(Received for publication, June 15, 1995; and in revised form, October 24, 1995)
From the
Previous transgenic mouse experiments localized the mammalian rhodopsin gene promoter to a region just upstream of the mRNA start site, and also suggested the existence of a second more distal regulatory region. A highly conserved 100-base pair (bp) sequence which is homologous to the red and green opsin locus control region is located 1.5-2 kilobases upstream of the rhodopsin gene (depending on the species). In order to test the activity of this 100-bp region, transgenic mice were generated with bovine rhodopsin promoter/lacZ constructs which differed only by the presence or absence of the sequence. Of 11 lines generated, all demonstrated photoreceptor-specific expression of the transgene, but the lines with the putative regulatory region showed significantly higher expression. Additional transgenic lines in which the region was fused to a minimal heterologous promoter did not show transgene expression in the retina. Gel mobility shift and DNase I footprint assays demonstrated that bovine retinal nuclear extracts contain retina-specific as well as ubiquitously expressed factors that interact with the putative regulatory region in a sequence-specific manner. These results indicate that the 100-bp sequence can indeed function in vivo as a rhodopsin enhancer region.
The neural retina is a specialized part of the central nervous system which both transduces light energy into neurochemical signals and begins initial information processing. It has a complex laminar structure in which there is segregation of form and function. Morphological and thymidine labeling studies have demonstrated that the different types of neuronal and glial cells that make up the retina are born and differentiate in a defined temporal and spatial sequence(1) . Cell lineage studies, utilizing both retroviral (2, 3) and fluorescent dextran (4) markers, indicate that most, if not all, of these cells arise from common progenitors. However, despite these and other important advances in the cell biology of retinal development(5) , the actual molecular mechanisms which regulate cell fate determination and the development of committed progenitors into mature retina cells remain poorly understood.
Since development and differentiation of the retina are thought to involve a cascade of events in which different genes are turned on and off in a precisely regulated manner, one approach to studying retinal development is to analyze the mechanisms that control gene expression within the retina. Identification of transcription factors which regulate cell type and lineage-specific gene expression could, for example, lead to the discovery of master regulatory factors analogous to those controlling other lineages, such as the MyoD/myogenin/myf-5 family involved in muscle development(6) .
Efforts to define the cis-acting DNA elements and trans-acting factors which regulate retina-specific gene
expression have so far focused primarily on photoreceptor-specific gene
products such as
rhodopsin(7, 8, 9, 10, 11, 12, 13) ,
red and green opsins(14, 15) , blue
opsin(16, 17, 18) , interphotoreceptor
retinoid-binding protein(19, 20) ,
S-antigen(21, 22) , arrestin(23) , and
-transducin(24) . Rhodopsin provides a particularly
attractive model system for these studies because: 1) both the gene and
the protein are well characterized(25) ; 2) its expression is
tightly regulated both in terms of cell-type specificity and
developmental timing, and it shows diurnal modulation(26) ; 3)
it is expressed at high levels; and 4) its similarity with the color
opsins allows useful homology comparisons (27) . Moreover,
approximately 30% of cases of autosomal dominant retinitis pigmentosa,
a currently untreatable disease in which photoreceptor degeneration
leads to blindness, are due to mutations in the rhodopsin
gene(28, 29, 30) . Development of effective
gene therapy for autosomal dominant retinitis pigmentosa will require
thorough understanding of rhodopsin regulation(31) ,
particularly since even wild type rhodopsin can lead to retinal
degeneration when abnormally expressed(32, 33) .
The induction and regulated increase in rhodopsin expression seen
during rod development is largely controlled at the transcriptional
level(34, 35, 36) . Transgenic mouse studies
utilizing overlapping sets of promoter-lacZ fusion constructs
have identified some of the DNA elements that regulate
photoreceptor-specific expression of rhodopsin(37) . Bovine
upstream fragments from -2174 to +70 bp, ()from
-735 to +70 bp, from -222 to + 70 bp, and from
-176 to +70 bp (relative to the mRNA start site) (7) (
)as well as murine 4.4 kb and 0.5 kb 5`
fragments (8) all direct photoreceptor-specific expression.
There are, however, important differences between the various
constructs. Although position effects can cause considerable variation,
the level of transgene expression is generally higher with the larger
constructs than with the smaller ones. In addition, a superior-temporal
to inferior-nasal transgene expression gradient is seen with the longer
but not with the shorter constructs, which show either a spotty or
diffuse pattern of expression.
These results suggested that there may be at least two classes of elements regulating rhodopsin expression: a ``proximal region'' in the vicinity of the mRNA start site (within -176 to +70 bp in the bovine gene) that serves as a minimal promoter capable of directing photoreceptor-specific expression, and a ``distal region,'' located further upstream, which serves as an enhancer. In addition, the finding of a gradient of expression which is unique to mice with the longer constructs raised the possibility that either the putative enhancer or a different distal sequence might function as a topological element controlling spatial expression across the retina.
In this paper we directly address the identity of the putative rhodopsin enhancer. Although the deletion series employed in the initial bovine transgenic experiments suggested that the enhancer and topological regulatory elements were located between -2174 and -734 bp, the mapping was not detailed enough to define a specific location. Based on sequence comparison of the mouse, cow, and human rhodopsin upstream regions, we hypothesized that the enhancer activity might be contained within a highly conserved 100-bp region and have generated transgenic mice that contain promoter-reporter fusion constructs that differ only by the presence or absence of this candidate region. Characterization of these mice indicates that the 100-bp candidate region displays many of the properties of an enhancer. It is not, however, required to establish an expression gradient across the retina. We also present biochemical evidence that bovine nuclear extracts contain both retina-specific and ubiquitously expressed proteins which bind to areas within the enhancer region in a sequence specific manner.
The bovine RER/heterologous promoter fusion construct was generated using the 0.3-kb BamHI/NcoI fragment from plasmid pRed2 (kindly provided by Jeremy Nathans and Yanshu Wang, Johns Hopkins University, Baltimore, MD) which contains the region from -88 to +230 bp from the hsp70 A1 gene(38) . The fragment, which was gel purified after filling-in the BamHI site with Klenow, was directionally cloned into the plasmid Rho -2174/placF which had been previously digested with XhoI, filled-in with Klenow, cut with NcoI, and then gel purified. The resulting plasmid was cut with HindIII and KpnI to generate the approximately 4.4-kb fragment that was used for microinjection.
Transgenic mice
were generated at the Johns Hopkins University School of Medicine
Transgenic Mouse Facility by pronuclear microinjection of B6AF1
(female) C57BL/6J (male) embryos using established techniques (39) , as described previously(7) . Animals were
screened by PCR as described previously (7, 37) except
that 30-bp primers (5`-GATGTGGCGAGATGCTCTTGAAGTCTGGTA3`,
5`-CAAGGCAACTCCTGATGCCAAAGCCCTGCCC-3`, and
5`-AGCTGAGCGCCGGTCGCTACCATTACCAGT-3`) were used instead of the
previously described 21-bp primers and the digestion buffer contained
2% Triton X-100 instead of 2% Nonidet P-40.
For method 1,
restriction fragment probes corresponding to the region -2143 to
-1895 bp were generated using plasmid
p(-2143/-1895)/rho, which contains the PCR product
amplified with the above primers cloned into the SalI
restriction site of pBluescript II KS. To prepare
probe labeled at its upstream end, p(-2143/-1895)/rho was
digested with HindIII, phosphatased with calf intestine
alkaline phosphatase, kinased with [
-
P]ATP
and T4 polynucleotide kinase, and digested with Asp718. The
desired end-labeled fragment was then gel purified. Probe labeled at
its downstream end was prepared similarly except that it was first
digested with Asp718 and cut with HindIII after the
kinase step.
DNase I footprint reactions were carried out using
standard procedures (44) . Binding reactions contained
approximately 10 fmol of probe and 50 µg of bovine nuclear extract
in a 50-µl reaction volume for method 1 and 25 µl for method 2.
For method 1, binding buffer consisted of 12.5 mM HEPES, pH
7.6, 100 mM KCl, 5 mM ZnSO, 0.5 mM dithiothreitol, 2% (w/v) polyvinyl alcohol, 10% glycerol, and 1
µg of poly(dI-dC). For method 2, binding buffer consisted of 12.5
mM HEPES, pH 7.6, 60 mM KCl, 5 mM MgCl
, 0.5 mM dithiothreitol, 10% glycerol,
and 1 µg of poly(dI-dC). After 15 min incubation on ice, the
reaction tubes were transferred to room temperature, incubated for 1
min, MgCl
and CaCl
were added to give final
concentrations of 5 and 2.5 mM, respectively, and then DNase I
(Worthington) at the appropriate concentration (see Fig. 7and Fig. 8) was added. Digestion was carried out for the times
indicated and then terminated by the addition of 90 µl of stop
solution (20 mM EDTA, pH 8.0, 1% (w/v) SDS, 0.2 M NaCl, and 250 µg/ml glycogen) and 10 µl of 2.5 mg/ml
proteinase K (Sigma). After incubation at room temperature for 5 min,
samples were extracted with phenol/chloroform, precipitated with
ethanol, and washed with 75% ethanol. Samples were resolved on standard
6% sequencing gels.
Figure 7: DNase I footprint of the RER with retina nuclear extract. A and B, footprint pattern of the RER using method I (see ``Experimental Procedures''). C, footprint pattern using method II. In lanes 1-3 and 7-9 the top template strand was labeled; in lanes 4-6 the bottom strand was labeled. The major protected regions on the top strand are labeled I, II, III and IV; the major protected regions on the bottom strand are labeled I`, II`, and III`. Hypersensitive sites are indicated with an ``*''. In panel C regions I and IV are indicated for comparative purposes but are shown in parentheses because significant protection is not evident. The sequences corresponding to each of the protected regions are indicated in Fig. 1. The reactions in lanes 1, 4, and 7 did not contain nuclear extract. The reactions in lanes 2, 3, 5, 6, 8, and 9 each contained 50 µg of bovine retina nuclear extract (R). The amounts of DNase I used per 50-µl reaction in lanes 1-9 were 3.3, 80, 40, 3.3, 80, 40, 5, 50, and 50 ng, respectively, and the digestion times were 1 min for lanes 1-6 and 5, 2, and 5 min for lanes 7-9, respectively.
Figure 8: Tissue specificity of RER DNase I footprint activity. A and B, comparison of RER footprint patterns with nuclear extracts from bovine retina (R), cerebellum (C), cerebral cortex (CC), liver (L), and skeletal muscle (M). The templates in A and B were labeled on the top and bottom strands, respectively. All lanes contained 50 µg of the indicated extract except for lane 1 which did not contain any extract. The nomenclature for labeling protected areas is the same as in Fig. 7. The protocol employed was method I (see ``Experimental Procedures''). The amounts of DNase I used per 50-µl reaction in lanes 1-12 were 3.3, 80, 80, 80, 80, 40, 3.3, 80, 80, 80, 80, and 40 ng, respectively, and the digestion time was 1 min.
Figure 1: Sequence comparison of distal homology regions from cow, human, mouse, and rat rhodopsin genes. RER sequences from cow, human, mouse, and rat rhodopsin genes were aligned to show maximal homology using GeneWorks 2.3 (Intelligenetics, Mountain View, CA). The 37-bp conserved sequence in the red/green LCR (15) is also shown and aligned for maximal homology. Bases that are identical in all four rhodopsin genes are enclosed with black lines. Bases that are identical in at least three of the rhodopsin genes are shaded. The base position relative to the mRNA start site of the 3`-most base of each sequence is shown on the right of the figure. The numbers on the bottom of the figure refer to base position for the bovine sequence. The RER, which extends from -2044 to -1943 bp in the bovine gene, is indicated by a bracket. The positions of the four DNase I protected regions (FPI, FPII, FPIII, and FPIV) are shown. The top arrows refer to protection seen with the top strand labeled and the bottom arrow refers to protection seen with the bottom strand labeled. The CGATGG core sequence is underlined. The positions of oligomer pair A (EMSA-A), oligomer pair B (EMSA-B), the putative rhodopsin homeodomain binding site-1 (RHBS-1), and the ret-3 binding site (10) are also shown. (Note: the bovine sequence shown contains a correction from the previously published sequence (7) indicating that at residues -2031 to -2030 there should be two Cs rather than one.)
The RER also shows homology to the highly conserved 37-bp sequence in the color opsin locus control region (LCR), an element involved in regulation of the red and green visual pigment gene cluster (15) (Fig. 1). This area of homology contains a sequence, CTAAT (-1985 to -1981 bp in the bovine sequence), that is similar to the homeodomain consensus binding sequence(45) , and henceforth will be referred to as ``rhodopsin homeodomain binding site-1'' (RHBS-1). The LCR sequence has a 6-bp deletion, relative to the RER, that is located just upstream of the putative homeodomain binding site. This 6-bp sequence and the putative homeodomain site both appear to be involved in sequence-specific DNA-protein interactions (see below).
Figure 2: Map of the upstream rhodopsin-lacZ fusion constructs. Schematic diagram of the constructs used in this study. A, construct rho-2045, which consists of bovine rhodopsin sequences extending from -2045 to +70 fused to the lacZ cassette from placF. The positions of the RER and proximal promoter region are indicated. The lacZ cassette from placF contains the 3`-untranslated region from the mouse protamine gene in order to provide an intron and poly(A) addition site (7, 63) . B, construct rho-1923, which consists of bovine rhodopsin sequences extending from -1923 to +70 fused to the lacZ cassette from placF. The RER is not included in this construct. C, construct RER-hsp70, which consists of bovine rhodopsin sequences extending from -2174 to -1620 fused to the -88 to +230 bp promoter fragment from the hsp70 A1 gene, which in turn is fused to the lacZ cassette from placF.
Six independent lines were obtained with construct rho-2045
(2045-2, -15, -19, -21, -35, -65) and five with rho-1923
(1923-8, -15, -21, -39, -45). Fig. 3shows the results of
solution assays for -galactosidase activity on eyes from each of
the lines at 22-26 days of age. Eyes from the lines containing
the RER had on the average 10-fold higher activity than eyes from the
lines that did not contain the RER; this difference is statistically
significant (p
0.026, Wilcoxon Rank sum test).
Figure 3:
-Galactosidase activity of eyes from
transgenic mice that differ by the presence or absence of the rhodopsin
RER. Eyes from 22-26-day-old transgenic animals from the
indicated lines were enucleated and tested for total
-galactosidase activity. Activity is expressed in milliunits (1
milliunit will hydrolyze 1.0 nmol of o-nitrophenol-
-D-galactopyranoside to o-nitrophenol and galactose per min). The rho-2045 lines
contain the RER and the rho-1923 lines do not. The previously described
2174-31 line, which includes the RER, is included as a positive control
for a high expressing line(7) . The copy number for lines
2045-2, -15, -19, -21, -35, -65, and 1923-8, -15, -21, -39,
-45 were approximately 1, 2, 2, 5, 40, 15, 50, 25, 1, 7, and 30,
respectively.
Analysis of each of the transgenic lines by quantitative Southern
analysis did not show evidence of a correlation of copy number with
expression level. Copy number for lines 2045-2, -15, -19, -21,
-35, -65 were approximately 1, 2, 2, 5, 40, and 15, respectively. Copy
number for lines 1923-8, -15, -21, -39, -45 were approximately
50, 25, 1, 7, and 30, respectively (data not shown). The variation in
-galactosidase activity between the lines which contain the same
construct presumably reflects position effects related to the transgene
integration site.
Figure 4: Comparison of transgene expression patterns in rho-2045 and rho-1923 mice. A-C, retinal whole-mount preparations from animal lines 2045-2, 2045-19, and 2045-65, respectively, stained with X-gal. D and E, retinal whole-mount preparation from animal lines 1923-39 and 1923-45, respectively, stained with X-gal. All eyes were from animals that were approximately 1 month old. The retinas in A, D, and E are from right eyes and the retinas in B and C are from left eyes. F, in situ hybridization of retinal whole-mount preparation from line 2045-65 using a lacZ antisense riboprobe. After hybridization the eye whole-mount was embedded in JB-4 and sectioned at 10 µm.
X-gal staining, a function of -galactosidase enzyme
activity, cannot per se elucidate whether the observed
expression gradients reflect mechanisms operating at the protein level,
such as translational control or differences in protein stability, or
differences in transgene mRNA levels. We therefore performed whole
mount in situ hybridization in order to visualize lacZ mRNA directly. The resulting patterns were essentially identical
to those seen with X-gal staining. Fig. 4F shows a
histological section through an in situ whole mount
demonstrating an area of spotty transgene expression.
Whole mount in situ hybridization was also performed with a rhodopsin probe to determine whether there were any developmental stages in which the endogenous rhodopsin gene was expressed in a gradient pattern similar to that seen with the transgenes. Eyes were examined daily or every other day from postnatal day 1, before rhodopsin mRNA could be detected, to postnatal day 20, at which time there was strong and uniform expression throughout the retina. Gradients in expression patterns similar to those seen with the transgenics were not observed at any of the developmental stages studied (data not shown).
Figure 5:
EMSA
with -2005 to -1986 bp probe: sequence and tissue
specificity. P-Labeled DNA oligomer pair A (-2005 to
-1986 bp), with or without cold competitor oligomer, was
incubated with bovine nuclear extract and the resulting DNA-protein
complexes were analyzed by nondenaturing polyacrylamide gel
electrophoresis. Individual complexes are labeled A-F. Lane 1 does not contain nuclear
extract; lanes 2-23 contain 3 µg of retina extract.
Each set of three lanes from 3 to 23 contains cold competitor at
increasing ratios of competitor to labeled oligomer of 5:1, 25:1, and
100:1. Lanes 3-5 contain wild-type cold competitor
oligomer (-2005 to -1986 bp). Lanes 6-23 contain cold competitor oligomers (-2005 to -1986 bp)
in which single base pair mutations have been introduced into the
CGATGG core sequence: As changed to Cs, Cs to As, Gs to Ts, and Ts to
Gs. The competitor nomenclature consists of a number corresponding to
the position in the core sequence and a letter denoting the base
mutation. For example, competitor 1A contains a C to A transversion in
the first position (C) of the core sequence; competitor 2T
contains a G to T transversion in the second position (G) of
the core sequence. Lanes 24-27 contain nuclear extract
from bovine cerebellum (C, 7 µg), cerebral cortex (CC, 7.5 µg), liver (L, 4 µg), and kidney (K, 7 µg), respectively. All lanes (1-27)
contain 1.0 µg of poly(dI-dC), except lane 24 which
contains 0.5 µg.
The
sequence specificity of the DNA-protein interactions with the
-2005 to -1986 bp sequence was explored using direct
binding and cold oligomer competition with a series of oligomers
containing site-specific mutations. The entire sequence was first
scanned with oligomers in which successive groups of 3 bp were mutated
one group at a time. The sequence CGATGG was identified by this
analysis as an important core sequence that was required to generate
the wild-type mobility shift pattern. Mutations in the sequence
flanking this core did not significantly affect the shift pattern (data
not shown). To further analyze the CGATGG region, each of the 6 bp in
the core sequence was mutated individually and used as a cold
competitor (Fig. 5, lanes 3-23). Wild-type
oligomer efficiently inhibited all bands, except for band F which was
only partially inhibited at the concentration used (lanes
3-5). The oligomers containing single base changes
dramatically altered the shift patterns, demonstrating a high degree of
sequence specificity in protein interactions with the CGATGG core.
Moreover, the specificity of interaction was present at the level of
individual shifted bands. For example, the oligomer with a C to A
mutation at position 1 (A) showed nearly wild-type ability
to inhibit all bands (lanes 6-8). In contrast, the
oligomer with a G to T mutation at position 5 (T
) showed
essentially no ability to inhibit bands D, E, and F, although it still
inhibited bands A, B, and C effectively (lanes 18-20).
The oligomer with a G to T mutation at position 6 (T
)
behaved similarly to T
, except that it was less effective
at inhibiting the B and C bands and, at high concentrations, it
slightly inhibited bands D, E, and F (lanes 21-23). The
oligomer with an A to C mutation at position 3 (C
) was less
effective at inhibiting the B, C, D, E, and F bands, but was
essentially as effective as the wild-type oligomer in inhibiting band A (lanes 12-14). Since the C
mutation makes
the bovine core sequence identical to that of the mouse and rat
sequences (Fig. 1), this result suggests that the putative rat
and mouse core binding proteins may display binding preferences that
are distinct from the bovine protein(s).
The tissue specificity of the proteins which interact with the -2005 to -1986 probe was examined by comparing EMSA patterns generated with bovine retina, cerebellum, cerebral cortex, kidney, and liver extracts (Fig. 5, lanes 2 and 24-27). Band D appears to be neuron-specific since it is observed with retina, cerebellum, and cerebral cortex extracts but not with kidney or liver extracts. Bands A-C appear restricted to retina, and perhaps cerebellum, but since they are weaker the differences between the tissues may be less significant.
EMSA was also performed with an overlapping sequence, oligomer pair B, which spans the region from -1995 to -1973 bp (Fig. 1), to analyze the putative CTAAT homeodomain binding site, RHBS-1, together with surrounding DNA (Fig. 6). Five shifted bands were observed (A-E, lane 2), which were effectively competed by unlabeled oligomer B (lanes 3-5) but not by an unrelated cold oligomer (lanes 6-8). Comparison of the shift pattern generated with retina, cerebral cortex, cerebellum, liver, and kidney nuclear extracts suggested that bands B and C might be retina-specific (lanes 2 and 9-12). Band D, or a band of similar mobility, was present with liver as well as retina extract. In addition, several bands were seen with the non-retinal extracts that were not present with retina extract.
Figure 6:
EMSA with -1995 to -1973 bp
probe. P-Labeled DNA oligomer pair B (-1995 to
-1973 bp), with or without cold competitor oligomer, was
incubated with bovine nuclear extract and the resulting DNA-protein
complexes were analyzed by nondenaturing polyacrylamide gel
electrophoresis. Individual complexes are labeled A-E. It should be noted that the intensity of band D varied with different retina extract preparations. Lane 1 does not contain nuclear extract. Lanes 2-8 contain retina extract (5 µg). Lanes 3-5 contain cold oligomer B competitor at increasing ratios of
competitor to labeled oligomer of 5:1, 25:1, and 100:1, respectively. Lanes 6-8 contain an unrelated oligomer competitor
(TCTTCACCTTGACCTCT) at increasing molar ratios of 5:1, 25:1, and 100:1,
respectively. Lanes 9-12 contain cerebral cortex (CC, 7.5 µg), cerebellum (C, 7 µg), liver (L, 4 µg), and kidney (K, 7 µg) extract,
respectively. All lanes contain 1.0 µg of poly(dI-dC), except lane 10 which contains 0.3 µg.
The positive regulatory activity of enhancers is generally position independent. Although the position independence of the RER was not directly tested, it is suggested by phylogenetic analysis which shows that despite the high sequence conservation between the mouse, rat, cow, and human RERs, there is significant variation in their position relative to the mRNA start site (Fig. 1). Furthermore, comparison of the bovine RER with those of the other species revealed a conserved 25-bp sequence that appears to have been inverted and transposed downstream(7) . The 37-bp core sequence in the red/green opsin LCR, which shows sequence homology with the RER (Fig. 1), also exhibits similar variation in position and orientation(15) .
The striking sequence homology between the RER and the 37-bp conserved sequence in the red/green opsin LCR probably reflects evolution of the rod and cone opsins from a common visual pigment progenitor gene. The lack of correlation of expression level with copy number in the rho-2045 mice argues that in the rhodopsin gene the RER does not function as a LCR, and suggests that the acquisition of such activity took place after the divergence of the genes. Whether the red/green LCR has the ability to act as a rhodopsin enhancer remains to be determined.
The DNase I footprint experiments provide data that is complementary to but not identical with that obtained with the EMSAs. Protected regions I, II, and III all correspond to highly conserved sequences (Fig. 1). Protected region IV is less highly conserved. The protection and mobility shift assays both provide evidence for protein interaction with RHBS-1. Protection regions II and III flank both sides of the CGATGG core sequence, and correspond to areas showing significant homology to the red/green LCR 37-bp sequence; however, there is no significant protection over the CGATGG sequence itself. This may partially result from difficulty in detecting a footprint over the region due to the relative lack of bands corresponding to the CGATGG sequence in the absence of nuclear extract, a reflection of the non-random nature of DNase I cleavage. It may also reflect a low abundance of the CGATGG binding protein(s), since EMSAs are generally more sensitive than footprint assays because a detectable signal in an EMSA requires a shift of only a small fraction of the labeled probe whereas in a footprint assay a large fraction of the labeled template needs to be protected. Alternatively, variation in the binding conditions in the two assays or the greater complexity of protein-DNA interactions involved in the footprint assay may account for the differences.
Other binding regions within the RER include the ret-3 site(10) , which overlaps region IV (Fig. 1), and a sequence that is homologous to the proposed chick homologue of the Drosophila glass binding site(11) . The binding site for the putative transcription factor Bd, TGACCT, which was identified upstream of the arrestin gene(23) , is also present within the RER, in footprint region II. However, although these in vitro studies of DNA-protein interaction are suggestive, they do not demonstrate that the individual interactions are biologically significant. Future functional analyses, such as additional transgenic, retinal cell culture, and retinal in vitro transcription assays, as well as cloning of the factors involved will be required to establish and characterize the biological role of the individual DNA elements within the RER.
The finding that superior-temporal to inferior-nasal expression gradients occur only in transgenic lines carrying longer upstream fragments suggested the hypothesis that the RER might also function as a topological element regulating retinal spatial expression patterns. Our results, however, argue against a simple model in which the RER is necessary and sufficient for gradient expression. The rho-2045 and the rho-1923 lines both exhibit superior-temporal to inferior-nasal gradients. Although variations of the gradient between lines are seen, there is no correlation between a particular type of pattern and the presence or absence of the RER. Essentially identical superior-temporal to inferior-nasal gradients can be seen with both rho-2045 and rho-1923 mice. It therefore appears that the DNA sequences required for the gradient pattern are located downstream of the RER.
Although the rhodopsin promoter and RER appear to account for most aspects of rhodopsin transcriptional regulation, they do not account for all aspects. Transgenic lines containing 2.2 kb of bovine rhodopsin upstream DNA as well as lines carrying 4.4 kb of murine upstream DNA show low level leaky expression in cones(60, 61) . Since endogenous rhodopsin is not thought to be expressed in cones, this finding suggests that the 2.2- and 4.4-kb constructs, both of which contain the promoter and the RER, may be missing a negative regulatory element which binds to a factor which ``silences'' expression in cones. The finding that transgene expression in mice carrying a 11-kb BamHI genomic mouse rhodopsin fragment, which contains 5 kb of upstream sequence, is rod-specific is consistent with such a hypothesis and suggests that the putative silencer element may be located between 4.4 and 5 kb upstream, within intron sequence, or in 3` DNA(60) . It is interesting to speculate that the putative silencer protein may repress a number of rod-specific proteins in non-rods and thus may be analogous to the recently cloned neuron-restrictive silencer element which inhibits neuronal gene transcription in non-neuronal cells(62) .