From the Department of Biochemistry and Molecular
Biology and
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
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ABSTRACT |
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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 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 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.
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
( 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 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).
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).
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). 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).
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
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
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 ( 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 ( 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.
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 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, 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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
,
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).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
-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.
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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.
-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
-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
-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.
-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.
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.
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.
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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
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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REFERENCES |
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