From the Departments of Internal Medicine and
Physiology & Biophysics, The University of Iowa College of Medicine,
Iowa City, Iowa 52242 and the § Department of Molecular and
Cellular Biology, Roswell Park Cancer Institute,
Buffalo, New York 14263
Received for publication, September 12, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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Previous studies demonstrate that the mouse
renin gene is regulated by a complex enhancer of
transcription located 2.6 kilobases upstream of the transcription start
site which is under both positive and negative influence. We
demonstrate herein that a positive regulatory element (Eb) is repeated
10 bp upstream (Ec), and both are required for baseline activity of the
enhancer. The Eb and Ec core sequences are identical to the consensus
sequence for the nuclear hormone receptor superfamily of transcription
factors, and transcriptional activity of constructs containing the
enhancer is increased after treatment with retinoic acid. Maximal
induction requires both Eb and Ec. Expression of endogenous
renin and a renin-promoter controlled transgene
in As4.1 cells, and kidney renin mRNA in C57BL/6J mice
was induced after retinoid treatment. Gel mobility supershift analysis
revealed the binding of RAR The renin-angiotensin system is a critical regulator of
arterial pressure and electrolyte homeostasis and is required for continued development of the kidney after birth. The cleavage of
angiotensinogen by renin is thought to be the rate-limiting step in the
biosynthesis of angiotensin II and is tightly regulated. Transcription of the renin gene, storage and processing of
renin in juxtaglomerular cell secretory granules, and secretion of
renin into the systemic circulation, each dictate the level of
angiotensin II produced. Although the regulation of the
renin gene has been studied for many years, the molecular
mechanisms controlling its cell-specific expression and regulation in
response to physiological cues remains incomplete.
Recent studies have identified an enhancer of transcription located
upstream of the renin gene which can markedly induce
transcription of renin promoter reporter constructs when
transfected into As4.1 cells, a renin expressing tumor cell
line isolated from the kidney thought to be derived from
juxtaglomerular cells (1, 2). This enhancer, located ~2.6
kb1 upstream of the mouse
renin gene is partially homologous to a sequence located
~12 kb upstream of the human renin gene (3). We previously
used mouse/human chimeric enhancers spanning the conserved and
nonconserved region to identify important sequences controlling
expression (4). Those studies revealed that a 40-bp segment (m40) in
the promoter proximal region of the mouse renin enhancer was
required for maximal activity. The m40 segment contained two regulatory
elements. The first sequence, element a (Ea), bound the factor NF-Y and
acted as a transcriptional repressor because mutations abolishing
binding of NF-Y significantly stimulated enhancer-mediated
transcription. The second sequence, element b (Eb), was required for
maximal activity of the enhancer, and its mutagenesis essentially
abolished enhancer activity. Given that Ea and Eb overlapped, we
hypothesized that NF-Y blocks enhancer activity by preventing the
binding of transcription factors to Eb. This is supported by
experiments in which the spacing between Ea and Eb is
altered.2
Based on the observation that the m40 sequence is insufficient to
stimulate transcription on its own, we speculated that additional sequences further upstream of m40, but within the 242-bp enhancer are
required for maximal induction. Herein we demonstrate that a third
element, a direct repeat of Eb, termed Ec, lying upstream of m40 is
also required for baseline enhancer activity. This sequence when
multimerized can strongly stimulate renin promoter activity on its own. Moreover, the Ec-10 bp-Eb sequence matches the consensus binding site for members of the nuclear hormone receptor superfamily. This sequence can bind the RAR Plasmids--
The luciferase (LUC) reporter vectors m4.1kLUC,
mE2.6kLUC, mEµa2.6kLUC, mEµb2.6kLUC, mEµba2.6kLUC, and mE117LUC
were described previously (4) (Fig. 1A). mE represents the
242-bp mouse renin enhancer sequence. m4.1k
represents a 4.1-kb 5'-flanking sequence of mouse renin
(
The µc mutation in mEµc2.6kLUC was generated using the
oligonucleotide
5'-CTCAGAGGTCAGAGTACAGCCAGGAAACCATCTG-3' containing two substituted bases in Ec (underlined). The µbc double mutant in
mEµcb2.6kLUC was generated with the oligonucleotide
5'-CTCAGAGGAAAGAGTACAGCCAGGAAACCATCTG-3' containing two substituted bases in both Ec and Eb. The triple µcba
mutant construct mEµcba2.6kLUC was made by using standard DNA
cloning taking advantage of an RsaI restriction digestion site present between Ec and Eb at the junction of
mEµa117LUC and mEµcba117LUC were generated by swapping the DNA
fragments between mE117LUC and mEµa2.6kLUC or mEµcba2.6kLUC, respectively. EcEb117LUC, 3EcEb117LUC, and 3 µcµb117LUC were
generated by insertion of synthetic double-stranded oligonucleotides
containing EcEb or µcµb in the forward orientation directly to the
5'-end of m117. SmaI to HindIII fragments
containing EcEb117, 3EcEb117, and 3 µcµb117 were then excised from
the corresponding subclones and inserted into pGL2-basic.
Cell Culture and Transient Transfection--
Cell culture and
transient transfection of the As4.1 cell line (American Type Culture
Collection, CRL2193) was as described previously (4). In brief, As4.1
cells were cultured in reduced-serum Opti-MEM supplemented with 2%
FBS, 1 mg/ml Albumax-II (Life Technologies, Inc.), penicillin (100 units/ml), and streptomycin (100 mg/ml) for 2 days before transient
transfection. The conditioned cells were transfected with plasmid DNA
by electroporation using equal molar amounts of each plasmid balanced
with pUC19. 2.5 × 107 cells were used in transfection
for nondrug treatment studies. For ligand treatment studies,
108 cells were transfected and then split into four equal
dishes containing Opti-MEM supplemented with 2% charcoal-treated FBS. A RSV-LUC vector was used as a standard positive control, and 0.1 µg
of cytomegalovirus- Hormone Treatment--
In our ligand treatment study,
transfected As4.1 cells were cultured in the Opti-MEM supplemented with
charcoal-treated fetal bovine serum (FBS) to minimize lipophilic
hormones. To deplete small lipophilic compounds from FBS, 2 g of
dextran-coated charcoal (C6197, Sigma-Aldrich Co.) was mixed with 100 ml of FBS. The mixture was gently rotated at 4 °C for 16 h, and
the charcoal was removed by centrifugation. The charcoal-treated FBS
was sterilized by filtration and stored at
Eleven C57BL/6J mice (4 months of age) were fed an vitamin A-deficient
diet (Formula TD 88407, Teklad, Madison, WI) for 2 months. Three
littermates were fed a normal diet as control. All animals had access
to water ad libitum. tRA was administrated by subcutaneous
injection (10 mg/kg). The test group received 5 injections, with 2 injections on each of the first 2 days (in the morning and evening) and
1 injection in the morning of the third day. The control group received
vehicle. The animals were sacrificed 6 h after the last injection.
The kidney was collected and frozen on dry ice immediately. Care and
use of the mice met the standard procedures approved by the University
Animal Care and Use Committee at the University of Iowa
Electrophoretic Mobility Shift (EMSA) and Supershift
Assay--
Preparations of the nuclear extract from As4.1 cells and
probes for EMSA were as described previously (4). To label probes, 5'-GATC overhangs at both ends of the annealed double-stranded oligonucleotides were filled with [
Rabbit polyclonal antibodies against human RAR subtypes: RAR RNA Isolation, RNase Protection Assay, and RT-PCR--
Total
cellular RNA was isolated from mouse As4.1 cells using TRI-REAGENT
(Molecular Research Center, Inc., Cincinnati, OH) using the
manufacturer's protocol. Total renal RNA was isolated from mouse
kidneys using our standard procedure (6). T7 RNA polymerase was used to
prepare antisense RNA transcripts as RNase protection assay probes. The
full-length and protected probe for mouse Ren-1c
mRNA was 235 and 175 nucleotides, respectively. The full-length and
protected probe for mouse 18 S rRNA was 140 and 80 nucleotides, respectively. RNase protection was performed using the Hyb-speed kit
(Ambion Inc., Austin, TX). The protected RNA probes were resolved on
6% polyacrylamide denaturing gel (containing 8 M urea) and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RT-PCR was performed as described previously (7). The following
synthetic oligonucleotides were used: RAR Construction of Wild-type and Dominant Negative
RAR Statistical Analysis--
All data are presented as mean ± S.E. Multiple comparison of data was analyzed by one-way ANOVA using
SigmaStat (SPSS Scientific). When the test for normalization failed,
the analysis was performed nonparametrically. Single comparisons are
performed using Student's t test.
Using chimeric enhancers derived from the divergent regions of the
mouse and human renin enhancer we previously identified two
regulatory elements in the promoter proximal portion of the mouse
renin enhancer. These studies were performed using As4.1 cells, which express renin and are likely derived from juxtaglomerular cells. Ea acted as a negative regulatory element and bound the factor
NF-Y, and Eb acted as a positive regulatory element (4). Mutation of Eb revealed that it is required for maximal enhancer activity, but that Eb and Ea alone were insufficient to stimulate renin promoter activity, suggesting the presence of other
transcription factor-binding sites in the enhancer. The purpose of the
current study is to further examine the requirements for
enhancer-mediated transcriptional activation and identify the
stimulatory factors.
Mutational analysis of Eb revealed that it has the core sequence
TGACCT. Sequence analysis of the 242-bp mE revealed two other TGACCT
motifs which lie upstream (more distal) of Eb. The first motif, termed
Es is present at the 5' terminus of the enhancer at coordinates and RXR
to oligonucleotides
containing both Eb and Ec. Reverse transcriptase-polymerase chain
reaction analysis revealed that As4.1 cells express both receptor
isoforms, along with RAR
, but do not express RAR
, RXR
, or
RXR
. Co-transfection of an expression vector encoding wild-type
RAR
increased enhancer activity, whereas a dominant negative mutant
of RAR
significantly attenuated retinoic acid-induced activity of
the enhancer. These results demonstrate the importance of the Eb and Ec
motifs in controlling baseline activity of the renin
enhancer, and suggest the potential importance of retinoids in
regulating renin expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and RXR
transcription factors and
is required for induction of the renin promoter by retinoic acid. That retinoic acid can stimulate endogenous renin
mRNA in As4.1 cells and mouse kidney suggests they may play a
potentially important role in regulating renin expression.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4.1 kb to +6) containing mE in its native position. m2.6k represents
a 2.6-kb 5'-flanking sequence of mouse renin lacking mE.
m117 is a minimal mouse renin promoter spanning from
117
bp to +6 relative to the transcription start site. mE2.6k is a 2866-bp
continuous sequence with a SphI site separating mE from
m2.6k. Mutations in elements a and b previously identified are
indicated by µa and µb. Oligonucleotide-mediated mutagenesis was
performed using the GeneEditor in vitro Site-directed
Mutagenesis System (Promega). The selection oligonucleotides came with
the kit. The mutagenesis oligonucleotides were generated by Genosys Biotechnologies Inc. and were phosphorylated before use. The sequence of all mutants was confirmed by direct fluorescent DNA sequencing.
2662 and
2661. We
excised the distal part containing µc (from
2866 to
2662) from
mEµcb2.6kLUC using SmaI and RsaI and the
proximal part containing µba (
2661 to
2625) from mEµba2.6LUC
using RsaI and SphI. The two fragments were then
cloned into pGEM-7zf(
) to generate mEµcba/pGEM-7 that was used to
make the final mutant construct. The µca mutant construct,
mEµca2.6kLUC, was generated by mutagenesis of mEµc2.6kLUC using the
oligonucleotide 5'-TGTACTCTGACCTCTTCGCTGCTGGTTGTG-3' containing four substituted bases in Ea (underlined). All
of the m4.1k-based mutants were generated by recovering the
BstXI to KpnI fragment (
2677 to
1217 bp)
harboring the mE mutations from mE2.6k-based constructs, and ligating
the fragments into a variant of the wild type m4.1kLUC construct
containing a deletion of the corresponding BstXI to
KpnI segment.
Gal was co-transfected as an internal control to
monitor transfection efficiency. Cells were harvested and assayed for
luciferase and
-galactosidase activity 48 h after initial
transfection. Activity assays were performed as described previously
(4, 5). Luciferase activity was normalized to
-galactosidase
activity from the same extract and presented as a percentage of
luciferase activity of the Rous sarcoma virus promoter transfected
separately in each experiment. All activity assays were performed in
duplicate and the average of 2 readings was used as 1 data point.
20 °C. Hormone or
vehicle was added to the media 24 h after transfection. The
hormones used in the study were all-trans-retinoic acid
(tRA), 9-cis-retinoic acid, 3,3',5-triiodo-L-thyronine (T3), and
1,25-dihydroxyvitamin D3 (D3) (Sigma-Aldrich
Co.).
-32P]dATP
(PerkinElmer Life Sciences) and 3 other cold nucleotides using Klenow
DNA polymerase. The probes were purified through Sephadex G-50. Each
binding reaction contained 0.02 pmol of labeled probe (about 60,000 dpm), 3 µg of nuclear extract, 1 µg of poly(dI-dC) (Roche Molecular
Biochemicals), and binding buffer with the final concentration of (in
mmol/liter): Tris-HCl (pH 7.5), 10, EDTA 1, dithiothreitol 1, MgCl2 1, and KCl 60, as well as 5% glycerol in a total
volume of 20 µl. For competition assays, cold competitor oligos were
preincubated with nuclear extract and binding buffer for 15 min on ice
before the addition of probes. The binding reactions were then
incubated on ice for another 15 min, and the products were resolved on
a 5% nondenaturing polyacrylamide gel.
,
RAR
, and RAR
, and RXR subtypes: RXR
, RXR
, and RXR
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The human antisera cross-reacts with the specific RAR and RXR subtypes in the
mouse. Universal monoclonal antibody against the mouse RXR family
(including all three subtypes of
,
, and
) was the generous gift from Dr. Pierre Chambon (CNRS, INSERM, Universite Louis Pasteur, Strasbourg, France). 10 µg of polyclonal antibodies (Santa Cruz) and
the indicated amount monoclonal antibodies (Chambon) were added
following the initial incubation of probe, nuclear extract, and binding
buffer, and were left on ice for 60 min before electrophoresis.
,
5'-CACTACGAACAACAGCTCAGAACA-3' and 5'-GTCCGTGTGTCGAGGTGGTCAT-3' (784 bp); RAR
, 5'-CAGCCCTGGAATTTGTGGAT-3' and
5'-CATGGAGTGGAGGCAGGGAGAGTC-3' (405 bp); RAR
,
5'-CTCGCCCGACAGCTATBAACT-3' and 5'-CCCGGCAAAGGCAAAGAC-3' (424 bp);
RXR
, 5'-TCGAGCCCAAGACTGAGACATACG-3' and
5'-TCTCCCTCAACGCCTCCACCTCAG-3' (459 bp); RXR
,
5'-CAGCAGCCCTCAGATCAACTCCAC-3' and 5'-CCCCATCTCCATCCCCGTCTTT-3' (404 bp); and RXR
, 5'-GGTCTGCCTGGGATTGG-3' and
5'-CATGTCACCGTAGGATTCTGTCTT-3' (420 bp).
--
The full-length cDNA encoding mouse RAR
1 was PCR
amplified using the primers 5'-GACTTGCTAGCCTGTTTGCCTG-3'
(NheI site underlined) and
5'-CTGAATTCCGTGTGTCGAGGTGGT-3' (EcoRI site
underlined). This cDNA clone contains 1441 bp including the entire
mRAR
coding sequence. The PCR product was digested with
NheI and EcoRI to expose the restriction ends and
was cloned into the mammalian expression vector, pCI (Promega, Madison,
WI) under the control of the cytomegalovirus enhancer/promoter. To make
the dominant negative mRAR
403 expression vector, we first amplified
a segment of cDNA, from +634 to +1209 from the full-length RAR
1
using the primers 5'-CACTACGAACAACAGCTCAGAACA-3' and
5'-GGTCTAGACTACGGGATCTCCATCTTCAATG-3' (XbaI site as underlined). This segment of cDNA
does not have the C-terminal AF-2 domain. This cDNA has an
intrinsic EcoRV site in the center of the RAR coding region
and a synthetic XbaI site at the 3' end. Therefore, the
EcoRV to XbaI fragment was isolated and ligated
into the pCI vector that contained all the N-terminal sequence upstream
from the EcoRV site.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2847
to
2852. The other TGACCT motif, termed Ec, is located 10 bp upstream
of Eb from
2668 to
2673, and thus lies upstream of the original m40
sequence containing Eb and Ea (Fig.
1B). Since our previous study
demonstrated that enhancer function requires Eb, we performed transient
transfection analysis in mouse renin expressing As4.1 cells
to test whether Es or Ec were also required for enhancer activity.
Site-directed mutagenesis was performed to convert the GA in
TGACCT to TT, which in our previous study caused a loss of
function of Eb. Both mutations were individually generated in
mE2.6kLUC, which contains the mouse enhancer fused upstream of a
2624-bp mouse renin promoter (Fig. 1A). Mutation
of Ec significantly reduced transcriptional activity (Fig.
2), whereas mutation of Es had no effect
(data not shown). Interestingly, mutation of Ec caused a significantly
greater drop in enhancer activity than did mutation of Eb. Moreover,
the increase in enhancer activity caused by mutation of the negative
regulatory Ea required both Eb and Ec (Fig. 2). The importance of Ec
and Eb was confirmed by mutagenesis of 4.1kLUC which contains the enhancer in its native position. Mutation of Eb and Ec lowered activity
of the promoter to that of an enhancerless mutant (data not shown).
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Fig. 1.
Schematic map of the renin
gene. A, schematic representation of the
renin gene and constructs used in this study are shown. The
location of mE is indicated by the crosshatched arrowhead.
B, the cis-acting elements in the promoter proximal portion of mE
and specific mutations are shown. The location of Ed, Ec, Eb, and Ea
are underlined and the consensus sequences for their cognate
factors are aligned. The dotted overlined sequence indicates
the sequence employed in the Ec + Eb constructs and the double stranded
oligonucleotides probes and competitors used for EMSA. The
arrowhead indicates the 5' most nucleotide of the original
m40 sequence containing Eb and Ea.
View larger version (26K):
[in a new window]
Fig. 2.
Identification of Ec. Reporter vectors
containing 2.6kLUC without (no mE) or with mE, or mE mutants were
transiently transfected into As4.1 cells. The motifs (a, b,
or c) after the µ indicate which sequences were mutated.
For example, mEµcb lacks both Eb and Ec. Fold induction over
enhancerless control vector are indicted in parentheses. *,
p < 0.05 versus no mE; ,
p < 0.05 mEµc and mEµcb versus mEµb
(n = 6).
The mouse renin enhancer acts in a position-independent
manner and can strongly stimulate (>100-fold) a minimal mouse
renin promoter when placed directly upstream. To
characterize whether Ec + Eb has intrinsic enhancer activity on its
own, we placed one or three copies of the Ec + Eb sequence directly
upstream of the 117-bp promoter. Although one copy of the Ec + Eb
sequence only slightly increased promoter activity (5.2-fold), three
tandem copies of Ec + Eb markedly increased promoter activity
(110-fold) to nearly the same level as mE (Fig.
3). Mutation of Eb and Ec in the 3X(Ec + Eb) construct abolished this induction.
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We previously reported that Eb specifically interacted with
unidentified nuclear proteins from As4.1 cells by EMSA. Since both Ec
and Eb consisted of a TGACCT stretch we hypothesized that they
possessed the same protein binding activity. We identified two major
DNA-protein complexes (L and S) formed on Ec + Eb (Fig. 4A). The two complexes were
efficiently competed by Ec + Eb as well as mutants lacking either Ec or
Eb. On the contrary, a mutant lacking both Eb and Ec was not able to
compete. The results suggest that both Ec and Eb have the ability to
form both complexes. This was confirmed by demonstrating that mutants
lacking either site, but not both, could still form complexes L and S
when used as probes in EMSA (Fig. 4B). The two complexes
formed on both Ecµb and µcEb were efficiently competed by
competitor DNAs containing either one or two TGACCT stretches (data not
shown).
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To facilitate our identification of the Ec + Eb-binding factors, we determined which bases in the TGACCT stretch were essential to its binding activity. To accomplish this we first demonstrated that complexes L and S could be efficiently competed by DNAs containing a single TGACCT motif from either Eb or Ec (Fig. 4C). We then examined the requirement of each base in the binding activity by using double stranded oligonucleotides mutated one base at a time as competitors in EMSA (Fig. 4C). Mutation of any base within the TGACCT motif resulted in loss of competition, whereas a base-change mutation outside of the TGACCT stretch did not affect competition.
A direct repeat of the TGACCT motif separated by a spacer of variable
length is the consensus recognition sequence for transcription factors
in the thyroid/retinoid superfamily of nuclear hormone receptors. As a
candidate approach to identify which nuclear receptor could bind to Ec + Eb, we transfected As4.1 cells with the 3X(Ec + Eb)117LUC reporter
vector, and then treated the cells with four different common nuclear
receptor cognate ligands. To eliminate potential effect of lipophilic
hormones, the transfected cells were grown in culture medium
supplemented with charcoal-treated fetal bovine serum. Thyroid hormone
did not affect promoter activity, while 1,25-dihydroxyvitamin
D3 modestly, but significantly reduced promoter activity
(Fig. 5A). In contrast,
promoter activity was significantly increased by
9-cis-retinoic acid and tRA, suggesting that Ec + Eb may be
a retinoic acid responsive element (RARE). The increase in
transcription caused by tRA was dose responsive (Fig.
6).
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To order to further confirm that the observed retinoic acid response
was directly mediated by Ec + Eb, we transiently transfected As4.1
cells with a vector containing 3 tandem repeats of a mutant Ec + Eb
sequence. Both baseline and tRA-induced transcription was abolished by
mutation of Ec + Eb (Fig. 6A). Similarly, transcriptional activity of the 4.1kbLUC construct (which contains the enhancer in its
native position) was induced by retinoic acid, and required an intact
Ec + Eb sequence (Fig. 6B). Loss of the Eb and Ec sequence in 4.1kbLUC had the same effect as eliminating the enhancer. Given our
observation that both Ec and Eb could bind the same nuclear factors we
determined whether both sites were required for the tRA-induced
response. Interestingly, although the induction was lower than
wild-type mE, tRA treatment still induced (2-fold) the promoter
activity of constructs containing a mutation of either Eb or Ec (Fig.
7). As above, loss of both Eb and Ec
abolished tRA-mediated induction.
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To evaluate the physiological relevance of our observation we examined
the effect of tRA on the level of endogenous renin in As4.1
cells. Renin mRNA level in As4.1 was modestly increased after 24 h treatment with tRA (data not shown). tRA also modestly increased the level of the renin promoter SV40 T antigen
transgene mRNA present in those cells (data not shown). We next
examined the effect of tRA on the level of endogenous renin
mRNA in mouse kidney. Mice received subcutaneous injections of tRA
for 3 days after being fed a vitamin A-deficient diet for 2 months. The
treatment significantly increased (3-fold) the level of endogenous
renal renin mRNA (Fig.
8).
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Based on these results we hypothesized that RAR bind to Ec + Eb in mE
to stimulate transcription from the renin promoter in As4.1
cells. We therefore performed supershift EMSA using antibodies specifically-targeting either retinoic acid receptors (RAR, -
, or
-
), or RXR. The anti-RAR
antisera caused the appearance of a
supershifted complex, whereas the antibodies against other subtypes of
RAR did not shift the DNA-protein complex (Fig.
9). The anti-RXR monoclonal antibody also
caused the appearance of a supershifted complex. Subtype-specific
antisera for RXR revealed that RXR
bound to Ec + Eb (data not
shown). That both RAR
and RXR
antisera only partially
supershifted the complex suggests that other proteins, perhaps other
members of the nuclear hormone superfamily may also bind to mE. RT-PCR
and DNA sequencing verified the expression of RAR
, RAR
, and
RXR
, but not RAR
, RXR
, and RXR
in As4.1 cells (data not
shown).
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Finally, we constructed expression vectors encoding mouse RAR1, and
a dominant negative mutant of mRAR
(mRAR
403) which lacked the
C-terminal ligand-dependent AF-2 transactivation domain, to
specifically test the role of RAR
in the Ec + Eb-mediated retinoic
acid-induced activation of the mouse renin promoter (8, 9).
Both cDNAs were placed under the control of the cytomegalovirus promoter/enhancer and were transiently cotransfected into As4.1 cells
along with the 4.1kbLUC reporter vector. Co-transfection of the
wild-type mRAR
expression vector significantly increased promoter
activity of m4.1kLUC whether induced with tRA or left untreated (Fig.
10). On the contrary, co-transfection
of the mRAR
403 expression vector significantly attenuated the
tRA-induced promoter activity of m4.1kLUC vector, but did not alter
baseline expression. The stimulatory effect of the wild-type mRAR
was completely abolished when Ec and Eb were mutated (data not shown).
Our results suggest an important role of the Ec and Eb sequence in
controlling baseline activity of mE and demonstrate a potential role
for retinoids in regulating expression of the renin
gene.
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DISCUSSION |
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The renin gene enhancer is a potent enhancer of transcription that works in a position- and orientation-independent manner in renin-expressing As4.1 cells (2). Its ability to stimulate up to a 100-fold increase in transcriptional activity of the renin promoter makes it an important candidate in renin gene regulation. We previously identified two transcription factor-binding sites which mechanistically oppose each other (4). The binding of NF-Y to Ea acts as a negative regulator because it blocks the binding of transcription factors to Eb. Studies where the spacing between Ea and Eb is altered strongly suggests that NF-Y sterically blocks the binding of factors to Eb.2 The importance of Eb was shown by a loss of enhancer activity after mutagenesis. In the present study, we demonstrate that a second TGACCT motif located 10 bp upstream of Eb termed Ec is also required for maximal enhancer activity. Mutation of either Ec or Eb in mE essentially eliminates enhancer function. Mutation of a third TGACCT motif (Es) did not effect enhancer function.
The Eb and Ec sequences are clearly required for baseline activity of
the enhancer. In addition, their importance is supported by the
observation that they fit the consensus sequence for a member of the
nuclear hormone receptor superfamily, are required to mediate induction
of the renin promoter by retinoic acid, and bind RAR and
RXR
. There was no induction when cells were treated with thyroid
hormone or vitamin D3. Within the superfamily, selective recognition of different ligand/receptors is determined by the number
of intervening base pairs between the two TGACCT motifs. In general,
heterodimers of TR/RXR selectively bind to DR4 and heterodimers of
VDR/RXR selectively bind to DR3. Interestingly, heterodimers of RAR/RXR
preferentially bind to DR2 or DR5 (10). There are 10 intervening base
pairs between Ec and Eb in mE. Our EMSA and supershift studies clearly
demonstrate the ability of the DR10 sequence to bind RAR
and RXR
.
Despite the preference for DR2 or DR5, the retinoic acid responsive
F-crystallin and medium chain
acyl-coenzyme A dehydrogenase genes contain DR8, and the
oxytocin and laminin B1 genes contain DR14
(11-14).
It is interesting to note that constructs containing mutations in
either Eb or Ec, but not both, still responded to tRA stimulation, but
with reduced responsiveness. This is consistent with our EMSA results
showing that probes containing one intact TGACCT motif were still
capable of forming two complexes that had the same mobility as those
with probe Ec + Eb. Imperfect motifs for RAR/RXR exist in many genes,
such as apolipoprotein A1, oxytocin, F-crystallin, medium chain acyl-coenzyme A dehydrogenase, phosphoenolpyruvate carboxykinase, and
-crystallin (11, 13-17). Each
can still bind heterodimers of RAR/RXR and mediate retinoic
acid-induced gene transcription. This may be an important consideration
because the human renin enhancer (hE) has one perfect TGACCT
motif (Ec) and one variant motif (TGGCCT, Eb). Baseline
transcriptional activity of hE was considerably lower than that of mE
(4), but nevertheless retained modest retinoic acid-induced
transcription (data not shown).
When a heterodimer of RAR/RXR binds to a TGACCT direct repeat, RAR selectively binds to the upstream TGACCT while RXR selectively binds to the downstream TGACCT (18). Forcing RAR/RXR to bind RARE in the opposite direction abolished its transactivation activity. In our study, mutation of Ec always had a stronger effect than mutation of Eb, suggesting the possibility for asymmetric binding of RAR/RXR to the RARE. In a model described in Westin et al. (19), RAR plays a pivotal role to initiate the hierarchical assembly of transcriptional proteins on RARE. If this is true for RAR/RXR binding to Ec + Eb in mE, RAR will bind to Ec and RXR will bind to Eb. Since RAR/RXR-mediated transactivation depends on coactivators, and the interaction between RAR/RXR and coactivators initiates from RAR, disturbance of RAR binding to Ec should cause a marked loss of the functional RAR/RXR-ligand-coactivator complex.
Despite the retinoid-mediated induction of the renin promoter (and endogenous renin gene) mediated by RAR, it is likely that other transcription factors bind to the Ec + Eb sequence. EMSA revealed two major protein-DNA complexes, and supershift does not cause a reduction in either the L or S complex. Indeed, Eb and Ec are both required for the induction by retinoids and for baseline activity of the enhancer in the absence of ligand. These data suggest that other transcription factors, perhaps other members of the hormone receptor superfamily may also play a role in regulating renin gene expression. It is possible that the requirement for Ec + Eb in mediating baseline transcriptional activity of mE may occur independently of RAR/RXR, while the retinoic acid induced activity requires RAR/RXR. We are currently attempting to identify other Ec + Eb binding factors using yeast one-hybrid analysis and have preliminary data implicating an orphan nuclear receptor. Some orphan receptors have been reported to bind the TGACCT as a monomer to regulate RARE function (20).
In addition to Eb and Ec, Gross and colleagues3 have identified a fourth transcription factor-binding site located 16 bp upstream of Ec termed Ed. This sequence is similar to the consensus binding site for members of the CREB/ATF-1 family of transcription factors. Interestingly, the transactivation function of both RAR/RXR and CREB/ATF-1 requires an interaction with coactivators, and both are able to interact with p300/CBP (21, 22). Therefore, it is possible that transcription factors binding to Ed and Ec may interact. Recall that the human renin enhancer retains an intact Ed and Ec, but lacks Eb. Mechanistically, the situation may be analogous to the pit-1 gene where RAR binds to a single core recognition motif to activate retinoic acid-dependent transcription (23). In the pit-1 gene, the RARE is immediately adjacent to a Pit-1-binding site. Retinoic acid-dependent transcription of pit-1 requires Pit-1, which like RAR and CREB/ATF-1 requires CBP as a coactivator (24).
In closing, our results pose the obvious question as to the relevance
of renin gene regulation by retinoids in vivo. As
this is the first study to implicate this pathway, its importance in adults remains unclear. However, it is now clearly recognized that
retinoids are critical signaling molecules during development. Vitamin
A deficiency during development leads to fetal vitamin A deficiency
syndrome, which includes abnormalities in the urogenital tract
including the kidney (25). Moreover, retinoic acid is thought to be the
active metabolite of vitamin A during development. Knockout mice
deficient in specific subtypes of both RAR and RAR
or RAR
and
RAR
develop severe renal malformations generally characterized by
renal agenesis and aplasia (26). Histological analysis revealed the
defect to be a failure of the Wolffian or mesonephric duct to contact
the metanephric blastema. This interaction is critical for the
differentiation of the tubular and eventual development of the vascular
system in the kidney. Interestingly, mice lacking genes in the
renin-angiotensin system also develop severe renal abnormalities
although at a much later stage in development (27). Renin expression is
first visible in the metanephric kidney at 15.5 days postcoitum
and is localized in the developing arterial tree (28, 29). Expression
of renin coincides with the growing branches of the arterial
tree suggesting it may play an important developmental role. Therefore,
it is possible that as retinoids are critical in very early events in
renal development, so to are they needed to induce renin
expression developmentally. The expression of RAR and RXR in As4.1
cells which are believed to be derived from juxtaglomerular cells is
consistent with this notion. Clearly, additional studies examining the
coexpression of RAR with renin during renal development
would seem necessary.
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ACKNOWLEDGEMENTS |
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We acknowledge the outstanding technical assistance of Deborah Davis and Xiaoji Zhang.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL48058, HL61446, and HL55006 (to C. D. S.) and HL48459 (to K. W. G.). DNA sequencing was performed at the University of Iowa DNA Core Facility.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.
¶ To whom correspondence should be addressed: Molecular Biology Interdisciplinary Program, Director, Transgenic and Gene Targeting Facility, Dept. of Internal Medicine and Physiology & Biophysics, 2191 Medical Laboratory, The University of Iowa College of Medicine, Iowa City, IA 52242. Tel.: 319-335-7604; Fax: 319-353-5350; E-mail: curt-sigmund@uiowa.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008361200
2 Q. Shi and C. D. Sigmund, submitted for publication.
3 T. A. Black and K. W. Gross, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: kb, kilobase(s); bp, base pair(s); Ea, element a; Eb, element b; LUC, luciferase; RAR, retinoic acid receptor; RXR, retinoic X receptor; FBS, fetal bovine serum; tRA, all-trans-retinoic acid; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcriptase-polymerase chain reaction; RARE, retinoic acid receptor element.
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