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(Received for publication, June 12, 1996, and in revised form, August 26, 1996)
From the Departments of ABSTRACT In vitro DNA binding
assays and transient transfection analysis with monkey kidney cells
have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP
transcription factors, and the Nrl response element (NRE) in the
regulation of rhodopsin expression. We have now further explored the
role of the NRE and surrounding promoter elements. Using the yeast
one-hybrid screen with integrated NRE and flanking DNA as bait, the
predominant clone obtained was bovine Nrl. Recovery of
truncated clones in the screen demonstrated that the carboxyl-terminal
half of Nrl, which contains the basic and leucine zipper domains, is
sufficient for DNA binding. To functionally dissect the rhodopsin
promoter, transient expression studies with primary chick retinal cell
cultures were performed. Deletion and mutation analyses identified two
positive regulatory sequences: one between INTRODUCTION Rhodopsin is the visual pigment of vertebrate rods, and its
activation by light initiates the phototransduction process (1). In
recent years there has been increasing interest in understanding the
mechanisms regulating rhodopsin gene expression, motivated both by the
fundamental importance of the protein in visual transduction and by its
role as a prototype for the study of photoreceptor-specific gene
regulation (2). In addition, since mutations in the rhodopsin gene can
lead to retinal degeneration (3, 4), understanding of its regulation
will have implications for designing effective retinal gene therapy
strategies (5).
Nuclear runoff experiments have demonstrated that the expression of
rhodopsin is primarily regulated at the transcriptional level (6, 7).
Transgenic studies have defined two regulatory regions, a rhodopsin
proximal promoter region (RPPR) and a more distal rhodopsin enhancer
region (RER).1 The rhodopsin proximal
promoter region, located within Sequence analysis also disclosed the presence of a putative binding
site for the Maf/Nrl family of transcription factors in the rhodopsin
promoter. Nrl is an evolutionarily conserved basic motif-leucine zipper
(bZIP)-containing DNA binding protein, which is homologous to the v-Maf
oncogene product (16). The Maf-Nrl subfamily has been implicated in
cell type-specific regulation of genes in tissues as diverse as the
hematopoietic system (17, 18), cerebellum (19), and developing
hindbrain (20, 21, 22). Maf and Nrl proteins bind an extended AP-1-like
sequence, and form both homodimers and heterodimers with other bZIP
family members (23, 24, 25, 26, 27). Nrl itself is specifically expressed in
retinal cells, including photoreceptors, in which its expression
precedes that of rhodopsin during development (28). Recently, we have
shown by electrophoretic mobility shift analysis that Nrl can bind
in vitro to an oligomer containing the Nrl response element
(NRE) present in the rhodopsin promoter, and that cotransfection of
CV-1 monkey kidney cells with an Nrl-expression vector can stimulate
expression of rhodopsin promoter-reporter fusion constructs (29).
In this report we provide more direct evidence supporting a role for
Nrl in the regulation of rhodopsin gene expression. In the first
part of the study, using the yeast one-hybrid system, we demonstrate
that Nrl can bind to the NRE in vivo and regulate expression
of a reporter gene. We then use transient expression analysis of
primary chick retinal cultures (30) to further analyze Nrl, the NRE,
and related cis-elements in the rhodopsin promoter. The cells in these
primary cultures, unlike those in transformed lines, express a highly
differentiated phenotype resembling closely their in vivo
counterparts (31, 32). Using transfection and cotransfection studies,
we show that Nrl can activate rhodopsin expression in retinal cells in
an NRE-dependent manner and identify a positive regulatory
region that is located just upstream of the NRE and acts
synergistically with it.
EXPERIMENTAL PROCEDURES A retinal cDNA fusion
library for one-hybrid screening was generated from
poly(A)+ bovine retinal RNA. Oligo(dT)-primed cDNA was
directionally inserted into the EcoRI and XhoI
sites of plasmid pACTII (Steve Elledge, Baylor College of Medicine)
just downstream of the GAL4 activation domain. The ligation products
were electroporated into Escherichia coli strain DH10B and
plated onto LB/carbenicillin plates. The average transformation
efficiency was 1010 transformants/µg of DNA.
Approximately 2 × 106 independent clones were
obtained. Testing of individual colonies revealed that the average
insert size was 2 kilobases. Bacteria from the plates was harvested by
scraping, and DNA was prepared using Qiagen Maxi columns according to
the manufacturer's directions. The resulting plasmid library was
amplified twice before use. Bait constructs containing tetramers of the
Reporter constructs were
generated by cloning appropriate DNA fragments into the multiple
cloning site of the pGL2-basic luciferase-containing plasmid (Promega,
Madison, WI). For the generation of bRho-2174, gBR200pLacF (34) was
digested with Asp-718 and BamHI, and the appropriate
gel-purified fragment was ligated into pGL2 predigested with Asp-718
and BglII. To generate mRho-4907, the plasmid gMR4-4, containing the mouse rhodopsin upstream sequence (34), was digested with SalI, filled in with Klenow, and digested with
XhoI, and the appropriate gel-purified fragment was ligated
into pGL2 that had been digested with NheI, filled in, and
then digested with XhoI. For mRho-1609, -1488, and -270, mRho-4907 was digested with Asp-718 and NheI,
MscI, and ApaI, respectively, and then filled in
and recircularized with ligase. The bovine and murine proximal element
deletion constructs were generated by polymerase chain reaction
amplification of appropriate regions of bRho-2174 and mRho-4907,
respectively; for each construct, a unique upstream primer containing
an XhoI site and a common downstream primer in the
luciferase gene (GLprimer 2; Promega) were used. The polymerase chain
reaction products were then digested with XhoI and
HindIII and ligated into pGL2 predigested with
XhoI and HindIII. hRho-85 was generated
similarly, using gJHN7 (35) as a template for the polymerase chain
reaction. The construction of pMT-NRL was described previously (29).
The human collagenase promoter construct and c-Fos and c-Jun expression
vectors (36) were gifts from Dr. Daniel Nathans (The Johns Hopkins
University); CMV-lacZ was a gift from Dr. Kuan Teh-Jeang
(NIAID); and RSV-Luc was a gift from Dr. Mark Showers
(Harvard University).
DNA for transfection was obtained using the alkaline lysis protocol and
double banding through CsCl2 (37). The supercoiled DNA was
precipitated twice with ethanol, resuspended in 2-4 ml of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) and filtered
through a Microcon-100 column (Amicon, Beverly, MA).
Embryonic day 8 (E8) and E17 chick retinal
cultures were prepared as described previously (32, 38). Photoreceptors
in these cultures develop and maintain a polarized phenotype, form outer segment processes, express a number of photoreceptor-specific genes, including rhodopsin, undergo photomechanical movements in
response to light, and respond to neurotransmitters and retinoids (31,
32). Conditions for transient expression were optimized using E8
retinal cultures. Pilot studies revealed that the calcium phosphate
method yielded higher transfection efficiencies than transfection with
Transfectam (Promega), TransfectACE (Life Technologies, Inc.),
Lipofectin (Life Technologies), LipofectAMINE (Life Technologies), or
calcium phosphate with glycerol shock. The calcium phosphate procedure
was then optimized with respect to the amount of reporter and carrier
DNA, the volume of calcium phosphate precipitate, cell density, the
number of days in vitro prior to transfection, and the
interval between transfection and harvesting (data not shown). These
studies resulted in the protocol described below. Using this protocol
and a Rous sarcoma virus long terminal repeat-luciferase reporter
construct, transfection efficiencies as high as 15% could be obtained
(data not shown).
E8 cultures were transfected after 8 days in vitro. E17
cultures were transfected after 3-5 days in vitro. DNA in
80 µl of solution I (0.25 M CaCl2) was added
dropwise to 80 µl of solution II (25 mM HEPES, pH 7.1, 140 mM NaCl, and 14 mM
Na2HPO4) while vortexing at a slow speed. The
mixture was incubated at room temperature for 15 min and then added
dropwise to the appropriate cell culture dish. For cotransfection
assays, all plasmids were added to solution I before mixing with
solution II. mRho-4907 was transfected at 9 µg of DNA/plate; for all
other reporter constructs, equivalent molar amounts of DNA were used.
Each transfection included 1 µg of CMV-lacZ as an internal
control, to measure and correct for differences in transfection
efficiency. Transfections were performed in duplicate during each
experiment, and all experiments were done three independent times.
Cell lysates were prepared 40-48 h after
transfection by scraping the cells from individual plates into 200 µl
of lysis buffer following the manufacturer's instructions (Promega).
The lysates were either used immediately for luciferase and
Fifty µl of cell lysate was
mixed with an equal volume of 8 mg/ml chlorophenyl
red- RESULTS Sequence
comparison of the mouse (34), rat (10), Chinese hamster (39), cow (40),
and human (35) rhodopsin proximal promoter regions demonstrate that the
NRE and flanking DNA are highly conserved (Fig. 1). The
NRE itself, spanning the bovine sequence between
Fig. 2 shows the predicted amino acid sequence of the bovine Nrl
protein, as determined by the sequence of one of the full-length library clones and its comparison with the previously determined human
and murine Nrl sequences (16, 43). The bovine sequence is 92 and 85%
identical to the human and murine sequences, respectively. Interestingly, the majority of the sequence differences are present in
the immediate carboxylterminal region, just beyond the leucine zipper motif.
Procedures for transient expression analysis using primary
chick retinal cultures were developed (see "Experimental
Procedures") to examine whether Nrl can trans-activate rhodopsin
promoter activity in retinal cells and to explore the ability of DNA
elements flanking the NRE to modulate Nrl activity. To determine
possible differences in promoter activity associated with the
differentiation stage of the retinal cells, transfections were
performed with chick retinal cultures derived from both E8 and E17.
E8-derived cultures are free of glial and retinal pigment epithelial
cells. They contain a mixture of photoreceptors and nonphotoreceptor
neurons; the photoreceptors, which constitute approximately 10-20% of
the cell population, differentiate largely in vitro (31,
44). In E17 cultures, photoreceptors represent 30-60% of the
population, and many aspects of their differentiation occur in
vivo, prior to their isolation for culture.
Chick retinal cells were transfected with a series of luciferase
fusion constructs containing DNA from the region upstream of the bovine
(bRho) and murine (mRho) rhodopsin. (The nomenclature used for reporter
constructs includes abbreviations for the species and gene name,
followed by a number that refers to the position of the 5
To look specifically at the activity of the NRE and flanking DNA, the
additional constructs bRho-38, bRho-84, bRho-130, mRho-40, mRho-84,
mRho-130, mRho-176, and mRho-222 were generated. As shown in Fig. 3,
B and C, the bovine (murine) constructs showed
evidence of positive regulatory elements between To further investigate the functional role of Nrl in
rhodopsin regulation, E8 and E17 retinal cell cultures were
cotransfected with bRho constructs together with an expression vector
containing the human NRL cDNA (pMT-NRL). Cotransfection
of pMT-NRL resulted in induction of reporter gene activity with
constructs that contained an intact NRE but had little effect with
constructs that lacked the NRE or contained a mutated NRE, in both E8
(Fig. 4A) and E17 (Fig. 4B) cells.
In E8 cultures, but not in E17 cultures, the Rho-130 constructs showed
substantially greater stimulation with pMT-NRL cotransfection than did
the Rho-84 constructs, even though there are no NRE-like sequences
between
Since the NRE is an extended AP-1 element, which binds
to c-Fos-c-Jun heterodimers, we investigated whether c-Fos and c-Jun could also trans-activate the rhodopsin promoter. As shown in Fig.
5A, cotransfected Nrl, c-Fos, and c-Jun
expression plasmids stimulated reporter gene activity from the bRho-130
construct by 3.2-, 1.9-, and 0.9-fold, respectively. When the
expression constructs were cotransfected as pairs, Nrl-c-Fos and
Nrl-c-Jun were both more potent than c-Fos-c-Jun. In contrast, in
control experiments with a collagenase reporter construct (hCol-71),
which contains a canonical AP-1 site but lacks an NRE, the maximal
stimulation was seen with c-Fos, and Nrl was only minimally effective
(Fig. 5B). Taken together, these results indicate that in
primary chick retinal cells Nrl preferentially trans-activates the
rhodopsin NRE compared with c-Fos and c-Jun. The results are also
consistent with in vitro studies that have shown that the
central cytidine in the consensus Maf binding site
(TGCTGA
DISCUSSION Using the yeast one-hybrid system together with transient
expression analysis of primary chick retinal cultures, we have obtained evidence supporting an important in vivo role for the bZIP
transcription factor Nrl in the regulation of rhodopsin expression.
Independent one-hybrid assays consistently demonstrated that Nrl is the
predominant gene obtained using the rhodopsin The one-hybrid analysis also provided a deletion analysis of the
regions of Nrl required for DNA binding. The finding that the smallest
5 In a complementary set of studies, transient expression analyses were
carried out to explore the rhodopsin promoter regulatory interactions
in the context of retinal cells. Primary chick retinal cell cultures
were chosen for these studies, because they allow transfection
efficiencies as high as 15% and contain photoreceptor cells that
exhibit many of the molecular, structural, and functional properties of
their in vivo counterparts (31). These characteristics are
particularly appealing, since they allow analysis of the results of
transient expression studies in the context of other differentiated cell behaviors, which is seldom possible using transformed cell lines.
Mouse retinal cells would theoretically provide another attractive
system for transfection analysis, since they also undergo extensive
differentiation in culture (46); however, pilot studies with mouse
cells demonstrated transfection efficiencies that were so low as to
preclude meaningful promoter analysis.
The deletion analysis did not demonstrate any activity associated with
the rhodopsin RER. Although the reason for this is unclear, it is
perhaps not surprising, given that presently there is no evidence to
suggest the existence of a chick version of the RER. Additionally,
enhancer regions often require chromatin structure for their activity.
Related to this, the RER does not show activity with a bovine in
vitro transcription system that also relies on naked DNA templates
(13).
The deletion analysis did provide evidence of the presence of
cis-acting elements located between The experiments presented here indicate that Nrl can stimulate
rhodopsin expression in an NRE-dependent manner in yeast
cells and chick retinal cell cultures, respectively, but they do not by
themselves demonstrate that Nrl plays a similar role in
vivo. Several lines of evidence, however, are consistent with this
possibility and favor the involvement of Nrl compared with other bZIP
proteins: 1) in the adult, Nrl is specifically expressed in the retina, including photoreceptors (16); 2) Nrl expression precedes rhodopsin expression during development (28); 3) the position and sequence of the
rhodopsin NRE is evolutionarily conserved; 4) supershift experiments
with retinal nuclear extracts indicate that the activity that binds to
a rhodopsin NRE oligomer contains Nrl, or an immunologically similar
molecule, but not Fos or Jun (29); 5) although Fos and Jun have been
shown to be expressed in the retina, they have not been identified in
photoreceptors (47, 48); 6) Nrl is more specific than either Fos or Jun
for the rhodopsin promoter; and 7) neither Fos nor Jun was detected in
the one-hybrid screen.
It is also clear that Nrl by itself is not sufficient to turn on
rhodopsin expression in vivo. Rhodopsin expression is
limited to rod photoreceptors, but Nrl is expressed in a number of
different retinal and neuronal cell types (16). Additionally,
retinoblastoma cell lines express Nrl but not rhodopsin (16, 49). Like
other genes that are regulated in a cell type- and developmentally
restricted manner, control of rhodopsin expression is likely to be
mediated by a combinatorial array of transcription factors, some of
which are retina- and/or cell type-specific, whereas others are more ubiquitously expressed (2). On the other hand, Nrl may also be involved
in the regulation of other photoreceptor-specific genes that contain
NRE-like sites, such as the human red and green opsin (50) and bovine
interphotoreceptor retinoid-binding protein genes,3 and nonphotoreceptor genes, such as
the quail gene QR1, a developmentally restricted gene expressed in
retinal Müller cells (51). Future experiments will be necessary
to define more precisely this apparently complex role of Nrl in
vivo.
FOOTNOTES Acknowledgments We thank Drs. Jeremy Nathans, Daniel Nathans,
Steve Elledge, Kuan Teh-Jeang, and Mark Showers for generously
providing gJHN7, the human collagenase promoter construct, pACTII,
CMV-lacZ, and RSV-Luc, respectively.
REFERENCES
Volume 271, Number 47,
Issue of November 22, 1996
pp. 29612-29618
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
§,,,,,
,¶¶ and§§
Ophthalmology,
Neuroscience, and
§§ Molecular Biology and Genetics, The Johns
Hopkins University School of Medicine,
Baltimore, Maryland 21287-9277, ¶ Departments of Ophthalmology,
Cell Biology, and Anatomy, Cornell University School of Medicine,
New York, New York 10021, and Departments of 
Radiation
Oncology, and ** Ophthalmology and Human Genetics, University of
Michigan, Ann Arbor, Michigan 48105
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
40 and 84 base pairs
(bp) and another between 84 and 130 bp. Activity of the 40 to
84 region was shown to be largely due to the NRE. On co-transfection
with an NRL expression vector, there were 3-5-fold increases in the
activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or
deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The 84-
to 130-bp region acted synergistically with the NRE to enhance both
the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin
expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary
retinal cell cultures for characterizing both the cis-acting response
elements and trans-acting factors that regulate photoreceptor gene
expression.
176 to +70 and 400 to +80 bp in the
bovine (34)2 and murine (8) genes,
respectively, provides low level, yet photoreceptor-specific,
expression. The RER, a 100-bp sequence located approximately 2 kilobases upstream from the mRNA initiation site, acts as an
enhancer (9). Biochemical analysis has identified a number of putative
cis-acting DNA regulatory elements; they include the Ret1-PCE1 (10,
11), Ret2, Ret3 (12), Ret4 (13), and Mash-1 (14) binding sites and a
site homologous to the Drosophila glass response element
(15).
73- to 30-bp sequence from the bovine rhodopsin promoter in the
vectors pHISi-1 and pLacZ were prepared (Clontech). The pHISi-1 and
pLacZ bait constructs were then linearized with XhoI and
NcoI, respectively, and integrated into the genome of
YM4271. The resulting strain with the integrated pHISi-1-bait construct
was used for library screening according to Clontech's protocol.
Positive library clones were defined as those that, on
retransformation, led to growth of pHISi-1-bait cells on
His
medium containing 60 mM
3-amino-1,2,4-triazole and generated a blue color on
5-bromo-4-chloro-3-indoyl-
-D-galactopyranoside-containing plates on transformation of cells harboring the pLacZ-bait construct. Library plasmid DNA minipreparations were prepared using standard methods (33).
-galactosidase assays or stored on ice for a few hours. Twenty µl
of cell lysate was mixed with 50 µl of reconstituted luciferin
(Promega). Luciferase activity was measured with a TD-20e luminometer
(Turner Designs, Inc., Mountain View, CA) with an integration time of
10 s.
-Galactosidase Assays
-D-galactopyranoside (Boehringer Mannheim) in
duplicate in a 96-well plate format, and reactions were measured using
an EL312e enzyme-linked immunosorbent assay reader (Bio-Tek Instruments, Inc., Winooski, VT). Serial dilution of a
-galactosidase enzyme standard on the same plate was used to
determine the linearity of the reaction and to calculate concentrations
based on the rate of reaction (Kineticalc software; Bio-Tek
Instruments). The resulting -galactosidase values were used to
correct the luciferase values for transfection efficiency.
68 and 56 bp, is
conserved in 12 of its 13 nucleotide positions. We had previously shown
that an oligomer pair containing the rhodopsin NRE can bind in
vitro to proteins in a bovine retinal nuclear extract and that Nrl
is one of the proteins in the bound complex (29). To identify
transcription factors that can bind to the rhodopsin NRE and its
flanking DNA in vivo, in the context of chromatin structure,
we used the yeast one-hybrid system (19, 41, 42). A bovine retinal
cDNA-GAL4 activation domain fusion library was generated and
transformed into yeast cells containing as bait a stably integrated
tetramer of the bovine rhodopsin promoter sequence from 73 to 30 bp
cloned upstream of a HIS3 reporter construct. A total of 1.7 × 105 yeast transformants were screened. Thirty-one positive
colonies that grew on His plates were identified in the
first round. On retransformation, plasmids from 11 of these 31 clones
were still positive with both the HIS3 and lacZ reporter assays.
Sequence analysis revealed that seven of the clones contained
full-length Nrl open reading frames, whereas three others contained
5
-Nrl truncations. The 11th clone was highly homologous to aspartate
aminotransferase and probably represented a false positive. In a second
round of library screening, involving 3.2 × 106
transformants, of 44 confirmed positives that have been characterized, 42 represent Nrl clones. The smallest 5-truncation, present in four
identical clones, encodes a fusion protein in which the Nrl sequence
begins at residue 129 (see Fig. 2), demonstrating that residues 129-236 are sufficient for DNA binding.
Fig. 1.
Sequence comparison of rhodopsin proximal
promoter regions. 5-flanking sequences from the rat, Chinese
hamster, mouse, cow, and human rhodopsin genes were aligned using
GeneWorks 2.3 software. Shaded boxes, regions conserved in
all five species. Positions of the transcription start site, TATA box,
Ret1, and NRE are indicated above the alignment. The position(s) of the mRNA start sites are also underlined.
[View Larger Version of this Image (40K GIF file)]
Fig. 2.
Sequence comparison of bovine, human, and
murine Nrl proteins. The predicted bovine Nrl sequence represents
translation of sequences obtained from the yeast one-hybrid clones
described in the text. *, position of residue 129, which is the
predicted amino terminus of the shortest 5-truncated clone. Sites at
which the human and murine sequences (16, 43) differ, as well as the
position of the extended homology (EHD), basic, and leucine zipper domains, are indicated. - in the bovine sequence, point of a
one-amino acid residue deletion relative to the human and murine
sequences.
[View Larger Version of this Image (15K GIF file)]
-end of the
construct relative to the mRNA start site.) The bRho-2174,
bRho-225, bRho-176, mRho-4907, mRho-1609, mRho-1488, and mRho-270
constructs demonstrated similar levels of activity, ranging between 66 and 107 relative light units (Fig. 3A). These results demonstrate that the bovine and murine rhodopsin proximal promoter regions, consisting of less than 300 bp of upstream DNA, are
as active in chick retinal cultures as longer constructs containing the
RER. (The RER spans the region from 2044 to 1943 and from 1575 to
1477 bp in the cow and mouse genes, respectively (9).)
Fig. 3.
Expression of murine and bovine rhodopsin
constructs in E17 cultures. E17 chick retinal cultures were
transfected with equal molar amounts of the indicated constructs by the
calcium phosphate method as described under "Experimental
Procedures." The constructs in A were designed to assess
the activity of the RER and other upstream regions, whereas the bovine
and murine constructs shown in B and C,
respectively, were designed to dissect the rhodopsin proximal promoter
region (RPPR). In B, the construct bRho225(m62-66), which contains a mutated NRE in which the central 5 bp (CTGAT) have been mutated to GTCGAG, is also included. Relative light units (R.L.U.) were corrected for transfection
efficiency with a -galactosidase internal control and represent the
ratio of the activity of each construct relative to the activity of the
promotorless construct pGL2-basic. The values are means of three
independent experiments performed in duplicate. Bars, 1 S.E.
[View Larger Version of this Image (14K GIF file)]
38 (40) and 84
(85) bp, which contain the NRE, and between 84 (85) and 130 bp. The activity of the 84- to 130-bp region, particularly for the bovine constructs, is significantly greater than the NRE-containing region. The results also suggest the possibility of a negative element
located between 130 and 176 bp. The importance of the NRE within
the 38- to 84-bp sequence was confirmed by the finding that
bRho-225(m62-66), which contains a mutated NRE, demonstrated significantly reduced reporter activity (Fig. 3B).
84 and 130 bp. This suggested that in the E8 cultures there
are factors that can interact with sequences in the 84- to 130-bp
region and increase NRL-mediated trans-activation. The human reporter
construct hRho-85, which contains a homologous NRE, also showed
approximately 3-fold stimulation on cotransfection with pMT-NRL (Fig.
4B), as did analogous murine rhodopsin constructs (data not
shown).
Fig. 4.
Stimulation of rhodopsin promoter activity by
cotransfection with an Nrl expression vector. E8 (A)
and E17 (B) cultures were transfected with equal molar
amounts of the indicated bovine rhodopsin-luciferase constructs either
alone or together with 1 µg of the Nrl expression plasmid pMT-NRL.
The -fold stimulation represents the ratio of the corrected luciferase
activity with pMT-NRL cotransfection to the activity without pMT-NRL
cotransfection. The values shown represent the means of three
independent experiments performed in duplicate ± S.E.
(bars).
[View Larger Version of this Image (17K GIF file)]
TCAGCA) is important in determining the specificity
of DNA interaction with bZIP proteins (25). Maf, Jun homodimers, and
Fos-Jun heterodimers were all found to bind equally well to the
consensus sequence; however, when the central cytidine nucleotide was
altered (TGCTGA
TCAGCA), as occurs in the rhodopsin NRE,
Maf still bound strongly to the oligomer, whereas the binding of both
Jun homodimers and Fos-Jun heterodimers was abolished.
Fig. 5.
Activities of Nrl, c-Fos, and c-Jun on
NRE-containing and AP-1-containing promoters. E17 chick retinal
cultures were cotransfected with either bRho-130 (A) or
collegenase reporter construct hCol-71 (B) and 1 µg of
each of the indicated bZIP expression vectors. The data are represented
as -fold stimulation as in Fig. 6. Bars, S.E.
[View Larger Version of this Image (14K GIF file)]
73- to 30-bp sequence
as bait, thus suggesting that Nrl may indeed be the primary
transcription factor binding to this region in vivo. The
observation that Nrl can bind to the NRE and flanking DNA in the
one-hybrid system adds to our previous demonstration that Nrl can bind
to the rhodopsin NRE in an in vitro eletrophoretic mobility
shift assay (29) by demonstrating that the interaction can take place
in an in vivo situation in the context of chromatin
structure, which is often more stringent than naked DNA. In addition,
since in some of the full-length constructs the Nrl coding region is
out of frame with the GAL4 activation domain, the activation of
reporter gene expression is presumably due to the activity of the Nrl
trans-activation domain itself. In an analogous analysis of
c-Maf, it was similarly found that the exogenous activation
domain was not needed when the full-length coding region was used
(19).
-truncations obtained in the screen corresponded to amino acid
residues 129-236 is consistent with the structure of Nrl and previous
biochemical studies with the Maf-Nrl family, which indicate that in
addition to the basic region and leucine zipper motifs, a highly
conserved sequence just amino-terminal to the basic region (extended
homology domain; see Fig. 2) is also required for specific DNA binding
(19, 23, 24, 45). Consistent with the model that the trans-activating
activity is associated with the amino-terminal part of the molecule,
the Nrl coding region in the deletions was in frame with the
GAL4 activation domain.
130 and 40 bp, a region that is
highly conserved among the mouse, rat, Chinese hamster, cow, and human
rhodopsin genes. Within this region, two separate domains could be
defined, one between 40 and 84 bp and another between 85 and
130 bp, that contain positive regulatory elements. A third domain,
located between 130 and 176 bp, may possess a negative regulatory
element. At least part of the activity of the 40 to 84 bp region
derives from the NRE present between 56 and 68 bp. The 85 to
130 bp region, which contains part of the rhodopsin ret1-PCE1 element
(10), is particularly active, stronger than the NRE-containing region.
It appears to act synergistically with the NRE because it leads to an
increase in the trans-activating activity of co-transfected Nrl, even
though it lacks a binding site for Nrl. The synergism could be mediated
by a regulatory protein that binds to a sequence between 85 and 130
bp and stabilizes the interaction between Nrl and the NRE or interacts
with bound Nrl and increases its trans-activating activity.
Alternatively, Nrl could independently stimulate the synthesis or
activity of another protein that binds to and acts directly through the
85 to 130 bp sequence. Regardless of its nature, the synergistic activity appears to be developmentally regulated, since it is seen with
E8 but not with E17 cultures (Fig. 4, A and B).
Future studies will better define the mode of interaction and
regulatory element(s) within the 85 to 130 bp region.
§
Present address: Bioelectronics Group, David Sarnoff Research
Center, Princeton, NJ 08543.
¶¶
A Senior Investigator from Research to Prevent
Blindness, Inc.
To whom correspondence should be addressed: The Johns
Hopkins University School of Medicine, 809 Maumenee, 600 North Wolfe St., Baltimore, MD 21287-9277. Tel.: 410-550-5230; Fax: 410-550-5382; E-mail: don_zack{at}qmail.bs.jhu.edu.
1
The abbreviations used are: RER, rhodopsin
enhancer region; bp, base pair; NRE, Nrl response element; bRho, bovine
rhodopsin; mRho, murine rhodopsin; E, embryonic day.
2
S. Chen and D. J. Zack, unpublished
results.
3
D. E. Borst, J.-S. Si, and J. M. Nickerson,
GenBank accession number M32733[GenBank].
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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