA Analysis-- Total RNA from adult male Xenopus retina, brain, liver, and olfactory bulbs were isolated by the acid-guanidinium method (50), and poly (A)+ RNA was isolated using an oligo(dT) column. Poly(A)+ (0.2 µg) or total RNA (10 µg) was resolved by denaturing agarose gel electrophoresis. After transfer, the blot was hybridized to the radiolabeled SmaI-KpnI cDNA fragment (Probe 1, Fig. 1). The blot was washed at a final stringency of 1 × SSPE (1 × SSPE = 0.18 M NaCl. 10 mM NaPi, 1 mM EDTA, pH 7.7) and 0.1% SDS at 60 °C. Primer extension reactions were carried out at 50 °C for 1 h using avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals) and 15 µg of total RNA with 0.5 pmol of radiolabeled 5'-ctgagatccagtaaggtaacccgttagc-3'. As a control, RNA was omitted from the reaction. The products were resolved on 8% acrylamide gels and exposed using a PhosphorImager (Molecular Dynamics).

In Situ Hybridization-- A fragment encoding nucleotides 178 to 720 of the cDNA was cloned into the EcoRV sites in pBSII (Stratagene). The plasmid was linearized with HindIII to generate the antisense probe using T7 RNA polymerase. XbaI-linearized plasmid was used with SP6 polymerase to generate the sense probe. Sense and antisense probes were synthesized using a digoxigenin labeling kit according to the manufacturer's instructions (Roche). Hybridization to 10-µm paraffin sections was carried out as described previously (51), and the hybridized probe was detected immunocytochemically using an antidigoxigenin and alkaline phosphatase-conjugated second antibody (Roche). Slides were visualized and photographed using a Zeiss Photomicroscope III.

Genomic Analysis-- Southern analysis was performed as described previously (43). The blot was hybridized using a 32P-labeled BamHI-AvaI fragment (Probe 2, Fig. 1), and final washes were performed at 62 °C in 0.1 × SSC (1 × SSC = 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS. The blot was exposed using a PhosphorImager. To amplify intron 2, PCR was carried out using exon-specific primers P2 and forward primer 5'-tgtcatgtataagaaaacc-3' as described previously (43) except that 30 cycles of annealing (58 °C), extension (72 °C for 2 min), and denaturation (95 °C for 1 min) were performed.

DNase I Footprinting-- Nuclear proteins were extracted from adult Xenopus retina as described (52). Glutathione S-transferase-tagged bovine Crx homeodomain and flanking region (residues 34-107, GST-CrxHD) and a hexahistidine-tagged mouse Nrl (residues 16-237, His-Nrl) were overexpressed in Escherichia coli and purified as described (35, 53). 32P end-labeled DNA fragments (-256/+25) were produced by PCR and footprinting reactions were carried out as described (35). Poly(dI-dC) was included only in the reactions containing retinal extracts. Varying concentrations of purified protein (as indicated in the Fig. 4 legend) were used in the footprinting reaction.

Construction of Arrestin Reporter Constructs-- The arrestin reporter constructs generated for promoter analysis contained upstream, exon, and intron 1 sequences. For clarity, upstream and exon sequences are numbered relative to transcription start site, + and -, respectively, whereas intron 1 sequences (nucleotides 1417-4741 in AF053942) are numbered from the 5' splice donor nucleotide without -/+. XAR1 (-1287/+130, in 1/3324, +131/+155), containing the 5' portion of the arrestin gene, was assembled in pBSII (Stratagene) from a 4.3-kbp PstI-XbaI fragment and a 450-bp XbaI-HindIII, fragment generated by PCR. The PCR fragment was sequenced after cloning. The 4.8-kbp XAR1 fragment was transferred into either pGL2 (Promega Corp) or pEGFP(-) (45) to generate pXAR1luc or pXAR1gfp respectively. pXAR2luc (-2600/+130, in 1/1383) was constructed by ligating a 4.0-kbp BamHI fragment into the BglII site of pGL2. To prepare plasmids containing intronic fragments, a 2.8-kbp BamHI fragment was first cloned into pBSII. The 5' end of this clone, XAR3luc (in 1383/2896) was cloned into pGL2 using KpnI from the multicloning site and XbaI. XAR3gfp was cloned as a KpnI and XbaI (filled in) fragment into the KpnI and BamHI (filled in) sites of pEGFP(-). pXAR4luc (in 1732/2896) was generated by removing the 350-bp 5' end at NcoI from pXAR3luc and recircularization of the blunted ends. XAR5 (-1287/+113) was cloned as an EcoRI fragment into pBSII. This fragment from cloned using KpnI and XbaI from the multicloning site into the KpnI-NheI sites of pGL2, generating pXAR5luc. pXAR5gfp was generated by directly cloning the 1.4-kbp EcoRI fragment into pEGFP(-). XAR6 (+113/+130, in 1/1325) was generated by ligating an EcoRI fragment into pBSII and cloned using KpnI and XbaI from the multicloning site into the KpnI-NheI sites of pGL2 to generate pXAR6luc. pXAR6gfp was generated by cloning the 1.3-kbp EcoRI fragment into pEGFP(-). pXAR7luc (-287/+113) was constructed by digesting pXAR5luc with KpnI and NheI, blunting with Klenow, and religation. pXAR7gfp (-289/+113) was generated by digestion of pXAR5gfp with HindIII and NheI, filling with DNA polymerase, and religation. The orientation of the inserts and junctions of all the constructs were verified by sequencing. Plasmid DNA was prepared using the Qiagen protocol (Qiagen, Chatsworth, CA).

Embryo Transfections-- Xenopus embryos were transfected as described previously (43), except that 10 µg of DNA was mixed with 30 µl of DOTAP (Roche) before transfection. Protein extracts from transfected heads or trunks were prepared in 80-100 µl of lysis buffer, and duplicate aliquots of 10-µl each (equivalent to one head or trunk) were assayed for luciferase activity. Groups of 8-10 heads were transfected and assayed for luciferase activity, and 4-6 independent groups (indicated as N in the Fig. 5 legend) were used for determining average activities. Statistical comparisons were performed using ANOVA (Excel). Average activities were expressed as relative light units/embryo, where 1 pg of purified luciferase gave 8.5-9.0 × 104 relative light units measured using a single-channel luminometer (Berthold) for 30 s. Protein determinations were performed using the Coomassie dye binding assay (Bio-Rad).

Production of Transgenic Xenopus-- Arrestin GFP plasmids were linearized using XhoI and transgenic Xenopus laevis embryos were produced as described (45). GFP-positive transgenic tadpoles were fixed overnight in phosphate-buffered 4% paraformaldehyde. Frozen sections (10-15-µm thickness) were prepared, and confocal images were obtained using a slit-scanner instrument (Meridian Insight). The figures were produced using Photoshop (Adobe).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and Characterization of Xenopus Arrestin-- An adult Xenopus retinal cDNA library (43) was screened using a 1.6-kbp bovine rod arrestin cDNA fragment (5). The sequence obtained from three partial overlapping cDNA clones (Fig. 1) exhibited 69% homology at the nucleotide level to bovine rod arrestin cDNA. The longest clone (Xarr10) contained 1835 nucleotides coding for amino acids 6-396 and the entire 3'-UTR. The 5' end of the cDNA was obtained by RACE, which had 2 changes compared with the corresponding sequence (236 bp) from the genomic region (see below), probably reflecting errors introduced during PCR. The lengths and sequence of the 5' UTR of Xenopus rod arrestin exhibited no homology to the mammalian arrestins; in fact, it was significantly shorter than both mouse and human (130 compared with 358 and 387 bp, respectively).


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Fig. 1.   Xenopus rod arrestin genomic and cDNA clones. A, restriction map of partial genomic clone gXArrA. The exons 1, 2, 3, and 4 are indicated by solid boxes. Restriction sites shown are BamHI (B), EcoRI (E), NotI (No), HindIII (H), PstI (P), SmaI (S', additional SmaI sites exist in the fragment but were not mapped), AvaI (A), KpnI (K), and NcoI (N). B, structure of the 1978-bp transcribed product with the coding region (solid box) and the UTR (open boxes) indicated. The positions of first four introns are indicated. cDNA clones (Xarr10, 9, and 11) (C) and a 250-bp RACE product used in sequence determination (D) are shown. Probes used for genomic screening and Northern analysis (Probe 1) and Southern analysis (Probe 2) (see "Experimental Procedures") are indicated by hatched boxes.

The full-length transcript (1978 bp) encodes a predicted polypeptide of 396 amino acids. Comparison of this sequence with other arrestins revealed a high degree of similarity with rod arrestins from other vertebrates (3-6). beta -Arrestins (54, 55) have 58% identity (75% similarity) to Xenopus rod arrestin, whereas invertebrate photoreceptor arrestins (12-14) exhibited more divergence (40% identity and 60% similarity). The Xenopus rod arrestin polypeptide was most closely related to amphibian rod arrestin (6) from Rana pipiens (86.1% identity and 92.4% similarity) and Rana catesbiana (86.1% identity and 92.6% similarity). In contrast, comparison with sequences from Xenopus cone arrestin (11) revealed only 54.7% identity (75.5% similarity) at the amino acid level. Comparison of the Xenopus polypeptide with cone arrestin sequences from other vertebrates showed an overall identity of only 50-55%. Phylogenetic analysis (using maximum parsimony analysis, data not shown) clearly places this Xenopus sequence with the rod arrestin group.

Characterization of Arrestin Genomic Clone-- To obtain the potential promoter regions of Xenopus rod arrestin, a genomic library was screened, and one clone (gXArrA, Fig. 1) containing a 6.5-kbp sequence upstream of the cDNA, was partially sequenced. Exon 1 (+1/+130), 2 (+131/+224), and 3 (+225/+285) were identical to the cDNA (except at two positions in the RACE product, see above). Like other mammalian arrestins, the 5'-UTR of the Xenopus mRNA was encoded by exons 1 and 2, with the translation initiation codon in exon 2. Although exon 1 exhibited significant variation in size amongst various species, exon 2 sizes were quite similar (94 versus 105 bp in mouse and human). In addition, exons 3 and 4 of Xenopus rod arrestin showed conservation of splice junctions (data not shown).

Arrestin Transcript Analysis-- Two major primer extension products, differing by one nucleotide, were obtained with retinal RNA, whereas no products were found using brain RNA (Fig. 2A). The shorter of the two corresponds to that obtained by RACE PCR, and the 5'-most nucleotide (T) was designated as +1. A minor doublet at +3 and +4 may reflect products from minor start sites, incomplete primer extension products because of secondary structure at the 5' end, or expression from additional alleles (see below). The presence of multiple start sites has been reported for a number of arrestin genes including human (56).


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Fig. 2.   Arrestin transcript analysis. A, primer extension of total RNA (15 µg) of retinal (R), brain (B) RNA, or no RNA (-). A sequencing ladder using the same primer is shown. The major transcription start site is indicated by 1, and the corresponding nucleotides are represented in capital letters. 2 represents minor extension products. B, Northern analysis. 0.2 µg of Xenopus adult retinal poly(A)+ RNA (R) and 10 µg each of total RNA from brain (B), liver (L), and olfactory bulb (O) were resolved on a 1% denaturing agarose gel, and hybridization was performed using probe 1. Molecular markers corresponding to ribosomal RNA are indicated. C, localization of rod arrestin mRNA by in situ hybridization. Fixed sections of adult Xenopus retina were hybridized with a digoxigenin-labeled antisense (top) or sense (bottom) Xenopus rod arrestin probe under high stringency conditions. Bound probe was visualized using anti-digoxigenin antibody. Staining with the antisense probe was detected exclusively in rods (large arrows, A). No staining was detected in cones (small arrow, A) or with the sense probe (bottom). RPE, retinal pigment epithelium; ONL, outer nuclear layer.

Northern blot analysis showed a single retina-specific band of 1.9 kbp (Fig. 2B). No cross-reactivity was observed in RNA from brain, liver, or olfactory bulb under high or low stringency conditions. These results are in contrast to a previous report that detected rod arrestin transcripts in brain and testes RNA in addition to the retina (11). The differences in the observations most likely reflects cross-reactivity with non-rod arrestins in these tissues. In situ hybridization was used to identify the retinal cell expressing the cloned arrestin mRNA. Antisense probes for arrestin cDNA hybridized specifically to the abundant, principal rod (large arrows, Fig. 2C). No hybridization was seen in cones (small arrow, Fig. 2C) or other retinal cell layers, whereas controls using the sense probe did not exhibit hybridization to rods or other retinal cells.

Copy Number Analysis-- Xenopus contains a pseudotetraploid genome (57) and has multiple alleles for rhodopsin (43) and transducin alpha -subunit genes.2 Southern blot analysis using an exon-specific probe (Probe 2, Fig. 1) showed more than the expected number of hybridizing bands (Fig. 3A). Cross-reactivity with other arrestin genes cannot explain this pattern, because the probe has only 61% nucleotide identity with Xenopus cone arrestin and similar expected levels of nucleotide identity for beta -arrestin. PCR of intron 2 also produced more than the expected ~1.0-kbp band (Fig. 3B). Again, the oligonucleotides used in the amplification have no homology to Xenopus cone arrestin or predicted beta -arrestin sequences and would not amplify a specific product. Taken together, the results from the Southern blot analysis and PCR analysis of genomic DNA suggest the presence of multiple alleles for rod arrestin in Xenopus.


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Fig. 3.   Xenopus arrestin alleles. A, Southern blot of Xenopus genomic DNA (15 µg) digested with PstI (lane 1), HindIII (lane 2), EcoRI (lane 3), or BamHI (lane 4), hybridized with the BamHI-AvaI cDNA probe (probe 2), and washed at high stringency. B, PCR of intron 2 using Xenopus genomic DNA (200 ng, lane 5) or genomic clone gXArrA (40 pg, lane 6) was carried out using exon-specific primers. Lanes 1-4 are controls in which no primers (lane 1), forward primer alone (lane 2), P2 alone (lane 3), and forward primer and P2 but no genomic DNA (lane 4) are shown. The molecular size markers in kbp are indicated on the left.

Upstream Sequence Analysis and DNase Footprinting-- The sequence upstream of the transcription initiation site was analyzed for potential transcriptional control elements (Fig. 4A). There is no apparent TATA motif in the upstream sequence, consistent with other arrestin genes (58, 56) and with Xenopus rhodopsin (43) and transducin alpha -subunit2 genes. However, a TATA-like sequence (ATATT) located at -25 could potentially function as a TATA-binding protein site (Fig. 4A). Upstream sequence comparison with mammalian arrestins did not reveal any significant regions of homology, although mammalian rod arrestins exhibit identity in the 300 bp of the immediate upstream region (32). A number of potential motifs were found in the immediate upstream region. The closest match to the consensus PCE I site (YCAATTAGS (32)), containing one mismatch, was found at -756. There were two regions with identity to a consensus element ((C/T)TAATC(C/A)) for Crx, a homeobox transcription factor (35, 59): on the sense strand at position -101/-108 and on the antisense strand at -79/-69. Crx also binds to a site, Ret4, in the rhodopsin promoter (consensus AGCTTACT (60)), and a potential Ret4 site (7/8 matches) was found at -163 in Xenopus arrestin upstream region. A potential AP-1 site was found at -120/-110 (consensus NTGAASTCAG (53)). Comparison to other Xenopus rod phototransduction genes did not show any significant homology.


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Fig. 4.   Analysis of the Xenopus rod arrestin proximal promoter. A, the nucleotide sequence of the proximal promoter (-287/+113, pXAR7) is shown with the major transcriptional start site numbered +1 (bold). Potential transcriptional regulatory sequences (Crx, Ret4, and AP1) identified by sequence comparisons are shaded, and the homeobox core sequences (ATTA) are underlined. B, DNase I footprinting analysis using purified GST-CrxHD. Both top (+) and bottom (-) strand of the proximal promoter were footprinted in the absence (-) and presence of 100, 10, 1, and 0.1 ng of protein. C, DNase I footprinting analysis using purified His-Nrl. Both the top (+) and bottom (-) strand of the proximal promoter were footprinted in the absence (-) and presence of 140, 1.4, 0.14 ng of protein. The major protected areas from both B and C are indicated by numbered lines, and the corresponding regions are underlined below the sequence in A. Nucleotide positions were determined by comparing to a sequencing ladder adjacent to the DNase-treated samples and representative size markers shown.

To locate binding sites for potential transcription factors, DNase footprinting was performed on the arrestin-proximal promoter. Using an adult retinal extract, numerous extended regions (-200 to +10) were protected from digestion, including those predicted from sequence comparisons in the proximal promoter (data not shown). At present, no Xenopus homologues of Crx, Nrl, or rod-specific transcription factors have been identified. Therefore, footprinting of the proximal promoter was done using recombinant mammalian proteins purified from E. coli: the bovine Crx homeodomain (GST-CrxHD (35)) and the DNA binding domain and surrounding bZIP regions of murine Nrl containing a hexahistidine tag (His-Nrl (53)). Both proteins protected the arrestin-proximal region (Fig. 4, B and C). GST-CrxHD produced four footprinted regions: strong protection of both Crx consensus sites (Crx-3, -119 to -99, and Crx-4, -86 to -61, sense strand and -109 to -94 and -85 to -71 antisense strand, respectively) and weak protection of Ret4 (Crx-1, -178 to -160, sense strand and -176 to -161, antisense strand) and a TAAT sequence (Crx-2, -151 to -139, sense strand and -152 to -144, antisense strand). The sense and antisense strands were not equally protected, particularly with Crx-2. Nrl protected a single major region (-145 to -103, sense strand and -143 to -100, antisense) that included the AP1 consensus region and was adjacent to two Crx sites. These results strongly suggest that the sequences in the arrestin-proximal promoter contain binding sites for members of the Otx2-related and leucine zipper families.

Arrestin Sequences Direct Expression in Transfected Embryos-- To characterize the functional role of the upstream region in cell-specific expression of arrestin, luciferase reporter constructs (Fig. 5A) were tested in transient transfection assays using Xenopus embryos (43). Constructs containing the upstream and intron 1 sequences (pXAR1) directed >100-fold activity in transfected heads as compared with the promoterless control, GL2 (Fig. 5B). The levels of reporter expression were comparable with that obtained from the transfection using the Xenopus rhodopsin (-508/+41) upstream sequence construct (Fig. 5B). Trunks containing no retinal or brain tissue transfected with pXAR1 or a promoterless control exhibited negligible levels (<0.01%) of luciferase expression when compared with the levels in heads. This was in contrast to high levels of luciferase activity observed in trunks transfected with a general promoter, such as cytomegalovirus (data not shown). An arrestin construct containing ~1300 bp of upstream sequence without intron 1, pXAR5, directed 3-5-fold higher levels of luciferase expression in transfected heads compared with pXAR1 (Fig. 5C). The increased expression in heads was derived from retinal cells, because heads and trunks transfected with either pXAR1GFP or pXAR5GFP exhibited fluorescence only in the everted eye structures (61). The proximal promoter (-287/+113, pXAR7) directed head-specific luciferase expression to levels comparable with the longer constructs (Fig. 5C), indicating that the major cell-specifying cis-acting elements are located in this region. A 1.6-kbp fragment containing sequences from the 3' end of intron 1 (pXAR3) and a 1.2-kbp subfragment derived from the same region of intron 1 (pXAR4) directed significant levels of luciferase expression in both heads and trunks, whereas a 1.4-kbp fragment from the 5' end of intron 1 produced no measurable reporter activity (Fig. 5C). This observation suggests that the 3' end of intron 1 may contain sequences that can support transcription in a non-cell-specific manner, although the significance to arrestin gene expression is uncertain. Constructs containing upstream plus intron 1 directed expression to levels different from the upstream region alone (pXAR1 versus pXAR5). A more dramatic difference was observed with pXAR2, containing 5' upstream sequences, exon 1 splice donor, and a portion of intron 1, which was inactive (Fig. 5C). This may be because of the utilization of a splice acceptor site located after the luciferase gene in the reporter construct, producing an mRNA without the luciferase coding region (Fig. 5A). Alternatively, the ~1.3 kbp of additional upstream sequence not present in pXAR1 or pXAR5 could contain negative regulatory elements that affect the luciferase expression levels in transfected heads and trunks. Alternative splicing could also explain the different levels of expression in heads transfected with pXAR1 compared with pXAR5 and pXAR7. Transcripts containing intron 1 (in the case of pXAR1) may be less stable or less efficiently translated than those without these sequences. The role, if any, of the nonspecific transcriptional control elements in intron 1 remains unclear but indicates that they are not essential for rod specific expression (see below).


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Fig. 5.   Transient transfections of Xenopus embryos with arrestin constructs. A, transfection constructs. The 5' end of genomic clone (gXArrA) showing the positions of exons 1 and 2 (solid boxes) and direction of transcription (arrow) is shown. Luciferase reporter constructs are shown above and below (pXAR1-7). The translational initiation codon encoded by exon 2 is indicated (atg). Restriction sites used in the generation of reporter constructs are BamHI (B), EcoRI (E), NcoI (Nc), PstI (P), SmaI (S), XbaI (X). Additional NcoI sites in this fragment have not been mapped. B, transient transfection of Xenopus embryos. Luciferase activity (relative light units (RLU)/embryo) from transfections in heads (solid bars) or trunks (open bars) using XOP (Xenopus opsin promoter, -508/+41), XAR (Xenopus arrestin promoter, -1287/+130, in 1/3324, +131/+155), or GL2 (promoterless control) were determined in 6 independent experiments. The activity measured from GL2 was not significantly different from controls without luciferase. Luciferase activity measured from heads transfected with XOP and XAR were 125- and 94-fold higher compared with activity measured from GL2 transfected heads. C, luciferase activities in heads (solid bars) or trunks (open bars) from various arrestin reporter constructs, normalized to XAR, were determined in 6-8 independent experiments. The sequence of pXAR7 is shown in Fig. 4A. Error bars represent S.E., and asterisks indicate values not statistically different (ANOVA, p < 0.05) from the promoterless control.

Arrestin Promoter Activity in Transgenic Xenopus-- To identify the cell-type specificity of reporter expression from arrestin promoter constructs, transgenic Xenopus tadpoles were generated as described previously (45, 61). GFP reporter constructs from either upstream (pXAR5gfp and pXAR7gfp) or upstream and intron 1 (pXAR1gfp) were used to generate several transgenic tadpole lines. In tadpoles transgenic for these constructs, GFP expression was observed only in the eye with no detectable GFP expression in the pineal or non-ocular tissues. XAR5 and XAR7 transgenic tadpoles appeared qualitatively brighter than siblings transgenic for XAR1 (data not shown). Confocal images through cryosections of fixed eyes from transgenic tadpoles showed GFP expression limited to rods (Fig. 6), whereas cones, other retinal cells, and lens did not express detectable GFP. A comparison of the cell-type expressing constructs containing the upstream alone (XAR5 and XAR7) and that encoding upstream and intron 1 (XAR1) showed that both constructs directed rod-specific expression and confirms that variation in levels of reporter expression observed in the transfection assay do not appear to be because of expression of GFP in different cell populations. These results show that the immediate upstream region of arrestin, in particular the proximal promoter, contains sufficient information to direct GFP expression to rods in vivo.


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Fig. 6.   Arrestin upstream/intron I sequences direct rod-specific expression of GFP in transgenic Xenopus tadpoles. A, bright field image of a radial cross-section through the central retina of a stage 56 transgenic tadpole generated using pXAR1gfp. The retinal pigment epithelium (RPE), photoreceptors (PR), ganglion cell (GC) layers, and lenses (L) are indicated, and cone cells in the PR layer are indicated by arrows. B, fluorescence image of the same section showing GFP expression limited to the rods. C, bright field and fluorescent lighting conditions showing no GFP expression in cone cells. GFP-positive rods are seen alongside GFP-negative cone cells (indicated by arrows). D, differential interference contrast image of a tangential cross-section near the peripheral retina of a stage-52 transgenic tadpole generated using pXAR7gfp. The darkly pigmented retinal pigment epithelium can be seen surrounding the rod outer segments; inner segments and photoreceptor cell bodies are in central portion of the section. E, fluorescence image of the same section. GFP expression can be seen in the rod outer segments and in the inner segments/soma of the rod cell mosaic. F, bright field and fluorescent images (D and E) were merged following acquisition of the same section. Nonexpressing photoreceptors can be observed adjacent to fluorescent rod cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand the mechanisms that govern the cell-specific expression of rod genes, we have isolated and characterized the Xenopus rod arrestin cDNA and genomic clone containing 5' regulatory regions. Analysis of genomic structure showed that the structural organization at the 5' end of Xenopus rod arrestin is conserved with mouse and human arrestin genes (56, 58). However, sequences in the immediate upstream region of the Xenopus rod arrestin gene do not exhibit significant overall homology with conserved mammalian upstream regions, in contrast to that observed for opsins (43). We found by sequence analysis and DNase footprinting that the proximal promoter contains four potential cis-acting elements: Crx-like, Ret4, PCE I, and AP-1-like. Although the Xenopus homologues of Crx or Nrl have not yet been identified, these results suggest that such related trans-acting factors may function in lower vertebrates.

In transient transfection assays, we observed levels of expression comparable with reporter expression driven by the rhodopsin promoter, although the endogenous transcript and protein levels of arrestin in the adult are about 10-fold lower than that of rhodopsin. This discrepancy may be because of differences in RNA stability or translation efficiency, caused by differences in the 5'-UTR regions. It may also be because of differences between the onset of arrestin expression relative to rhodopsin during photoreceptor differentiation. The developmental onset of photoreceptor cell-specific genes varies in different organisms. In mouse (28), arrestin expression precedes rhodopsin expression, whereas in the bovine retina they exhibit a similar temporal onset (27). The timing of gene expression in Xenopus is currently being investigated.

Transgenic mice created with a -1283/+163 bp upstream fragment of the mouse gene (18) exhibited expression of reporter genes in the retina, with lower levels in pineal, lens, cerebral cortex, and cerebellum. Within the retina, chloramphenicol acetyltransferase expression was observed in rods; expression in cones was not determined. Reporter activity directed by this fragment in brain and lens of transgenic mice was low and variable; transgenic mice that expressed high levels of chloramphenicol acetyltransferase in the retina, containing multiple copies of the transgene, showed increased levels of reporter expression in pineal, lens, and brain. This suggests that copy number or integration position might play a role in the extraretinal reporter expression in mice. Using GFP as the reporter gene in transgenic Xenopus, we observed fluorescence in rods but not in cones, lens, or other cells in the brain, although a very low level of GFP expression, below our limits of detection, is possible. In Xenopus, the pineal shrinks and changes during later stages of development (62), and this could contribute to the lack of detectable GFP expression in the older animals. It is also possible that the difference in expression pattern in the two transgenic systems reflects species differences in arrestin expression. The cell-specific expression in transgenic Xenopus is corroborated by our observations in transient transfection assays, which showed GFP-expressing cells limited to eye structures in transfected heads. Together, the two approaches show that the regulation of arrestin gene expression in rods is governed by cis regulatory sequences in the immediate upstream region largely unaffected by copy number or integration effects. Our data constitute a direct demonstration that the proximal promoter of rod arrestin contains sufficient regulatory sequences to drive high level of rod-specific reporter expression in vertebrates. Moreover, these results highlight the apparent conservation in vertebrates of transcriptional control mechanisms that determine rod-specific expression. Xenopus offers the unique advantage of combining embryo transfection (43) with a rapid and powerful transgenic approach (44, 45) to study the role of cis-acting elements in controlling cell-specific and coordinate regulation of phototransduction genes during retinal development in vertebrates.

    ACKNOWLEDGEMENTS

We thank Kristen Swenn, Lia Scalzetti, and Dr. Yemisi Oluwatosin for assistance in the library screens and Dr. Suchitra Batni for advice on the embryo transfections. We gratefully acknowledge Dr. Shiming Chen and Dr. Tom Kerppola for generously providing us with purified recombinant Crx and Nrl, respectively. Drs. David Turner, Robert West, and Marianna Max provided many helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants EY09409 (to B. E. K.) and EY02414 (to J. C. B.) and The Research to Prevent Blindness Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U41623 and AF053942.

§ Present address: Section of Genetics and Development, Cornell University, Ithaca, NY.

** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY HSC at Syracuse, 750 E. Adams St. Syracuse, NY 13210. Tel.: 315-464-8719; Fax: 315-464-8750; E-mail: knoxb{at}hscsyr.edu.

2 B. E. Knox, J. Liu, S. Noonan, and S. Batni, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: kbp, kilobase pair(s); bp, base pair(s); UTR, untranslated region of mRNA; RACE, rapid amplification of cDNA ends; GFP, green fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; Crx, cone-rod homeobox protein; Nrl, neural retinal leucine zipper protein; GST-CrxHD, glutathione S-transferase-tagged Crx homeodomain; His-Nrl, hexahistidine-tagged Nrl.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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