Department of Pharmacology, College of Medicine, the University of
Tennessee Health Sciences Center, Memphis, Tennessee
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Introduction |
-Adrenergic
receptors (-AR) are Gs-coupled receptors that
transduce the binding of catecholamines into activation of adenylyl cyclase and elevation of cyclic AMP. The 1-AR
seems to be the dominant subtype of the -AR family for mediating the
chronotropic, inotropic, and lusitropic actions of catecholamines in
the heart. Targeted disruption of the 1-AR
gene produced mice that lacked chronotropic and inotropic
responsiveness to catecholamines, further demonstrating the importance
of 1-AR for proper cardiac function (Rohrer et
al., 1996
). In addition to the 1-AR, two
additional subtypes of -AR have been cloned. The three subtypes of
-AR are products of separate genes and are unusual in that they are intronless or, as in the case of the 3-AR,
contain a very small intron (Kobilka et al., 1987; Machida et al.,
1990; Granneman et al., 1993; Searles et al., 1995). The 5'-flanking
region of mammalian -AR reveal G+C-rich sequences which lack
consensus TATA boxes to initiate transcription (Kobilka et al., 1987;
Searles et al., 1995; Evanko et al., 1998). The major transcriptional start site (TSS) of the rat 1-AR gene is at
253 relative to the first base of the initiation
codon,1 whereas
that of the human 2-AR gene is at 219,
suggesting that similarities exist in their transcriptional initiation
(Kobilka et al., 1987; Searles et al., 1995).
The expression of the 1-AR is very low in
tissues as well as in cultured primary ventricular myocytes (Bahouth et
al., 1997a) and in cell lines that express the
1-AR endogenously such as rat C6 glioma and
human neuroepithelioma SK-N-MC cells (Bahouth et al., 1997b, 2001;
Esbenshade et al., 1992). Promoter studies have shown that the 3-kb rat
1-AR promoter has very low inherent ability to
drive transcription compared with shorter promoter sequences (Searles
et al., 1995, Bahouth et al., 1997b). It is likely that low basal
expression of the 1-AR is caused by inhibitory domains or to a weak basal promoter. The first possibility was addressed by deletion studies from the 5' end of the promoter. Shortening the 3-kb promoter to less than 1 kb substantially increased its activity in a variety of transiently transfected cell lines (Searles et al., 1995; Bahouth et al., 1997a,b). To characterize the
basal promoter of the rat 1-AR gene and to
measure its relative transcriptional potency, we generated deletion
mutants of the 5'-flanking sequence and then linked them to the
expression of the gene for firefly luciferase. These constructs
extended to bp 126 to avoid interference by regulatory sequences
down-stream from bp 125 (Bahouth et al., 1997a,b). These analyses
localized the sequence of the core promoter of the rat
1-AR gene between 394 and 330 and
identified two transcription factors that are involved in reciprocal
regulation of the transcription of the 1-AR gene.
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Materials and Methods |
Culture of Ventricular Myocytes.
For each cell preparation,
ventricles were isolated from 1- to 3-day-old Sprague-Dawley rats under
aseptic conditions. Rat neonatal ventricular myocytes were dissociated
with pancrelipase and isolated by discontinuous Percoll gradient
centrifugation (Bahouth et al., 1997a). Using this procedure, highly
enriched populations of ventricular myocytes (>95%) were obtained.
Isolated myocytes were cultured on collagen-coated plates a density of 2 × 106 cells per 60-mm plate in 68% DMEM,
17% medium-199, 10% horse serum, and 5% fetal bovine serum and
antibiotics. The next day the medium was aspirated and replaced with
80% DMEM and 20% medium-199.
Construction of 1-AR Promoter-Luciferase
Chimera and Luciferase Assays.
Progressive deletions within the
KpnI-SacII genomic fragment of the rat
1-AR encoding the sequences between 1251 to
126 were generated either by restriction enzymes or by PCR and then ligated into the promoterless pGL3basic luciferase
expression plasmid. Each 60-mm plate was transfected with 5 µg of
plasmid DNA composed of 3.8 µg of the smallest vector
(pGL3basic), 1 µg of carrier
pGEM7ZF+ DNA, and 0.2 µg of CMV-pRL
(Renilla reniformis luciferase vector for correcting firefly
luciferase activity) by the calcium phosphate precipitation technique
(Bahouth et al., 1997a,b). In all transfections, the amount of each
1-AR-luciferase construct was increased to an
equivalent molar ratio of pGL3basic and the balance of the DNA was with pGEM7ZF+. In all experiments, the
pGL3control vector, which is a luciferase vector driven by
the SV40 promoter and enhancer sequences, was transfected in equimolar
amounts to the pGL3basic vector. Luciferase assays were
performed using the Dual Luciferase Assay System (Promega Corp.,
Madison, WI). The luminescence of each construct is expressed as a
percentage of the expression of pGL3control after
normalizing for transfection efficiency. Each construct was transfected
into three 60-mm plates and these transfections were replicated for each cell type in a minimum of three separate experiments
(n 
9). The values from all experiments were combined
and subjected to analysis of variance using Microsoft Excel (Microsoft
Corp., Redmond, WA). Significance was determined by Student's
t test (p = 0.05).
Culture and Transient Transfection of SL2 Cells.
Drosophila melanogaster SL2 cells were cultured at room
temperature in Schneider's Drosophila medium (SDM)
supplemented with 10% FBS. DNA (1.5 µg per 35-mm plate) was mixed
with 100 µl SDM and 9 µl of CellFECTIN solution (Invitrogen,
Carlsbad, CA) for 15 min. DNA consisted of 0.5 µg of
1-AR promoter luciferase vector, 0.1 µg
pRL-CMV R. reniformis luciferase vector and the balance was
composed of either carrier DNA or the appropriate Sp expression vector
as described in the legend of Fig. 3. To the DNA-CellFECTIN mixture,
0.8 ml of SDM with 1.5% FBS was added, and the DNA/liposome complexes
were layered over the cells for 5 h. After transfection, the DNA
containing medium was replaced with 2 ml of SDM with 10% FBS for
36 h. Thereafter, the cells were harvested and luminescence was
determined. The Sp1 and SP0 vectors under the control of the actin 5C
promoter were obtained from R. Tjian (Kadonga et al., 1987; Courey and
Tjian, 1988) and the other Sp vectors were provided by Guntram Suske
and are described in Dennig et al. (1995).
Site-Directed Mutagenesis of 1-AR Genomic
Fragments.
Mutagenesis of the KpnI-SacII
genomic fragment of the 1-AR gene was performed based on
the methods described in the Transformer site-directed mutagenesis
manual (CLONTECH, Palo Alto, CA). The sequence of the mutagenic primer
for site directed mutagenesis of the triplet between 380 and 378
was 5'-pGGGACACCATTGTAAAGGGGCGTGCCTTG-3'; for mutating the
sequence between 371 and 369 was
5'-pTTGTTCGGGGGCGAAACTTGGCGACGATTG-3'; and for mutating the
sequence between 374 and 372 was
5'-pCCATTGTTCGGGGAAATGCCTTGGCGACG-3'. The underlined
oligonucleotides indicate the mutated sequence. Plasmid DNA was
sequenced by automated dye-termination sequencing using a primer
5'-TCTGGAAGAAGCCTGAGCAG corresponding to the sequence between 519 and
500 in 5'-flanking region the 1-AR gene.
Using the mutagenized KpnI-SacII fragment as
template, the 3311 to 126 and 484 and 126 genomic fragments
were prepared and subcloned into pGL3basic to generate the
desired vector.
Gel Electromobility-Shift Assays (EMSA).
Three
double-stranded oligomers with CTAG overhangs representing the
wild-type and mutated sequences between 385 and 365 in the
1-AR promoter were synthesized. These were the
wild type sequence 5'-ATTGTTCGGGGGCGTGCCTTG-3'; the
mut-Egr-1 oligomer 5'-ATTGTaaaGGGGCGTGCCTTG-3', in which the
Egr-1 site was mutated; and the mut-Sp1 oligomer 5'-ATTGTTCGGGGGCGaaaCTTG-3', in which the Sp1-binding site was mutated.
These oligomers were labeled with Klenow enzyme and
[
-32P]dCTP and combined with nuclear
extracts for 20 min in a binding buffer composed of 80 mM KCl, 10 mM
HEPES, pH 7.1, 1 or 2 µg of poly(dI-dC), and 10% glycerol (Bahouth
et al., 1997a). The resulting complexes were resolved on 5%
nondenaturing acrylamide gels in 25 mM Tris, 200 mM glycine at 4°C
(Bahouth et al., 1997a). Nuclear extracts prepared from rat
neonatal ventricular myocytes were prepared as described in Bahouth et
al. (1997a). Sp1 was purchased from Promega Corp. Egr-1 cDNA cloned in
pRSET-A (Cui et al., 1996), was obtained from N. Mackman (Scripps
Research Institute, La Jolla, CA). Hexahistidine-tagged Egr-1 expressed
in bacteria was partially-purified by affinity chromatography with
nickel nitriloacetic acid resin (Cui et al., 1996).
DNase Footprinting Assay.
The PstI Genomic
fragment between 484 and +269 was 32P-labeled
on one end as described in Bahouth et al. (1997b). Each
32P-labeled DNA fragment (20,000 cpm) was
incubated with purified transcription factor (10 ng) in binding buffer
composed of 20 mM HEPES, pH 7.6, 0.1 mM EDTA, 1 mM dithiothreitol, 10%
glycerol, 50 mM NaCl, and 1 µg of poly(dI-dC) for 30 min at 0°C
(Bahouth et al., 1997b). Digestion with 0.03-0.1 units of DNase I was
allowed to proceed for 45 s, followed by adding 150 mM NaCl, 0.7%
SDS, 15 mM EDTA, and 30 µg of yeast tRNA. The samples were extracted and subjected to electrophoresis on 6% acrylamide in 8 M urea gels.
The protected DNA sequences were identified by running a separate lane
containing a G sequence ladder generated by cleaving the
32P-DNA fragment with piperidine as described
previously (Bahouth et al., 1997b).
Generation of HeLa Cells with Tetracycline-Regulated Expression
of Egr-1.
HeLa S3 cells harboring a stably integrated copy of the
regulator plasmid pTet-Off, were purchased from CLONTECH. pTet-Off contains a fusion of the Tet repressor and the carboxyl-terminal 130 amino acids of VP16 as well as a G418 resistance cassette (Gossen and
Bujard, 1992; Yin et al., 1996). Transcription of the gene under the
control of the tetracycline responsive element is inhibited by
tetracycline or its analog doxycycline. Conversely, transcription of
the gene of interest is active and maintained so long as tetracycline
is absent (Gossen and Bujard, 1992; Yin et al., 1996). pTet-Off-HeLa S3
cells were cultured in DMEM plus 10% FBS supplemented with 600 µg/ml
G418 and 5 ng/ml doxycycline.
Egr-1 and ETTL (ETTL contains Egr-1 cDNA minus 2 zinc fingers and is
therefore inactive) cDNAs were provided by Vikas Sukhatme (Sukhatme et
al., 1988). Egr-1 and ETTL cDNAs were excised with EcoRI and
cloned into the pTRE vector. The pTRE vector is a reconstituting vector
that contains the necessary components to obtain regulated expression
of Egr-1 or ETTL by doxycycline. HeLa S3 cells were transfected using
the LipofectAMINE transfection reagent (6 µl/µg of DNA; Invitrogen)
with Egr-1-pTRE and pTK hygromycin (at 10:1 ratio) for the selection of
double-stable Tet-Off HeLa cells stably expressing either the Egr-1 or
the ETTL gene under the control of the tetracycline responsive element.
Clonal cell lines for either Egr-1 or ETTL were selected by their
ability to suppress Egr-1 mRNA or ETTL mRNA expression in the presence
of doxycycline and to express these mRNAs in the absence of doxycycline.
Double-stable Tet-Off HeLa cells were cultured DMEM plus 10% FBS, 600 µg/ml G418, 200 µg/ml hygromycin, and 5 ng/ml doxycycline until
they were ~80% confluent, then were switched to DMEM supplemented with 1 ng/ml doxycycline and 0.5% FBS to suppress endogenous and exogenous Egr-1 (Cao et al., 1993). After 2 days, the cells were transiently transfected with 1.9 µg per 35-mm plate of the
appropriate wild-type or mutagenized
1-AR-promoter-luciferase plasmid and 0.1 µg
of CMV-pRL R. reniformis vector using LipofectAMINE. After 5 h, the medium was aspirated and the cells were cultured
overnight in DMEM + 0.5% FBS and 5 ng/ml doxycycline, to allow for
cell recovery. The cells were then washed extensively with
phosphate-buffered saline (to remove doxycycline) and recultured for
1.5 h in DMEM with 0.5% FBS, but without doxycycline to induce
Egr-1 transcription. After 90 min, 5 ng/ml of doxycycline was added to
suppress Egr-1 or ETTL transcription, and the cells were harvested
24 h later to measure the luciferase activity. A set of control
plates which contained 5 ng/ml doxycycline to continuously suppress
Egr-1 or ETTL expression were run in parallel.
Culture and Transient Transfection of SK-N-MC Cells.
SK-N-MC
cells are human neuroepithelioma cells that endogenously express the
1-AR (Esbenshade et al., 1992; Bahouth et al., 2001). A mammalian expression vector for Sp1 was generated from pPac
Sp1 as follows: a forward primer, 5'-GCAATGAACTCGTACTTTGGAACAGGC-3', and reverse primer, 5'-TCAGAAGCCATTGCCACTGATATTAAT-3', were used to
amplify the 2.1-kb Sp1 cDNA fragment (Kadonga et al., 1987). The
PCR-generated cDNA was cloned into the PCR 2.1 TOPO vector (Invitrogen), excised with KpnI and XbaI and
cloned into the mammalian expression vector pcDNA 3.1(Invitrogen). The
mammalian expression vectors for Egr-1, pCMV-Egr-1 and for ETTL,
pCMV-ETTL were described previously (Sukhatme et al., 1988).
SK-N-MC cells were cultured in DMEM supplemented with 10% FBS. For
transfection, SK-N-MC cells were cultured in DMEM and transfected with
5 µg of each vector per 150-mm plate (10 µg DNA per plate) by the
LipofectAMINE method for 6 h. After 6 h, the medium was
aspirated, and the cells were cultured in DMEM supplemented with 10%
FBS for 48 h. Total cellular RNA was extracted by the RNA-STAT 60 method and 25 µg or RNA was subjected to Northern blotting and
transferred to Nytran filters as described previously (Bahouth et al.,
2001). The blot was prehybridized for 6 h in Ultrahybrid solution
(Ambion, Austin, TX), then incubated in Ultrahybrid solution containing
2 × 106 cpm/ml of radiolabeled human
1-AR probe for 16 h at 42°C. The 1-AR probe was a 227-bp fragment from human
1-AR cDNA corresponding to the sequences +522
to +748 (Bahouth et al., 2001). To control for the variability in RNA
loading, the blot was stripped and reprobed with a 103-bp human
cyclophilin cDNA probe corresponding to the sequences between +38 and + 140 (Ambion). The cpm in each band was counted by electronic
autoradiography in the InstantImager (Packard Bioscience,
Meriden, CT) and the data for each condition subtracted from background.
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Results |
Transcriptional Regulation of the Rat 1-AR Gene by
Sp1.
To define the region within the 1-AR
gene that is involved in regulating basal expression, deletion mutants
of the 5'-flanking sequences between 1251 to 126 were constructed
and linked to the expression of the firefly luciferase reporter gene.
Progressive deletions from the 5' end indicated that the minimal
promoter of the 1-AR gene lies within a 65-bp
region between 394 and 330 (Fig. 1A).
The first 26-bp of the minimal promoter between 394 and 368
accounted for about 45 ± 5% of the basal activity. Deleting this
construct down to 349 resulted in an additional 35 ± 5%
decline in activity, and the remaining 20% of the activity was lost
when the promoter was truncated to 330.
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Fig. 1.
Characterization of the early promoter region of the
rat 1-adrenergic gene by transient transfections and
gel-shift assays. A, the sequences in the 1-AR promoter
shown in the left side were ligated into the multiple cloning region of
the promoterless firefly luciferase vector pGL3basic and
transfected into ventricular myocytes. The activity of luciferase
relative to the SV40-driven firefly luciferase vector
pGL3control was determined. Transfections were performed
in triplicate and replicated five times, n = 15. The sequence of the core promoter of the rat 1-AR gene
and the percentile activity of its segments are shown. B, a
32P-labeled oligomer corresponding to the sequence between
385 and 365 in the rat 1-AR promoter was incubated
with 1 µl of nuclear extract prepared from rat neonatal ventricular
myocytes that were cultured in serum-free medium (lanes 2-10) and with
IgG or immunoglobulins against Sp family members as indicated for 20 min. The complexes were resolved on 5% nondenaturing polyacrylamide
gels in Tris/glycine and visualized by autoradiography. * and **,
major and minor binding complexes of Sp1 and Sp3, respectively.
***, faster migrating band 3.
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To characterize the transcription factors that bind to the domain
between 394 and 368 we performed EMSA between a
32P-labeled double-stranded oligomer
corresponding to the sequence between 385 and 365 and nuclear
extract prepared from rat neonatal ventricular myocytes that were
cultured in serum-free medium (Fig. 1B). The nuclear proteins formed
three major complexes with the wild-type oligomer, whereby the more
slowly migrating complex was the major forming complex. Because the
domain between 394 and 373 is G+C rich, we used antibodies to
transcription factors with preference to high G+C clusters. Antibodies
to the Sp1 transcription factor supershifted the most slowly migrating
complex, suggesting that transcription factors related to the Sp family
were part of the complex over the oligomer between 385 and 365
(Fig. 1B, lane 4). The Sp family of transcription factors consists of
four members, Sp1, Sp2, Sp3, and Sp4 (Suske, 1999). Sp2 expression is
restricted to the CNS, whereas Sp1, Sp3, and Sp4 are widely distributed
(Dennig et al., 1995). In addition to Sp1, the Sp3 antibody disrupted
the minor band just below the Sp1 complex and, along with the anti-Sp1
antibody was able to supershift the entire Sp1 complex (Fig. 1B, lane
8). The antibody to Sp4 did not supershift, indicating that both Sp1
and Sp3 bind to the 385 to 365 region. Antibodies to Sp family
members did not disrupt the binding to the faster migrating band 3 (Fig. 1B). Binding of nuclear proteins to band 3 was dependent on the
concentration of poly(dI-dC) in the EMSA. Increasing the poly(dI-dC)
from 1 to 2 µg/assay disrupted the binding to band 3, indicating that
it represented a nonspecific protein-DNA complex.
The next experiment was designed to determine by DNase I footprinting
whether the Sp1 transcription factor could bind to any sites in the
basal promoter region of the 1-AR gene. A
753-bp PstI fragment containing the sequences between 484
and +269 was labeled on the top strand (5' end) and the binding of Sp1
to this fragment was analyzed (Fig. 2 A).
Protected footprints were localized to the region between 377 and
365 and to the region between 399 and 390. No other protected
regions were identified (data not shown). Therefore, as suggested by
the EMSA, Sp1 binds to a core sequence within the 394 and 368
domain. Based upon the results of transient transfection assays in Fig.
1, the activity of the 1-AR promoter was
unaffected when the region between 414 and 394 was deleted,
indicating that the footprint between 399 and 390 is not critical
for basal expression of the 1-AR gene.
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Fig. 2.
DNase I footprinting of the
1-adrenergic receptor promoter with Sp1 and Egr-1. The
32P-labeled PstI 484 to +268
1-AR genomic fragment (20,000 cpm) was incubated alone
(lanes 2 and 6), with 30 µunits DNase I (lanes 3 and 7), or with 1 µl of either Sp1 (lane 4) or histidine-tagged Egr-1 (lane 5) and
subjected to DNase I footprinting as described under Materials
and Methods. The localization of the DNase I footprints was
determined using Maxam-Gilbert G ladders (lanes 1 and 5). The sequences
for protected bands I and II are outlined along the figure.
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To determine the functional significance of Sp1 binding to the
1-AR promoter, we performed transient
transfection assays in D. melanogaster Schneider SL2 cells,
which do not express Sp1 or the other Sp family members (Kadonga et
al., 1987; Courey and Tjian, 1988; Dennig et al., 1995). SL2 cells were
transfected with the 394,-126-pGL3, which contains the Sp1-binding
site and with the 368, 126-pGL3 construct that lacks the
Sp1-binding site (Fig. 3A). The cells
were also transfected with the expression vector for Sp1, pPac Sp1, and
with its inactive control pPac O (Courey and Tjian, 1988; Dennig et
al., 1995). The data reveal that cotransfection of 0.1 or 0.3 µg of
pPac Sp1 per plate increased the expression of the 394, 126-pGL3
construct by 6- and 17-fold, respectively. The expression of the 368,
126-pGL3 construct in which the Sp1-binding site was deleted was not
stimulated by low concentrations of Sp1. At the highest Sp1
concentration used, there was a slight (~2-fold) stimulation of the
368, 126-pGL3 construct. This is expected because SL2 cells undergo
a slight activation of general transcription when transfected with high levels of pPacSp1 (Kadonga et al., 1987).
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Fig. 3.
Effect of transient expression of Sp family members
in Schneider D. melanogaster SL2 cells on reporter gene
expression by 1-AR-luciferase constructs. A total of 1.5 µg DNA per 35-mm culture plate consisting of 1 µg
1-AR-luciferase expression vector alone or with the
indicated amounts of pPacSp1, pPacSp3 or pPac0 with 0.1 µg of pRL-CMV
R. reniformis luciferase vector were transiently
transfected into D. melanogaster SL2 cells. The
expression of the 1-AR construct in response to Sp1 or
Sp3 relative to the expression of pPac0 are provided. The data
represent the mean ± S.E. for the combined results of four
transfections. Each transfection used triplicate samples.
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Figure 3B shows the effect of lower amounts of Sp1 and Sp3 on
transcription of the 394, 126-pGL3 construct in SL2 cells. Sp1
alone increased the transcription of by 10-fold, whereas Sp3 did not
appreciably increase the expression of this construct. Sp1 and Sp3
together were not additive. Coexpression of Sp3 with Sp1 reduced the
induction by Sp1 of 1-AR transcription from
10- to 4-fold.
Characterization of Egr-1 Binding to the Early Promoter of the Rat
1-AR Gene.
Another transcription factor that binds
to G+C rich elements is the early growth response gene 1 (Egr-1) factor
(Sukhatme et al., 1988; Rauscher et al., 1990), also known as Zif268,
NGF1-A, krox24, and TIS8. Egr-1 is a zinc-finger transcription factor that binds to a consensus sequence GCGGGGGCG (Lim et al., 1989). We
observed that the sequence between 385 and 365 contained overlapping binding sites for Sp1 and Egr-1 (Fig. 1). In this region,
the Sp1-footprint is between nucleotides 377 and 365, and the
putative Egr-1 binding motif is between 380 and 372 (Figs. 1 and
4). Each site is imperfect by one nucleotide from the consensus Sp1 and
Egr-1 binding sites.
We tested by EMSA whether Egr-1 could bind to the domain between 385
and 365. Partially purified hexa-histidine tagged human Egr-1 bound
to the 32P labeled oligomer corresponding to the
sequence between 385 and 365 in the 1-AR
gene (Fig. 4A, lane 3). Nuclear extracts were prepared from rat neonatal ventricular myocytes that either were
exposed or were not exposed to phorbol-12-myristate-13-acetate (PMA)
for 1 h, a condition known to induce Egr-1 expression in quiescent
heart cells (Chien et al., 1991; Knowlton et al., 1993). In control and
PMA-treated nuclear extracts a slowly migrating band corresponding to
Sp1 was present (Fig. 4A, lanes 1 and 2). In nuclear extracts prepared
from PMA-treated myocytes an additional faster migrating band that
comigrated with purified Egr-1 was evident (denoted with an asterisk in
Fig. 4A, lane 2). To identify the sequences in the
1-AR promoter which bind Egr-1, DNase
footprinting between the 32P-labeled 753-bp
PstI-fragment described earlier and purified hexa-histidine
human Egr-1 was performed (Fig. 2 B). A major protected footprint was
localized between 381 and 367, indicating that the Egr-1 binding
site overlapped with the Sp1 binding site between 377 and 365.
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Fig. 4.
Electromobility shift assay between nuclear extracts
prepared from phorbol ester-treated ventricular myocytes and wild-type
or point mutants of the sequence between 385 and 365. A, nuclear
extracts were prepared from rat neonatal ventricular myocytes that were
cultured in serum-free medium for 2 days and then exposed either to
DMSO or 50 ng/ml PMA for 1 h. Nuclear extracts (1 µl) were
combined with 20,000 cpm of 32P-labeled oligomer
representing the sequence between 385/365. Hexa-histidine human
Egr-1 purified from Escherichia coli was added in lane 3 to mark the migration of Egr-1. B, the sequences of oligonucleotides
representing either the wild-type sequence between 365/365 or those
with a 3-bp mutation between 371 and 361 (mut Sp1)
or with a 3-bp mutation between 380 and 378
(mut-Egr-1) are shown. C, represents competition between
increasing amounts of unlabeled wild-type, mut-Sp1, and
mut-Egr-1 oligonucleotides versus the
32P-labeled 385/365 oligomer in binding to nuclear
extracts prepared from PMA-treated ventricular myocytes. As indicated
above each lane, a 100-fold molar excess (100×), 10-fold molar excess
(10×), or equal amounts of unlabeled oligomer (1×) was added to each
binding reaction mixture.
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To determine the contribution of the individual Sp1 and Egr-1 sites to
transcription factor binding in EMSA assays, we mutagenized the
nonoverlapping sequences between the Egr-1 and Sp1 sites in the
fragment corresponding to the sequence between 385 to 365 (Khachigian et al., 1995; Cui et al., 1996). This approach involved mutagenesis of the three nucleotides in the Sp1-binding site that are
not shared with Egr-1 (between 369 and 371) to disrupt the Sp1
binding site without affecting the Egr-1 binding site (Fig. 4B).
Conversely, by mutagenizing the nucleotides between 380 and 378,
the Egr-1 binding site was disrupted without affecting the Sp1 binding
site. These oligomers were used in competition EMSA assays between
nuclear extract from PMA-treated cardiac myocytes and
32P-labeled wild-type sequence between 385 and
366 (Fig. 4C). In these experiments the concentration of poly(dI-dC)
was doubled to 2 µg/assay to disrupt the binding of the wild-type
32P-oligomer to band 3 that was described earlier
in Fig. 1B. Using this approach, three major bands were bound to the
wild-type oligomer, with the more slowly migrating band corresponding
to Sp1 factor binding (Fig. 4C, lane 1). The unlabeled wild-type
oligomer effectively competed Sp1 binding at 10 × molar excess,
while the oligomer mutagenized between 369/371 (mut-Sp1)
did not compete for Sp1 binding at 10 × molar excess (Fig. 4C,
compare lanes 3 and 6). On the other hand, the mut-Sp1
oligomer was equally effective to the wild-type oligomer in competing
with the 32P-labeled probe for binding to the two
faster migrating bands that comigrated with recombinant Egr-1.
Therefore, it seems that selective disruption of the Sp1 binding site
was achieved using this strategy. The oligomer in which the sequence
between 380 and 378 was mutated (mut-Egr-1) was equally
effective to the wild-type oligomer in competing Sp1-binding to the
32P-labeled probe. However, this mutant oligomer
was significantly weaker than the wild-type or the mut-Sp1
probes in competing for Egr-1 binding (Fig. 4C, compare lane 8 versus
lanes 2 and 5). These data suggest that Sp1 and Egr-1 binding can be
selectively inhibited by this approach.
Next, we examined the promoters of 1-AR genes
from several mammalian species to determine whether a shared
Sp1/Egr-1-binding element was present (Fig.
5). Our analyses revealed a strong
conservation of the overlapping Sp1/Egr-1 binding site in mammalian
1-AR genes both in terms of sequence and
localization within the promoter.
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Fig. 5.
Comparison of the Sp1 and Egr-1 binding region in
5'-flanking region of mammalian 1-AR. A sequence
comparison of the Sp1 and Egr-1 binding region in promoters of the
human, rat, mouse, monkey, and pig 1-AR genes. The
numbers are with respect to the translation initiation site. The
accession numbers of the genes are as follows: X75540, rhesus;
AF042454, porcine; D00634 and J05561, rat; X69168, human, and mouse
(Cohen et al., 1993).
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Transcriptional Regulation of the Rat 1-AR Gene by
Transient Expression of Egr-1.
The induction of Egr-1 mRNA in
neonatal rat ventricular myocytes in response to PMA or other
hypertrophic stimuli is transient in nature (Knowlton et al., 1993). As
illustrated in Fig. 6A, Egr-1 mRNA was
undetectable in neonatal rat ventricular myocytes that were cultured in
serum-free medium. Exposing these cells to PMA, maximally induced Egr-1
mRNA within 30 min and this effect was attenuated in 3 h even
though the concentration of PMA was sustained through out the
incubation period. These data indicated to us that proper kinetics of
induction and suppression of the Egr-1 gene are required to
appropriately assess its transcriptional regulatory effects.
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Fig. 6.
Comparison between the kinetics of transient Egr-1
induction in ventricular myocytes by phorbol-12-myristate-13-acetate
(PMA) and regulated Egr-1 expression in HeLa cells by the Tet-Off
system. A, rat neonatal ventricular myocytes were cultured in
DMEM/medium-199 (80:20) for 2 days and then exposed to 50 ng/ml of PMA.
B, HeLa S3 cells expressing pTet-Off and human Egr-1 cDNA that were
cultured for 2 days in medium containing 0.5% FBS and doxycycline to
suppress Egr-1 expression. Then doxycycline was removed and Egr-1 was
induced for 90 min, followed by the re-addition of 5 ng/ml doxycycline
(arrow) to suppress Egr-1. RNA was prepared before Egr-1 induction,
during Egr-1 induction, and 1, 2, 3, and 4 h after Egr-1 was
suppressed. RNA were subjected to Northern blotting and probed with
32P-labeled human Egr-1 cDNA probe. C, plasmid DNA (2 µg)
consisting of 1.9 µg of 1-AR-luciferase expression
vector and 0.1 µg of pRL CMV R. reniformis luciferase
vector were transiently transfected into 60-mm plates containing the
double-mutant HeLa S3 cells Eg-15 or Et-32. Two vectors were used,
394, 126-Luc with the Egr-1 binding site and 368, 126 Luc
without the Egr-1 binding site. After transfection, the cells were
cultured overnight in the presence of 5 ng/ml doxycycline to suppress
Egr-1 or ETTL. The next day, Egr-1 or ETTL were induced for 90 min,
followed by the readdition of 5 ng/ml doxycycline. After 24 h, the
cells were lysed for the measurement of luciferase activities. The
corrected luciferase activity in light units/10 µg of protein ± S.E. for the combined results of four transfections, each in triplicate
were calculated.
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We adopted two strategies to achieve transient induction of Egr-1. The
first was the classical approach that involves chemical treatment with
PMA to transiently induce Egr-1 expression (Table 1). This approach has a drawback in that
PMA is known to induce cardiac hypertrophy and induces a plethora of
protooncogenes including c-fos, c-jun, and others (Chien et
al., 1991; Knowlton et al., 1993). Nevertheless, in PMA-treated
neonatal rat ventricular myocytes, the luciferase activity of the
wild-type 494 to 126-pGL3 construct was reduced by 35 ± 11%
(n = 4) compared with cells exposed to vehicle alone.
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TABLE 1
Effect of PMA on the responsiveness of the wild-type and mutated
1-AR promoter in neonatal ventricular myocytes
The wild-type and mutagenized 1-AR promoters indicated below
were ligated to luciferase in pGL3basic. A total of 5 µg
of DNA per 60-mm culture plate consisting of 4.8 µg of
1-AR-luciferase expression vector with carrier DNA and 0.2 µg of pRL-CMV R. reniformis luciferase vector were
transiently transfected into neonatal rat ventricular myocytes. The
next day, the cells were washed with phosphate-buffered saline and
cultured for 48 h in 80:20 DMEM/medium-199 to suppress Egr-1
expression. PMA (50 ng/ml) was added and its effect on luciferase
activity was determined after 24 h. The corrected luciferase
activity in light units/10 µg of protein ± S.E. in the presence
or absence of PMA were calculated and divided by the luciferase
activity in the absence of PMA to determine the percentage change in
activity in response to PMA. The data represent the combined results of
four transfections where each transfection was in triplicate.
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The other approach involved regulated expression of Egr-1 by the
tetracycline responsive promoter of Bujard (Gossen and Bujard, 1992;
Yin et al., 1996). This approach selectively induces a single gene in
the absence of tetracycline and represses it in the presence of
tetracycline or its more active analog doxycycline. To examine the
effects of transient expression of Egr-1, the cDNAs for Egr-1 or of its
inactive analog ETTL, which lacks two zinc fingers (Sukhatme et al.,
1988), were stably expressed in a HeLa S3 cell line that contains a stably integrated copy of the Tet repressor plasmid. A
double-stable HeLa S3 cell line with proper induction and suppression of Egr-1 mRNA (Eg-15) and another for ETTL (Et-32) were selected by the
criteria outlined under Experimental Procedures. The
expression of Egr-1 in Eg-15 cells was undetectable in the presence of
doxycycline (Fig. 6B, lane 1) but was rapidly induced when doxycycline
is removed (Fig. 6B, lane 2). The levels of Egr-1 mRNA in the S3 system
were 3 ± 1-fold higher than those induced by PMA in neonatal cardiac myocytes. After 1.5 h, doxycycline was added and the
levels of Egr-1 mRNA declined rapidly and were undetectable after
4 h from the readdition of doxycycline (Fig. 6B, lanes 3-6). The
kinetics of induction of Egr-1 in the Tet-Off system in HeLa S3 cells
were comparable with those obtained by PMA treatment in heart cells because the half-life of Egr-1 mRNA is short (<15 min) allowing quick
down-regulation after transcriptional silencing by doxycycline (Gossen
and Bujard, 1992; Yin et al., 1996). The expression pattern of ETTL in
response to doxycycline was similar to that shown in Fig. 6B for Egr-1
(data not shown).
In the next series of experiments, the Eg-15 and Et-32 cell lines were
transfected with the 1-AR-luciferase vector
containing the putative Egr-1 site (394, 126) or that without it
(368, 125). Transient induction of Egr-1 for 90 min, followed by
silencing of Egr-1 expression for 24 h, reduced the luciferase
activity of the 394, 126 construct to 58 ± 16% (Fig. 6C).
The magnitude of Egr-1-mediated inhibition of reporter gene activity
was ~42% that correlates favorably with the percentile of basal
promoter activity imparted by the Sp-1 binding site. The luciferase
activity of the 368, 126-pGL3 was not affected whether Egr-1 was
induced or suppressed. In ETTL expressing Et-32 cells the luciferase
activities of the 394, 126-pGL3 and 368, 126-pGL3 under
conditions of transient ETTL induction or suppression were the same.
These data reveal that suppression of 1-AR
transcription by Egr-1 is dependent upon the 394 to 368 region in
the 1-AR promoter.
Reciprocal Regulation of 1-AR mRNA Expression by Sp1
and Egr-1 in SK-N-MC Cells.
To test the functional relevance of
Sp1 and Egr-1 in regulating the transcriptional activity of the intact
1-AR gene, we determined the effect of
transient expression of these transcription factors on
1-AR mRNA levels in SK-N-MC cells. SK-N-MC cells are human neuroepithelioma cells that express moderate amounts of cell-surface 1-AR (Esbenshade et al., 1992;
Bahouth et al., 2001). Transient transfection of a CMV-driven Sp1
expression vector increased the levels of 1-AR
mRNA by 235 ± 50% compared with cells that were transfected with
the control inactive vectors (Fig. 7).
The expression of CMV-Egr-1 on the other hand reduced
1-AR mRNA levels by 40%. Coexpression of
equal amounts of Sp1 and Egr-1 vectors reduced Sp1-mediated induction
of 1-AR mRNA by 60%. Therefore, under
these conditions the transcriptional activity of the
1-AR gene was reciprocally regulated by Sp1
and Egr-1.
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Fig. 7.
Effect of transient expression of Sp1 and Egr-1 on
1-AR mRNA levels in SK-N-MC cells. SK-N-MC
cells in DMEM were transiently transfected for 6 h with mammalian
expression vectors harboring pcDNA, Sp1, Egr-1, or its inactive
derivative ETTL by the LipofectAMINE method. After 6 h, the medium
was aspirated and the cells were cultured in DMEM + 10% FBS for
48 h. RNA was extracted and 1-AR mRNA
levels were determined by Northern blotting as described under
Materials and Methods. The values of
1-AR mRNA are the means ± S.E.M. of 3 separate determinations. The percentage of
1-AR mRNA in control (ETTL + pCDNA) was 100%;
in Sp1, 235% ± 50; in Egr-1 alone, 60% ± 20; in Egr-1 + Sp1, 140% ± 30. **p < 0.001 for
1-AR mRNA in control versus Sp1,
*p < 0.01 for 1-AR mRNA in
control versus Egr-1.
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A Shared Overlapping Site Is Involved in Reciprocal Regulation of
1-AR Expression by Sp1 and Egr-1.
To
determine the mechanism by which Sp1 and Egr-1 bind to their
individual sites in their common binding region between 380 and
369, the 32P 385/365 oligonucleotide was
incubated with increasing amounts of Egr-1 in the presence of a fixed
amount of Sp1 (Fig. 8A). As the
concentration of Egr-1 was raised, the amount of Egr-1 complexed to
32P 385/365 increased, whereas the Sp1
complexed to 32P 383/365 decreased. In the
next set of experiments, we performed EMSA using the
32P-labeled mut-Egr-1 oligomer to
determine whether the Egr-1 binding site is necessary for the
competitive interaction of Egr-1 with Sp1 (Fig. 8B). The binding of Sp1
to the 32P-mut-Egr-1 was not affected
by this mutation. However, the ability of Egr-1 to competitively
displace Sp1 was severely compromised. These data indicate that Sp1 and
Egr-1 do not simultaneously bind to 385/365 and that these two
transcription factors compete for binding.
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Fig. 8.
Competitive displacement of Sp1 binding of the 385
and 365 G+C-rich region in the 1-AR
promoter by Egr-1. EMSA was performed on
32P-labeled wild-type 385/365 oligonucleotide
(left) or 32P-mut-Egr-1-385/365
oligonucleotide (right), in which the Egr-1 binding site was mutated.
The oligomers were incubate with a constant amount of Sp1 (0.05 footprinting unit/incubation) and increasing amounts of
(His)6-Egr-1 fusion protein (2-50 ng/lane) for
20 min at room temperature. The resulting complexes were the resolved
by electrophoresis and visualized by autoradiography.
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Functional Relevance of the Egr-1 Binding Site in the
1-AR Promoter.
To determine whether reciprocal
regulation of the 1-AR gene described earlier
was caused by the interaction of Sp1 or Egr-1 with their respective
binding sites, the nonoverlapping Sp1 and Egr-1 binding sites were
mutated by site directed mutagenesis. Two rat genomic 1-AR
constructs were mutagenized, a long 1-AR promoter construct extending from 3311 and 126 and a shorter version extending from 494 and 126. To disrupt Egr-1 binding, the
sequences between 380/378 that are involved in Egr-1 binding were
mutated. To disrupt Sp1 binding, the sequences between 371/369 that
are involved in Sp1 binding were mutated. In addition we disrupted the
Sp1 and Egr-1 binding sites simultaneously by mutagenizing the GCG
sequence in the core Sp1/Egr-1 binding region between 374/372
(Table 2). Transient transfection of the
long 1-AR promoter constructs into ventricular
myocytes indicated that mutating the Egr-1 site between 380 and 378
did not inhibit the activity of the 1-AR
promoter (Table 2). However, mutating the Sp1-binding site between
371 and 369 or the core sequence between 374 and 372, caused in
both instances a 38% drop in the activity of these constructs.
Therefore, the Sp1-binding site seems to be the dominant site in
stimulating the expression of the 1-AR
promoter from this region.
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TABLE 2
Effect of disrupting the Sp1 or Egr-1 binding sites by site-directed
mutagenesis on the activity of the 1-AR promoter-luciferase
chimera in neonatal rat ventricular myocytes
Vectors and carrier DNA (4.8 µg) were transiently transfected into
neonatal ventricular myocytes with 0.2 µg pRL-CMV by calcium
phosphate precipitation. After 2 days, the activity of firefly
luciferase were measured and corrected for minor changes in
transfection efficiency. The relative expression of luciferase by the
mutated 1-AR promoter constructs relative to the expression
of luciferase by the intact 1-AR promoter are provided. The
data represent the mean ± S.E. for the combined results of four
transfections. Each transfection utilized triplicate samples.
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In follow-up experiments, we transfected the wild-type and mutagenized
versions of the short promoter extending from 494 to 126 into
D. melanogaster SL2 cells (Fig.
9) and into HeLa S3 cells (Fig.
10). It was necessary to use the short
promoter in these studies because the luciferase activity of the long
promoter was very low. The expression of the long-promoter is efficient in cells that express the 1-AR endogenously
and low in cells that do not express endogenous
1-AR, such as SL2 and HeLa cells (Bahouth et
al., 1997b). Coexpression of the wild-type 494 to 126 promoter with
the Sp1 expression vector in SL2 cells caused a 10-fold stimulation of
luciferase activity compared with the activity generated by the Sp0
vector (Fig. 9). Similarly, coexpression of Sp1 with the promoter in
which the Egr-1-binding site was disrupted resulted in luciferase
activity comparable with the wild-type vector. The luciferase activity
of the 1-AR promoter with a mutation in the
Sp1-binding site was 20% of that attained by the wild-type vector,
whereas no induction of luciferase activity by Sp1 was observed in the
vector with a mutation in the core region.
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Fig. 9.
Effect of transient expression of Sp1 in D.
melanogaster SL2 cells on reporter gene expression by mutated
1-AR genomic-luciferase constructs. 5'flanking sequences
containing point mutations within the rat 1-AR promoter
were subcloned into the luciferase expression vector and transfected
into SL2 cells as described in the legend of Fig. 3. The effect of Sp1
on the expression of the wild type 1-AR construct and
the constructs in which the Sp1-binding site
(494-mut-Sp1), the Egr-1-binding site
(494-mut-Egr-1) or the Sp1/Egr-1 binding sites
(494-mutSp1/Egr-1) were mutated are provided. The data represent the
expression of each vector in response to Sp1 relative to its expression
in response to pPacO. The data represent the mean ± S. E. for the combined results of three transfections. Each transfection used
triplicate samples.
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Fig. 10.
Effect of transient expression of Egr-1 or ETTL on
the expression of mutated 1-AR
genomic-luciferase constructs in HeLa cells. A total of 2 µg DNA per
60-mm culture plate consisting of 1.9 µg of the
1-AR-luciferase expression vectors (described
in the legend of Fig. 9) and 0.1 µg of pRL CMV renilla
luciferase vectors were transiently transfected into the double-mutant
HeLa S3 cells Eg-15 or Et-32 cells as described under Materials
and Methods. After transfection, Egr-1 or ETTL were induced for 90 min and the cells were processed as described in the legend of Fig. 6.
The corrected luciferase activity in light units/10 µg of
protein ± S. E. for the combined results of three
transfections each were calculated in triplicate.
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To determine whether the region between 380 and 378 was
functionally involved in Egr-1 mediated events, the activity of the
wild-type and mutagenized promoters in HeLa cells in response to
transient induction of Egr-1 or ETTL was determined (Fig. 10). Transient induction of Egr-1 in Eg-15 cells was associated with 45%
decline in the luciferase activity, whereas the induction of ETTL had
no effect on the luciferase activity of the wild-type 494 to 126
construct. Disruption of the Egr-1 biding site abolished the inhibitory
effect of transient Egr-1 expression. Transient induction of Egr-1 or
ETTL in cells expressing the 494-mut Sp1 construct was
associated with 50% decline in luciferase activity compared with the
wild-type construct. Moreover, the luciferase activity of the
construct with the disrupted Sp1-binding site was not further
diminished in response to transient Egr-1 expression. These experiments
were repeated in PMA-treated heart cells (Table 1). Mutagenesis of the
Sp1-binding was associated with 50% reduction in activity. Mutagenesis
of Sp1 or the Egr-1 binding sites abolished PMA-mediated inhibition of
1-AR promoter activity. These data demonstrate
the requirement for intact Egr-1 and Sp1-binding sites in the proximal
1-AR promoter sequence for the effects of
Egr-1 on 1-AR expression to be observed.
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Discussion |
In this work, we identified the core promoter region of the rat
1-AR gene and characterized a G+C-rich region
that has a consensus Sp1-binding site. An intact Sp1 site was required
for the full activity of the 1-AR promoter in
heart cells. The conservation of this binding site across mammalian
1-AR genes suggests that this element is
crucial to the regulated expression of the
1-AR gene (Fig. 5). Sp1 is a potent activator
of a wide variety of G protein-coupled receptor promoters, such as the
1B- and 1D-adrenergic receptor genes (Chen et al., 1997; Arai et al., 1999), the 
- and
µ-opioid receptor genes (Ko et al., 1998; Liu et al., 1999), and the
dopamine D2-receptor gene (Yajima et al., 1998).
The migration patterns of nuclear extracts from SH-SY5Y cells bound to
a 40-mer fragment containing the Sp1 site of the µ-opioid receptor
(Liu et al., 1999) were similar to those shown in Fig. 1B. In both instances, the more slowly migrating bands were supershifted by anti-Sp
antibodies, whereas the faster migrating band was not affected. In this
study, we inhibited the binding of nuclear factors to the faster
migrating band by increasing the concentration of poly(dI-dC) in the
EMSA. This result suggests that the binding of nuclear factors to the
faster migrating band had a lower affinity than their binding to the
more slowly migrating complex.
In the µ-opioid and the D2 dopamine receptor
genes, Sp1 and Sp3 were bound to the Sp-binding sites of these GPCR
promoters. The effect of Sp3 on Sp1-mediated activation of
transcription of these GPCR genes was promoter dependent. In the
µ-opioid receptor gene, Sp3 trans-activated the promoter
and its activity was additive with Sp1 (Liu et al., 1999). Whereas in
the D2 dopamine receptor gene, Sp3 alone failed
to affect transcription and repressed Sp1-induced trans-activation of transcription. The mechanism by which
Sp3 inhibits Sp1-mediated trans-activation of transcription
is unknown. In the collagen 2A1 promoter, Sp3 did not influence gene
transcription, but concomitant overexpression of Sp1 and Sp3 inhibited
the transcription of the collagen 2A1 promoter via Sp3-mediated
blockade of Sp1-mediated induction of the collagen 2A promoter activity
(Ghayor et al., 2001). These results indicate that the behavior of Sp3
over the 1-AR promoter resembles its activity
over the promoters for the D2 dopamine receptor
and collagen 2A1.
The binding of Sp1 to G+C boxes is often critical to achieving
significant levels of transcription from promoters that lack TATA and
CCAAT elements, such as the 1-AR gene (Araki
et al., 1991; Boisclair et al., 1993). The activity of the basal
promoter of the 1-AR gene relative to the
SV40-driven pGL3control vector was about 60%, indicating
that the 1-AR gene contained a strong minimal
promoter. However, the expression of 1-AR mRNA
is very low in tissues and cell lines that endogenously express
1-AR (Bahouth et al., 1997b, 2001; Searles et
al., 1995). Promoter studies have shown that the complete 3.3-kb rat
1-AR promoter has very low inherent ability to
drive transcription compared with the minimal promoter (Searles et al.,
1995; Bahouth et al., 1997b; Evanko et al., 1998). Therefore, it is
likely that the activity of the minimal promoter is modulated by
sequence specific enhancer and/or silencer binding proteins that
produce low-level as well as tissue-specific expression. Progressive
deletions and site-directed mutagenesis of the
1-AR gene revealed positive and negative
regulatory domains flanking both ends of the core promoter between
394 to 330 that played a critical role in regulating the expression
of this gene. A strong negative regulatory domain between the sequences
from 2870 to 2740 was found in the rat and human
1-AR promoters (Bahouth et al., 1997b; Evanko
et al., 1998). In addition, two hormonally responsive domains in the
5'-flanking region were identified. An element that inhibited the
transcription of the 1-AR gene in response to
glucocorticoids was localized in the sequence between 950 and 926
and a cyclic AMP-responsive element that inhibited the transcription of
the 1-AR gene in C6 glioma cells in response
to -agonists was localized within the sequences between 1258 and
1250 (Bahouth et al., 1996; Fitzgerald et al., 1996). The mechanism
of -agonist-induced repression of 1-AR gene
transcription is interesting. -Agonists rapidly stimulate the
expression of the inducible cyclic AMP early repressor which is a
member of the cyclic AMP response element modulator family of
transcription factors (Fitzgerald et al., 1996). Increased expression
of inducible cyclic AMP early repressor and cyclic AMP response element
modulator inhibited the expression of
1-AR-luciferase constructs that contained the
cyclic AMP-responsive element.
Kirigiti et al. (2000) and Searles et al. (1995) identified two
additional domains within the rat 1-AR
promoter that lie 5' and 3' to the TSS. The first domain situated
upstream from the TSS at 389 was necessary for expression and
possessed AP-2 like consensus elements which bound the recombinant AP-2
protein (Kirgiti et al., 2000). The domain 3' to the TSS consisted of two clusters between 1 to 159 and 186 to 211 that when deleted increased or decreased transcription, respectively (Searles et al.,
1995). In addition, Bahouth et al. (1997b) identified two additional
domains that lie 3' to the TSS. One domain was a thyroid hormone
responsive element between nucleotides 101 and 117 that mediated
transcriptional activation of the 1-AR gene in
response to thyroid hormones and the other was an adjacent domain
between nucleotides 118 and 125 that suppressed the expression of
1-AR-luciferase chimera in a tissue-specific
manner (Bahouth et al., 1997a,b). These data suggest that low
expression levels of the 1-AR gene were not
primarily caused by a weak promoter but more probably by inhibitory
sequences in the promoter of the 1-AR gene
(Machida et al., 1990; Searles et al., 1995; Fitzgerald et al., 1996;
Bahouth et al., 1997a,b; Evanko et al., 1998).
Our studies revealed that the protooncoge
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1-Adrenergic Receptor
Gene Transcription by Sp1 and Early Growth Response Gene 1: Induction
of EGR-1 Inhibits the Expression of the
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Abstract
Introduction
