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J. Biol. Chem., Vol. 276, Issue 49, 45530-45538, December 7, 2001
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From the
Received for publication, April 5, 2001, and in revised form, September 13, 2001
Mouse As4.1 cells, obtained after
transgene-targeted oncogenesis to induce neoplasia in renal renin
expressing cells, express high levels of renin mRNA from their
endogenous Ren-1c gene. We have previously
identified a 242-base pair enhancer (coordinates The renin-angiotensen system has long been recognized to play a
major physiological role in regulating systemic blood pressure and
fluid/electrolyte homeostasis. Renin, an aspartyl protease produced and
secreted by the juxtaglomerular cells of the kidney, initiates an
enzymatic cascade which results in the production of the vasoactive
peptide angiotensin II, the major effector molecule of the
renin-angiotensen system. Juxtaglomerular cells are modified smooth
muscle cells located at the distal end of the afferent arteriole and in
the normal mature kidney represent <.1% of the total kidney cell
population (1). Under normal physiological conditions, juxtaglomerular
cells are the principal source of active renin found in the circulation
(2, 3). In addition to kidney, there are several extra-renal sites of
renin mRNA expression (4).
Some mouse strains have only a single renin gene
(Ren-1c) whereas other strains have two copies
(Ren-1d and Ren-2) (5, 6). All these
mouse renin genes are approximately equivalently expressed in
juxtaglomerular cells, however, their expression is differentially
regulated in some extra-renal tissues (7).
A number of transgenic studies have presented compelling evidence
suggesting that sequences regulating the tissue, cell, and developmental expression of mouse renin are located between 2.5 and 4.6 kb1 upstream of the
transcription start site of the Ren-2 gene (8, 9). More
recently, studies employing a
Ren-1c-GFP transgenic reporter have
indicated that the major sequences required to correctly specify
Ren-1c expression spatially and temporally
within embryonic, extra-embryonic and adult tissues, and in response to
physiological perturbation of angiotensin II signaling, are resident
within 4.1 kb of the immediate 5'-flanking sequence (45).
A kidney-derived renin expressing cell line (As4.1), developed from a
mouse strain containing only a single renin gene by transgene-targeted
oncogenesis (11), facilitates the identification of important
cis-acting regulatory sequences controlling renin gene
expression. Consistent with results from transgenic studies, transient
transfection analysis of renin-chloramphenicol acetyltransferase reporter gene constructs have demonstrated that sequences located >2.6
kb upstream of the transcription start site are necessary for high
level reporter gene expression (10). In these studies, renin promoter
( Despite the high degree of sequence similarity, the mouse and human
enhancers differ in their ability to activate transcription of a renin
promoter. With respect to the transcription start site, the distal 202 bp of the mouse and human sequences demonstrate a higher degree of
homology (80%) than the more proximal 40 bp (45%) (12, 13). Recent
studies with chimeric enhancers have demonstrated that the functional
difference between the mouse and human enhancers is due to sequence
differences in the proximal 40 bp (13). Electrophoretic mobility shift
assays (EMSAs) detected sequence-specific binding to two overlapping
elements, Ea and Eb, within the proximal 40 bp of the mouse enhancer.
Mutagenesis and transfection analysis revealed that the higher
transcriptional activity observed with the mouse enhancer is due to the
stimulating effects of element Eb, whereas element Ea antagonizes the
positive influence of element Eb. In addition, the 40-bp element does
not stimulate basal promoter activity on its own, suggesting that additional upstream sequences within the 202-bp distal sequence are
also necessary for full transcriptional activity. A direct repeat of
Eb, named Ec, has been localized upstream of the 40-bp element
and 10 bp from Eb (14). Both Eb and Ec are important for the basal
expression of the mouse renin gene and bind the retinoic acid receptor
and retinoic X receptor to confer retinoic acid activation.
Here we report the further delimitation and characterization of
cis-acting elements in the distal 202 bp of the mouse
Ren-1c enhancer. Our results indicate that a
CRE-like element and an E-box are required for the enhancer activity.
Moreover, CREB/CREM and USF1/USF2 are identified as transcription
factors in As4.1 cells binding to these two sequences, respectively.
Mutation of either the CRE or E-box nearly abolishes
Ren-1c expression, supporting the critical roles
these elements play in regulating renin gene transcription. Also, our
results suggest that the high basal level of renin gene expression in
As4.1 cells is probably the result of constitutive activation of PKA
and that the Ren-1c CRE is a target sequence for
the cAMP/PKA pathway.
Plasmid Construction--
Plasmid 117R1 was
constructed by inserting a 123-bp fragment containing
Ren-1c peomoter sequence from Cell Culture and Transfection--
The generation and
characterization of the renin expressing As4.1 cell line (ATCC CRL2193)
was previously described (11). Both As4.1 and JEG-3 cells were
propagated in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and transfected using FuGENE 6 (Roche Molecular
Biochemicals). For each transfection in a 35-mm culture dish, 2.2 µg
of DNA including 0.5 µg of reporter plasmid, 0.5 µg of expression
plasmid if needed, nonspecific plasmid, and 0.2 µg of plasmid
containing Rous sarcoma virus promoter driving EMSA--
The EMSAs were performed as previously described (10).
For each reaction (10 µl), labeled DNA probe (20,000 cpm) was mixed with about 6 µg of nuclear extracts and 1 µg of poly(dI-dC) in 10 mM Hepes, pH 7.9, 10 mM KCl, 50 mM
NaCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 10% glycerol. When oligonucleotide containing E-box was used
as probe, 1 µg of nonspecific competitor polydG·polydC was chosen
instead of poly(dI-dC). Reaction mixture was incubated on ice for 15 min and then run on 5% polyacrylamide gel in 0.5 × Tris
borate-EDTA buffer. After electrophoresis at room temperature, the gels
were dried for autoradiography. In a competition or supershift assay, an excess amount of unlabeled DNA or 1 µl of antibody was added to
reaction mixture 15 min or 1 h, respectively, prior to the addition of labeled DNA probe. Antibodies against CREB-1, CREB-2, ATF-1, ATF-2, ATF-3, CREM, c-JUN, USF1, USF2, and c-MYC were purchased from Santa Cruz Biotechnology, Inc.
In Vitro Transcription and Translation--
Transcription factor
USF1 was in vitro transcribed/translated by TNT-coupled
wheat germ system (Promega). Two microliters of reaction were used in
each EMSA.
Northern Blot Analysis--
As4.1 cells were either
untreated or treated with 10 µM H-89 for 24 h. Total
RNA was isolated by TRIZOL reagent (Life Technologies, Inc.), separated
on 1.5% agarose formaldehyde gels, transferred to nitrocellulose
membrane, and hybridized as described (49). Radioactive signals from
blots were quantified by phosphorimagery.
Delimiting Enhancer Regions Necessary for High Level Reporter Gene
Expression in As4.1 Cells--
Using a renin expressing kidney-derived
tumoral cell line (As4.1) we have previously identified a 242-bp renin
enhancer (coordinates
To further localize sequences 3' to Sequence from
Search of the transcription factor data base identified an 8-bp
sequence within this motif which showed significant similarity (7/8 bp
match) to a consensus CRE. While the consensus CRE is defined by the
palindromic sequence TGACGTCA (15-18), the majority of CREs identified
thus far deviate from the consensus sequence by one or more base pairs
(19). The sequence identified in the renin enhancer is
TGACATCA. To further investigate the potential role of this
CRE-like element in the DNA-protein interaction(s) observed during EMSA
with As4.1 nuclear extracts, we performed competition assays using
double stranded oligonucleotides containing consensus CRE and mutated
CRE sequences (Fig. 4A). When
the 21-bp renin oligonucleotide was used as a probe, a single complex
was formed (Fig. 4B, lane 1) and was efficiently
competed by increasing molar excess (×25, ×50, ×100) of both
unlabeled renin (lanes 2, 3, and 4) and consensus
CRE (lanes 5, 6, and 7) oligonucleotides. The
mutated CRE oligonucleotide (Fig. 4B, lanes 8,
9, and 10), however, was unable to effectively
compete complex formation. In a reciprocal experiment, the consensus
CRE oligonucleotide was labeled and used in EMSA with As4.1 nuclear
extracts. This experiment also revealed a single complex (Fig.
4C, lane 1) which was efficiently competed by
increasing molar excess (×25, ×50, ×100) of both unlabeled CRE
(lanes 2, 3, and 4) and renin (lanes 5, 6, and 7) oligonucleotides. The mutated
CRE oligonucleotide (Fig. 4C, lanes 8,
9, and 10) was again incapable of competing the
DNA-protein interaction. These experimental data suggest that complex
formation is mediated by a CRE-like element.
CREs are known to form DNA·protein complexes with the ATF/CREB family
of transcription factors. Although each of these proteins can bind to
CRE as homodimers, some of these proteins can bind to CRE as
heterodimers, both within the family and with members of the AP-1
transcription family (20). To identify the specific nuclear factors
that contribute to the DNA·protein complex observed with the renin
CRE-like element and As4.1 nuclear extracts, we performed EMSAs using
antibody against CREB-1 (Fig.
5A). The results showed that
addition of this antibody almost completely abolished the formation of
As4.1 nuclear proteins·1c complex. The CREB-1 antibody
used can also react to ATF-1 and CREM. However, antibodies against
ATF-1 and other transcription factors including CREB-2, ATF-2, ATF-3,
and c-JUN did not attenuate or supershift the complex (Fig.
5B). Addition of anti-CREM-1 antibody resulted in a
partially supershifted complex (Fig. 5B, lane 6), indicating that the CREM transcription factor(s) can also bind to this
site. These results demonstrate that the CRE-like sequence located
between
To determine whether CREB/CREM can activate gene expression by binding
to this CRE-like element, a construct containing four copies of this
element fused to 117R1 was transfected into As4.1 cells. Addition of
Ren-1c CREs resulted in almost 50-fold increase
in transcription (Fig. 6). Moreover, the
induction was reduced 4-fold by co-transfecting cells with a dominant
negative CREB (A-CREB) (21), further suggesting involvement of CREB/ATF
transcription factors in renin gene regulation.
Sequence from
To examine whether the bHLH-leucine zipper transcription factors
USF1/USF2 bind to the Ren-1c enhancer E-box,
antibodies against USF1 and USF2 were used in supershift EMSA (Fig.
7D). To separate the USF1/USF2 homodimers and heterodimer in
EMSA, the specific RE/As4.1 nuclear extract complex shown in Fig. 7,
B and C, was run considerately further into the
gel. At least two complexes (L and S) were observed (Fig. 7D, lane 1). Addition of an increasing amount of
antibody against USF1 gradually attenuated both the L and S bands (Fig.
7D, lanes 2-6) and the formation of both L and S
complexes was completely abolished with more than 0.5 µl of USF1
antiserum (Fig. 7D, lanes 4-6). Addition of 1 µl of USF2
antiserum almost completely disrupted the formation of the S complex
(Fig. 7D, lane 7), whereas antibody against
c-MYC, which recognizes a similar E-box sequence, did not disrupt the
formation of either the L or S complex (Fig. 7D, lane
8). These results suggest that the S band most probably contains the USF1/USF2 heterodimer whereas the L band contains the USF1 homodimer. Considering that USF1 has lower molecular weight than USF2,
the USF1 homodimer would move faster than the USF1/USF2 heterodimer in
the gel. Thus, the USF1 homodimer in the L band is probably associated
with other unidentified proteins. In addition, given the decrease in
intensity of the L band in response to antibody against USF2 (Fig.
7D, lane 7), the L band may also contain
USF1/USF2 heterodimer associated with other proteins. In
vitro synthesized USF1 protein was able to bind to the RE probe
(Fig. 7E, lane 3), and the binding was completely
abolished by the addition of USF1 antibody (Fig. 7E,
lane 4). These results suggest a potential role for the USF
transcription factors in regulation of renin gene expression.
Synergistic Activation of Transcription by CRE and
E-box--
Since the progressive deletion experiments indicated that
CRE, E-box, and Ec in the Ren-1c enhancer are
all required for the enhancer activity, we wanted to test whether these
sites cooperatively activate gene expression. A construct containing
enhancer sequence from CRE and E-box Elements Are Crucial for Enhancer Activity and Gene
Expression of the Mouse Ren-1c Gene--
To test how
important the CRE, E-box, and other sites are in regulating
Ren-1c enhancer activity, a series of constructs
were made based on 117R1enh (enhancer sequence from
We then placed wild-type and site-specifically mutated
Ren-1c enhancer ( cAMP Responsiveness of the Ren-1c CRE in As4.1 and
JEG-3 Cells--
To test whether the CRE within the
Ren-1c enhancer is the target of cAMP/PKA signal
transduction pathway, As4.1 cells were transfected with constructs
2625enh and 2625enh/mcre. Treatment of transfected cells with
forskolin, an activator of adenylyl cyclase, did not cause any
significant increase in luciferase activity for either plasmid (Fig.
11A). Similar results were
obtained with plasmids Enh-TA and Enh/mcre-TA containing the
Ren-1c enhancer and the CRE-mutated enhancer,
respectively, inserted immediately upstream of an E1b TATA box. These
results suggest that either the Ren-1c CRE is
not a functional CRE or As4.1 cells are insensitive to cAMP treatment.
To test these possibilities, SSCRE, a construct containing a consensus
CRE, was transfected into As4.1 cells and treated with forskolin. No
induction by forskolin was observed for SSCRE, suggesting that the cAMP
responsiveness is lost in As4.1 cells.
The same plasmids were also transfected into JEG-3 cells, which do not
express renin but are highly responsive to cAMP. The basal
transcriptional activity for 2625enh was very low in JEG-3 cells (Fig.
11B). Moreover, mutation of the CRE within the enhancer (2625enh/mcre) did not cause any decrease in transcriptional activity, suggesting that the Ren-1c enhancer is not
functional when placed 2.6 kb upstream of the transcription start site.
Thus, JEG-3 cells appear to lack components needed for high level
expression of the Ren-1c gene. Both plasmids
2625enh and 2625enh/mcre responded to forskolin treatment with about a
3-fold increase in transcriptional activity. However, this increase in
activity is not mediated by the CRE. When JEG-3 cells were transfected
with Enh-TA, a 7-fold induction by forskolin was observed. Mutation of
the CRE in Enh-TA (Enh/mcre-TA) reduced the basal transcription by
17-fold. Forskolin only caused a 2-fold increase in luciferase
activity, which is similar to forskolins effect on the E1b TATA box
alone (data not shown). As a positive control, plasmid SSCRE responded
to forskolin with a 20-fold increase in transcriptional activity. These
results demonstrate that the Ren-1c CRE is a
functional target for the cAMP/PKA pathway when present in JEG-3 cells.
The PKA Inhibitor H-89 Reduces the Ren-1c mRNA
Level and this Inhibitory Effect Is Specifically Mediated by the
CRE--
It is possible that the lack of an overtly inducible cAMP
response for renin expression in As4.1 cells reflects constitutive and
maximal stimulation of the cAMP pathway under basal conditions. To test
whether PKA might be constitutively active, we treated As4.1 cells with
a PKA-specific inhibitor, H-89, and performed Northern blots to analyze
the effect of H-89 on renin mRNA level. The results show that the
H-89 treatment caused a more than 3-fold decrease in renin mRNA
level (Fig. 12A). These
results were confirmed by transfection assays. H-89 treatment of As4.1
cells transfected with 2625enh resulted in 3-fold reduction in
luciferase activity (Fig. 12B). However, H-89 had no
significant inhibitory effect on the transcriptional activity of
2625enh/mcre. To further demonstrate that the inhibitory effect of H-89
is mediated by the CRE, the construct 117R1-CRE4 containing four copies
of the Ren-1c CRE inserted upstream of the
Ren-1c promoter was tested for the H-89
response. The H-89 treatment caused a 6-fold decrease in the promoter
activity of 117R1-CRE4, whereas H-89 had no effect on 117R1. These
results demonstrate that the PKA-specific inhibitor H-89 is able to
decrease renin gene expression in As4.1 cells and this reduction is
mediated by the CRE. These results further suggest that the CRE within the Ren-1c enhancer is a target for the cAMP/PKA
pathway and that PKA is constitutively active in As4.1 cells.
In this report, we have identified several sets of protein-DNA
interaction within the mouse Ren-1c enhancer
that are required for its full activity. Sequences from Mouse renin gene expression is subject to cAMP stimulation in kidney
(24). Two regions located at Horiuchi et al. (27, 28) have identified a poor consensus
CRE at Lastly as reported here, the finding of a CREB/CREM transcription
factor-binding site in the enhancer region of mouse
Ren-1c provides another potential site for cAMP
regulation of renin genes. The effect of cAMP on basal renin gene
expression in As4.1 cells is not readily discernible since these cells
do not respond to cAMP or forskolin by increasing gene expression (Fig.
11A) (29, 50). However, the Ren-1c
CRE was responsive to forskolin stimulation in JEG-3 cells, indicating that the CRE is functional in cAMP-responsive cells. As4.1 cells were
selected by transgene-targeted oncogenesis with a Ren
promoter driving SV40 T antigen, and tumor outgrowth depended on one or more additional stochastic events. It would not be surprising if a
second cellular mutation were to have occurred in a signal transduction pathway (i.e. such as cAMP pathway) that
leads to maximal stimulation of the enhancer, driving oncogene
expression. We therefore investigated whether PKA activity is
constitutive and demonstrated that, in As4.1 cells, the PKA-specific
inhibitor, H-89, was able to decrease both renin mRNA level and
expression of 2625enh, a construct containing the
Ren-1c enhancer. Moreover, this inhibitory
effect was mediated by the CRE, suggesting that a component in cAMP
pathway may be mutated in As4.1 cells to result in the constitutive
activation of PKA. Constitutive activation of the cAMP pathway in tumor
cells has been reported previously. These studies have shown, for
example, that, in about 30-40% growth hormone-secreting pituitary
adenomas, a mutation in the Gsa gene results in
constitutively active adenylyl cyclase and elevated cAMP level (see
Refs. 51 and 52, for reviews).
The bHLH-leucine zipper transcription factors USF1 and USF2 were first
identified as activators of the adenovirus major late promoter by
binding to a USF consensus sequence containing the CACGTG E-box motif
(30). However, in addition to its consensus sequence, USF proteins have
also been reported to bind other sequences, including CGCGTG (31, 32),
CCCGTG (33), CAGCTG (22, 34), CACCTG (35), and CACATG (36, 37, 44). We
have demonstrated that USF1/USF2 binds to a CAGATG motif within the
mouse Ren-1c enhancer. Thus, USF is capable of
binding to various E-box motifs to regulate gene expression.
From mutational analyses of mouse Ren-1c
enhancer, the binding sites for CREB/CREM and USF1 appear to be crucial
elements. Deletion of either site results in almost complete loss of
the enhancer activity and renin expression in As4.1 cells. Thus
contribution of these two sites seems to lie at the foundation of the
renal enhancer. To determine whether CREB and USF1 can bind
cooperatively to an oligonucleotide spanning the
Ren-1c enhancer region from By contrast with the CRE and E-box, either element Ec or Eb, which
binds RAR and RXR as well as other unidentified orphan nuclear
receptors (14), contributes to enhancement to a lesser extent than
either the CRE or E-box. In addition to the above elements, the renin
enhancer also contains other important and as yet unidentified elements
residing 5' to the CRE site, including regions from A major contribution to the tissue specificity of renin
expression is very likely conferred by the HOX·PBX-binding site that we have recently identified in the proximal promoter region, at We thank Dr. Heinz Baumann for helpful advice.
*
This work was supported in part by National Institute of
Health Grants HL48459 and CA16056 (to K. W. G.) and HL48058,
HL61446, and HL55006 (to C. D. S.).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.
§
Supported by a Postdoctoral Fellowship from the National Institutes
of Health.
¶
Present address: School of Medicine and Biomedical Sciences,
40 CFS Bldg., 3435, Main St., Buffalo, NY 14214.
**
Present address: University Department of Medicine, Queen Elizabeth
II Medical Center, Nedlands, Western Australia.
§§
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton
Sts., Buffalo, NY 14263-0001. Tel.: 716-845-4572; Fax: 716-845-8169;
E-mail: gross@acsu.buffalo.edu.
Published, JBC Papers in Press, September 19, 2001, DOI 10.1074/jbc.M103010200
2
C. D. Sigmund and K. W. Gross,
unpublished data.
3
L. Pan and K. W. Gross, unpublished data.
The abbreviations used are:
kb, kilobase pair(s);
EMSA, electrophoretic mobility shift assay;
Luc, luciferase;
bp, base pair(s);
cAMP, cyclic AMP;
CRE, cAMP responsive element;
USF, upstream stimulatory factor;
CREB, cyclic AMP responsive
element-binding protein;
CREM, cyclic AMP responsive element modulator;
PKA, protein kinase A;
NRE, negative responsive element.
Critical Roles of a Cyclic AMP Responsive Element and an E-box in
Regulation of Mouse Renin Gene Expression*
§,¶,
,,**,
,,, and§§
Department of Molecular and Cellular
Biology, Roswell Park Cancer Institute, Buffalo, New York 14263 and the
Departments of Internal Medicine and Physiology & Biophysics,
University of Iowa College of Medicine, Iowa City, Iowa 52242
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2866 to 2625
relative to the CAP site) upstream of the mouse
Ren-1c gene. This enhancer, in combination with
the proximal promoter (117 to +6), activates transcription nearly 2 orders of magnitude in an orientation independent fashion. To further
delimit sequences necessary for transcriptional activation, renin
promoter-luciferase reporter gene constructs containing selected
regions of the Ren-1c enhancer were analyzed
after transfection into As4.1 cells. These results demonstrate that
several regions are required for full enhancer activity. Sequences from
2699 to 2672, which are critical for the enhancer activity, contain
a cyclic AMP responsive element (CRE) and an E-box. Electrophoretic
mobility shift assays demonstrated that transcription factors
CREB/CREM and USF1/USF2 in As4.1 cell nuclear extracts bind to
oligonucleotides containing the Ren-1c CRE and
E-box, respectively. These two elements are capable of synergistically
activating transcription from the Ren-1c
promoter. Moreover, mutation of either the CRE or E-box results in
almost complete loss of enhancer activity, suggesting the critical roles these two elements play in regulating mouse
Ren-1c gene expression. Although the
Ren-1c gene contains a CRE, its
expression is not induced by cAMP in As4.1 cells. This appears to
reflect constitutive activation of protein kinase A in As4.1 cells
since treatment with the protein kinase A inhibitor, H-89, caused a
significant reduction in Ren-1c gene expression
and this reduction is mediated through the CRE at 2699 to
2688.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
117 to +6) CAT constructs containing 5'-flanking sequence extending
to 2.6 kb did not increase basal transcriptional activity. However,
promoter constructs containing 4.0 kb of upstream sequence (4,100 to
117) increased transcriptional activity ~2 orders of magnitude.
Progressive deletion analysis localized equivalent activity to a 242-bp
fragment (2866 to 2625). This fragment acts as a classical enhancer
of transcription, stimulating promoter activity >50-fold in an
orientation and position independent fashion. Sequences showing ~98%
sequence identity to the Ren-1c enhancer have
been identified in the 5'-flanking regions of Ren-2 and
Ren-1d.2
Significantly, recent studies have identified a sequence homologous to
the mouse enhancer ~12 kb upstream of the human renin gene (12, 13).
The high degree of enhancer homology in the mouse renin genes and the
cross-species conservation suggests that this region plays an important
role in regulating renin gene transcription.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
117 to +6 into
BglII/HindIII sites in pGL2-basic (Promega).
Plasmid 2866/2625 containing intact Ren-1c
enhancer fused to the promoter region was prepared by inserting the
BamHI-digested enhancer fragment from plasmid
117CAT(2866 to 2625) (10) into the BglII site in 117R1
in correct orientation. All 5' deletion mutants of
Ren-1c enhancer in Fig. 1A were
constructed by inserting the corresponding polymerase chain reaction
synthesized fragments containing truncated enhancer plus the promoter
region into SacI/HindIII sites in pGL2-basic. Those 3' enhancer deletion mutants in Fig. 1B contain
polymerase chain reaction-synthesized truncated enhancer fragments
inserted into SacI/BglII sites in 117R1. Plasmids
117R1CEC, 117R1mCEC, 117R1CmEC, and 117R1CEmC were constructed by
inserting oligonucleotides containing wild-type
Ren-1c enhancer sequence from 2702 to 2663
(CEC), CRE-mutated CEC, E-box-mutated CEC, and Ec-mutated CEC into
SacI/BglII sites in 117R1. Plasmid 2625 was
constructed by inserting an
XhoI-BglII-SphI linker into
XhoI/SphI-digested pGL2-basic to create pGL-XBS,
and then by inserting a SphI fragment containing
Ren-1c sequence from 2625 to +6 from plasmid
4.1R1 (48) into SphI-digested pGL-XBS. The
XhoI/BglII-digested polymerase chain reaction
fragments containing Ren-1c enhancer and site
specifically mutated enhancers including Ea, Eb, Ec, Ea + Eb, E-box,
CRE, and E-box + CRE mutations were inserted into
XhoI/BglII sites in 2625 to create 2625enh,
2625enh/ma, 2625enh/mb, 2625enh/mc, 2625enh/mbc, 2625enh/me,
2625enh/mcre, and 2625enh/mce, respectively. All these enhancers
containing mutated sites were also inserted into
SacI/BglII sites in 117R1 to create 117enh, 117enh/ma, 117enh/mb, 117enh/mc, 117enh/mbc, 117enh/me, 117enh/mcre, and 117enh/mce, respectively. Plasmid 117R1-CRE4 was constructed by
inserting an oligonucleotide containing four copies of the Ren-1c CRE into SacI/BglII
sites of 117R1. Enh-TA and Enh/mcre-TA were constructed by inserting
the Ren-1c enhancer and CRE-mutated enhancer,
respectively, into SacI/BgII sites in a pGL2-basic-derived
plasmid containing the adenovirus E1b TATA box (46). Plasmid SSCRE
contains the somatostatin promoter and consensus CRE in pGL2-basic
(47). Full-length cDNA for USF1 was isolated by reverse
transcriptase-polymerase chain reaction from As4.1 cells and cloned
into BamHI/EcoRI sites in
pcDNA3.1/myc-His(+)A vector (Invitrogen) without incorporating any
epitope Tag. Sequences of all plasmid DNAs were verified by automated
sequencing at the Roswell Park biopolymer facility.
-galactosidase
(RSV-gal) were mixed with 4.4 µl of FuGENE reagent. Twenty-four h
after transfection cells were harvested and measured for luciferase
(Luc) and -galactosidase activities using the Luciferase Assay
System (Promega) and Galacto-Light PlusTM chemiluminescent
reporter assay (Tropix), respectively. In Figs. 11 and 12, 2 µg of
reporter plasmid were used in each transfection assay and cells were
harvested 48 h after transfection. Forskolin (10 µM)
and H-89 (10 µM) were added 24 h prior to
harvesting. The Luc activity is normalized with -galactosidase
activity to correct differences in transfection efficiency between
experiments. All transfection results represent the average + S.E. of
at least three separate experiments.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2866 to 2625 relative to the transcription
start site) which activates transcription >50-fold in combination with
the renin proximal promoter (117 to +6) (10). In an effort to more
precisely define sequence motifs necessary for high level gene
expression, a series of 5'-deleted fragments of the
Ren-1c enhancer sequence were fused to a renin
proximal promoter-Luc reporter gene construct (117R1) (Fig.
1A) and assayed for activities after transient transfection into As4.1 cells (Fig. 1B).
Deletions of sequences from 2866 to 2699 result in about a 10-fold
decrease in enhancer activity. Regions from 2829 to 2803, 2777 to
2751, 2738 to 2712, and 2712 to 2699 contribute to the
enhancer activity by about 1.6-, 1.4-, 1.6-, and 3.0-fold,
respectively. However, deletion of sequence from 2699 to 2684
results in a dramatic drop (10-fold) in the enhancer activity,
suggesting that the sequence within the region from 2699 to 2684 is
critical for maximal enhancer activity.
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Fig. 1.
Activation of the renin promoter-Luc reporter
construct by 5' truncated Ren-1c distal
enhancer sequences after transfection into As4.1 cells.
A, shown are constructs used in transfections, which contain
wild-type and 5' truncated Ren-1c enhancer
sequences fused to a 123-bp Ren-1c promoter.
B, As4.1 cells were transfected with the indicated plasmids.
The Luc activity is expressed relative to that of plasmid 2829/2625
(arbitrarily set to 1000). * and **, statistically significant
differences relative to 2777/2625 (p < 0.05) and
2738/2625 (p < 0.005), respectively, measured by
Student's t tests.
2684 which are important for the
enhancer activity, a series of 3' deletion mutants of enhancer were
fused to 117R1 (Fig. 2A).
Deletion of sequence from 2625 to 2663, which contains Ea and Eb
sites (13), does not significantly affect the enhancer activity (Fig.
2B). However, removal of Ec site (14) causes 6-fold
reduction in activity. Further deletion from 2672 to 2684 results
in another 6-fold decrease in activity, suggesting that this region may
contain a protein-DNA interaction site. Consistent with results
from 5' deletion mutants, deletion of sequence from 2684 to 2699
results in almost complete loss of enhancer activity.
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Fig. 2.
Activation of the renin promoter-Luc reporter
construct by 3' truncated Ren-1c distal
enhancer sequences after transfection into As4.1 cells.
A, shown are constructs used in transfections, which contain
3' truncated Ren-1c enhancer sequences fused to
a 123-bp Ren-1c promoter. B, As4.1
cells were transfected with the indicated plasmids. The Luc activity is
expressed relative to that of plasmid 2829/2625 (arbitrarily set to
1000).
2699 to 2684 Contains a CRE-like Element, Which
Binds CREB/CREM--
To identify the DNA-protein interaction site
between 2699 to 2684, we employed EMSA using double stranded
oligonucleotides containing 21 bp of renin enhancer sequence, 2702 to
2682 (oligonucleotide 1c in Fig.
3A), and nuclear extracts
prepared from As4.1 cells (Fig. 3B). These studies
demonstrated the formation of a single complex (Fig. 3B,
lane 1) which was efficiently competed with 50-fold molar
excess of unlabeled oligonucleotide (lane 2). To more
precisely define the specific base pairs critical for complex
formation, competition was performed using six double stranded mutant
oligonucleotides each containing sequential 3-bp mutations. Mutants
CM1, CM2, CM3, and CM4 (Fig. 3B, lanes 3-6) were
unable to effectively compete the DNA-protein interaction. However,
mutants CM5 and CM6 (Fig. 3B, lanes 7-8)
effectively competed complex formation. These results suggest that the
DNA-protein interaction is confined to the sequence motif
AATGACATCACT.
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Fig. 3.
EMSA with an oligonucleotide derived from the
Ren-1c enhancer and competition for complex
formation with mutant Ren-1c oligonucleotides
using nuclear proteins from As4.1 cells. A, shown are
the sequences of the double stranded oligonucleotides that were used in
EMSA and competition assays. 1c represents wild type mouse
Ren-1c sequence (coordinates 2702 to 2682).
CM1-CM6 contain selectively altered bases (indicated by
underlined lowercase letters). The sequence motif important
for the DNA-protein interaction in 1c is indicated by a
solid line. B, oligonucleotide 1c was
used as the probe in EMSA without competitor ( lane), or
competed with specific oligonucleotides as indicated. A 50-fold molar
excess of competitor over the probe was used. The specific complex
formed with As4.1 cell nuclear extract and 1c is indicated
by an arrow. Free probe is indicated by FP.
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Fig. 4.
Competition of consensus CRE and mutant CRE
oligonucleotides for complex formation in EMSAs with nuclear proteins
from As4.1 cells. A, sequences of the double stranded
oligonucleotides used in EMSA: 1c is the wild type renin
sequence (coordinates 2702 to 2682, the CRE like site is
underlined), CON CRE is an oligonucleotide
containing a consensus CRE element (indicated by underlined
letters), MUT CRE is an oligonucleotide containing a
mutated CRE element (CRE element underlined, mutated base
pairs indicated by lowercase letters). B,
oligonucleotide 1c was used as probe in EMSA without
competitor ( lane) or competed with 25-, 50-, and 100-fold
molar excess of unlabeled oligonucleotides 1c, CON CRE (CRE)
and MUT CRE (mutCRE). C, EMSA was performed using CON CRE as
probe and competitions were performed with 25-, 50-, and 100-fold molar
excess of unlabeled oligonucleotide CON CRE (CRE), 1c and MUT
CRE (mutCRE). The lane indicates no competitor. The
specific complex formed with As4.1 cell nuclear extract and
1c is indicated by an arrow. Free probe is
indicated by FP.
2699 and 2684 is a CREB/CREM-binding site.
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Fig. 5.
Supershift EMSAs of the
Ren-1c CRE-like element. Shown are the
results from EMSA and supershift analysis using the 1c
oligonucleotide (coordinates 2702 to 2682) as probe and nuclear
proteins from As4.1 cells. The reactions were set up with no
antibody ( lane), with antibodies against CREB-1 in
A and CREB-2, ATF-1, ATF-2, ATF-3, CREM, and c-JUN in
B. The specific complex formed with As4.1 cell nuclear
extract and 1c is indicated by an arrow. The
supershifted complex is indicated by SS. Free probe is
indicated by FP.
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Fig. 6.
Effect of a dominant negative CREB on
Ren-1c promoter activity induced by multiple
copies of the CRE-like element. As4.1 cells were transfected with
117R1 and 117R1-CRE4 containing four copies of the
Ren-1c CRE-like sequence fused 5' to 117R1 in
the absence or presence of a dominant negative CREB (A-CREB). The Luc
activity is expressed relative to that of plasmid 117R1.
2684 to 2672 Contains an E-box, Which Binds
USF1/USF2--
Sequence from 2684 to 2672, which is required for
the full enhancer activity, contains an E-box motif (CAGATG). To
determine whether this E-box binds nuclear proteins in As4.1 cells, an
oligonucleotide RE containing the E-box motif was used in EMSA (Fig.
7A). A complex was formed with
polydG·polydC as nonspecific competitor (Fig. 7B,
lane 1) but not with poly(dI-dC) (data not shown). This
complex is RE-specific since it was competed by 100-fold excess of RE itself but not by an oligonucleotide mRE containing a mutated E-box
(Fig. 7B, lanes 2 and 3). Also, the
E-box complex was effectively competed with 100-fold molar excess of an
oligonucleotide containing a USF1-binding site but not with a similar
oligonucleotide containing a mutated USF1-binding site (Fig.
7C).
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Fig. 7.
Binding of USF1/USF2 to the E-box motif
located within the Ren-1c enhancer.
A, sequences of the double stranded oligonucleotides used in
EMSA: RE is the wild type renin sequence (coordinates 2688 to 2669)
containing an E-box motif (indicated by a solid line), mRE
contains selectively altered bases (indicated by underlined
lowercase letters) within the E-box motif. USF1 is an
oligonucleotide containing a consensus USF1 binding element (indicated
by a solid line), mUSF1 is an oligonucleotide containing a
mutated USF1 element (mutated base pairs indicated by underlined
lowercase letters). B and C, oligonucleotide
RE was used as probe in EMSA without competitor ( lane)
or competed 100-fold molar excess of unlabeled oligonucleotides as
indicated. Nuclear extracts were prepared from As4.1 cells.
D, supershift EMSA was performed using RE as probe, As4.1
cell nuclear proteins and antibodies against USF1 (
USF1), USF2
(USF2), and c-MYC (MYC). The lane
indicates no antibody added. An increasing amount of antibody against
USF1 (0.125, 0.25, 0.5, 1, and 2 µl) was added in lanes
2-6, respectively. Antibodies against USF2 (lane 7)
and c-MYC (lane 8) were added 1 µl each. The nonspecific
complex (NS) and free probe (FP) are not shown.
E, EMSA was performed on the RE probe using in
vitro synthesized USF1. Lanes labeled with Lys contain
the in vitro transcription/translation lysate without any
DNA added. Antibody against USF1 (USF1) was used to supershift or
attenuate the DNA·protein complex. The nonspecific binding by
proteins from lysate is indicated by Lys and an
arrow. The specific complex formed with As4.1 cell nuclear
extract or USF1 protein and RE is indicated by an arrow.
Nonspecific binding with RE and As4.1 nuclear proteins is indicated by
NS. Free probe is indicated by FP.
2702 to 2663 (see Fig.
8 for positions of various regulatory
elements), which includes CRE, E-box, and Ec motifs, fused to 117R1
(Fig. 9A), was transfected into As4.1 cells. This construct activates luciferase expression 29-fold over 117R1 (Fig. 9B). However, similar constructs
containing either mutated CRE or mutated E-box (Fig. 8) was not capable
of activating the Ren-1c promoter whereas a
construct containing mutated Ec was still able to activate the promoter
activity by almost 7-fold. These results suggest that CRE and E-box
activate transcription cooperatively. Although Ec clearly participates
in activity of the enhancer, loss of this binding site still retains
some activity suggesting it may not be obligatorily required.
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Fig. 8.
Sequences of the proximal portion of
Ren-1c enhancer. Sequence from 2702 to
2637 of the Ren-1c enhancer is shown. The
cis-acting elements including CRE, E-box, Ec, Eb, and Ea are
indicated. The site-specific mutations contained in various constructs
in Figs. 9 and 10 are also indicated by lowercase
letters.
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Fig. 9.
Synergistic activation of
Ren-1c promoter activity by CRE and
E-box. A, shown are constructs used in transfections,
which contain wild-type and site-specific mutated
Ren-1c enhancer sequences from 2702 to 2663
fused to a 123-bp Ren-1c promoter. E-box and Ec
are indicated by e and c, respectively.
B, As4.1 cells were transfected with the indicated plasmids.
The Luc activity is expressed relative to that of plasmid
2829/2625 (arbitrarily set to 1000) used in Figs. 1 and 2.
2866 to 2625
fused 5' to 123 bp Ren-1c promoter) and contain
point mutations in CRE, E-box, Ec, Eb, and Ea motifs (Fig. 8). These
constructs were transfected into As4.1 cells and analyzed for their
expression capabilities (Fig. 10A). Mutation of Ea did not
significantly change expression while mutation of Eb resulted in a 40%
decrease in activity. Mutation of either Ec or the E-box reduced
expression by 90%. The CRE element was a crucial regulatory site in
the enhancer since mutation of the CRE site resulted in 97% reduction
in enhancer activity. Mutations of both CRE and E-box caused almost
complete loss of enhancer activity.
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Fig. 10.
Mutational analyses of
cis-acting elements within the
Ren-1c enhancer. A, As4.1
cells were transfected with 117R1 and constructs containing wild-type
and site-specific mutated Ren-1c enhancer
( 2866 to 2625) fused to 117R1 (see Fig. 8 for specific mutations).
The Luc activity is expressed relative to that of 117enh (arbitrarily
set to 100). B, As4.1 cells were transfected with plasmid
2625 containing 2625 bp of Ren-1c 5'-flanking
sequence and constructs containing wild-type and site-specific mutated
Ren-1c enhancer (2866 to 2625) fused to
2625. The Luc activity is expressed relative to that of 2625enh
(arbitrarily set to 100).
2866 to 2625) at 2625 bp.
This permitted testing how mutations of these regulatory sites in their
natural context would affect the Ren-1c gene
expression. A construct containing only 2625 bp of
Ren-1c 5'-flanking sequences fused to Luc had
3-fold more activity than 117R1 (data not shown). Addition of enhancer
to this construct resulted in a 23-fold increase in activity (Fig.
10B). Mutation of Ea did not have significant effect on the
enhancer activity, whereas mutations of Eb or Ec caused about 40 and
60% reduction in activity, respectively. When both Eb and Ec were
mutated, 86% of enhancer activity was lost. Mutations of either CRE or
E-box almost completely abolished the Ren-1c
enhancer function, again, demonstrating that both the CRE motif and
E-box are crucial for renin gene expression.
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Fig. 11.
cAMP responsiveness of the
Ren-1c CRE in As4.1 and JEG-3 cells.
As4.1 cells (A) and JEG-3 cells (B) were
transfected with reporter constructs 2625enh, 2625enh/mcre, Enh-TA,
Enh/mcre-TA, and SSCRE. Filled bars, untreated; open
bars, forskolin-treated. The Luc activity is expressed relative to
that of 2625enh in As4.1 cells (arbitrarily set to 100).
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Fig. 12.
Effect of PKA specific inhibitor H-89 on
Ren-1c expression in As4.1 cells.
A, Northern blot analysis of renin mRNA levels in
As4.1 cells either untreated (Control) or treated with 10 µM H-89 for 24 h. Results are shown in duplicates
as indicated at the top. The hybridization with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe
served as loading control. Radioactive signals from the Northern blots
were quantified by phosphorimagery and the relative renin mRNA
levels were normalized with GAPDH expression. B, As4.1 cells
were transfected with 2625enh, 2625enh/mcre, 117R1, and 117R1-CRE4 and
either untreated or treated with H-89 for 24 h. The H-89 effect is
expressed as normalized Luc activity from H-89-treated cells divided by
normalized Luc activity from untreated cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2699 to
2684 and from 2684 to 2674 were found to have the most effect on
activity. The region from 2699 to 2684 contains a sequence motif
homologous to the consensus CRE. Results from EMSAs using wild-type and
mutated oligonucleotides have demonstrated that this CRE-like sequence
motif binds As4.1 cell nuclear proteins identical with a consensus CRE.
Supershift EMSAs have identified the interacting proteins as CREB and
CREM, members of the CREB/ATF transcription factor family. A dominant
negative CREB mutant (A-CREB) was capable of inhibiting transcription
from a construct containing four copies of a
Ren-1c CRE in As4.1 cells, further suggesting
that the CRE is a binding site for CREB/ATF transcription factors. An
E-box motif is contained in the other critical region from 2684 to
2674. This E-box binds nuclear proteins in As4.1 cells which are
specific for this motif since an excess of wild-type E-box
oligonucleotide competes complex formation while a similar
oligonucleotide containing a mutated E-box cannot. Competition and
supershift EMSAs have demonstrated that bHLH-leucine zipper
transcription factors USF1/USF2 bind to this E-box. This is further
supported by the binding capability of in vitro
transcribed/translated USF1 to the E-box. Interestingly, USF1 has also
recently been reported to bind E-box motifs in actin and osteopontin
gene promoters in vascular smooth muscle (22, 23).
60 and 600 have been reported to
confer the cAMP responsiveness. By transfecting wild-type and mutated
Ren-1c promoter constructs into embryonic
kidney-derived 293 cells, which do not express renin, Tamura et
al. (25) observed that the RP-2 element (75 to 47) was capable
of directing the cAMP response. EMSA demonstrated that nuclear proteins
from 293 cells bound the RP-2 element and the binding activity was
enhanced by pretreatment with cAMP. Our results have shown that RP-2
element corresponds to a HOX·PBX·PREP1-binding site (48), which is
critical for the Ren-1c expression (10, 48). HOX
paralog groups 9 and 10 bind this site with high affinities (48). A
recent report by Saleh et al. (26) suggests that PKA can
activate a HOX·PBX complex to induce transcription through
recruitment of the CREB-binding protein. Thus, the cAMP response of the
RP-2 element is possibly mediated by the HOX·PBX complex.
600 of Ren-1d, which overlaps a
negative responsive element (NRE). A more recent report by Tamura
et al. (29) has demonstrated that the oligonucleotide containing both the CRE and NRE, termed CNRE, binds LXR, a member of
the nuclear receptor superfamily. In studies where the CNRE elements
have been fused to heterologous promoters, but not the native mouse
renin promoter sequence, they have identified LXR as the cAMP
responsive modulator of the renin gene. It remains to be seen whether
this element contributes to basal and cAMP-induced mouse renin
expression in vivo.
2702 to 2679 bp,
which contains both the CREB/CREM and USF-binding sites, we performed
EMSAs using in vitro translated CREB and USF1. No
cooperative binding between CREB and USF1 was observed (data not
shown). The results suggest that coactivators, such as CREB-binding
protein/p300 (38, 39), may be necessary for the indirect interaction
between these two transcription factors. It has been recently reported
that the activation of gene promoters by USF may be mediated by
CREB-binding protein/p300 (40, 41). Interestingly, transcription of
transforming growth factor-2 is also dependent on a CRE which binds
CREB/ATF-1 and an adjacent E-box which binds USF1/USF2 (42, 43).
Mutation of either site results in 60-80% reduction in transforming
growth factor-2 gene expression.
2829 to 2803,
2777 to 2751, and 2738 to 2699. Deletions of these regions
result in a 10-fold decrease in enhancer activity (Fig. 1). Moreover,
EMSAs indicate the As4.1 nuclear protein-DNA interactions in these
regions.3 These regions may
bind tissue or cell-specific transcription factors to contribute to the
highly regulated mouse renin expression. However, these elements are by
themselves not sufficient to enhance basal promoter activity,
suggesting that they may contribute to enhancer activity by interacting
with CRE/E-box core enhancer elements.
60
relative to the transcription start site (48). This finding strongly
implicates renin, which initiates a cascade leading to production of
the growth factor and pressor substance angiotensin II, as an
immediate downstream target of Class I Hox genes.
Interestingly, while this site is absolutely critical for expression
from the Ren-1c gene (10, 48), it confers no
transcriptional activity as an isolated element (10). Indeed, the
situation which appears to pertain in the case of mammalian renin gene
expression is highly reminiscent of recent insights gleaned from
studies of downstream targets of HOX regulation in
Drosophila (53-55). Enhancers regulating expression of such
target genes appear to be modular in structure. Their activity is
dependent upon the obligate and synergistic integration of information
from signal transduction pathways and selector genes through provision
of binding sites for the nuclear effectors of signaling pathways, such
as CREB, and selector genes, such as Hox. The molecular
basis for the obligate synergy between the selector and signaling
protein complexes in these systems is not as yet clear. Similarly, how
the distal renal enhancer communicates with the critical proximal
promoter element remains to be answered. It is possible that CREB/CREM
proteins interact with HOX·PBX·PREP1 indirectly by binding to a
common coactivator. Both CREB/CREM and HOX·PBX are capable of
interacting with coactivaters such as CREB-binding protein/p300.
Alternatively, direct protein-protein interactions between
CREB/CREM/USF and HOX·PBX·PREP1 may be responsible for the high
level expression of mouse Ren-1c gene in As4.1 cells.
ACKNOWLEDGEMENT
FOOTNOTES
Present address: Southern Australia Dental Service, Somerton
Park Dental Complex, Adelaide, Australia.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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