Molecular and Cellular Biochemistry 188: 169–176, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
169
An A/G-rich motif in the rat fibroblast growth
factor-2 gene confers enhancer activity on a
heterologous promoter in neonatal rat cardiac
myocytes
Karen A. Detillieux, Adrienne F.A. Meyers, Johanna T.A. Meij and
Peter A. Cattini
Gene Technology Group and Departments of Physiology and Pharmacology and Therapeutics, University of Manitoba,
Winnipeg, Manitoba, Canada
Abstract
We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5′-flanking DNA.
Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides –793 and –720 that was greater than
97% A/G-rich. A repeat of the sequence 5′-AGGGAGGG-3′ separated by 11 bp was located at the core of this domain. A 37 bp
A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes
simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal
rat cardiac myocytes. Promoter activity was stimulated ~3 fold in the presence of AGGG-repeat sequences. This effect was neither
tissue or species-specific since TK promoter activity was increased ~11 fold in both rat and human glial tumor cells. Four specific
complexes (C1–4) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility
shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very
high affinity/specificity interactions (C2 > C4) while C1 and C3 were of lower affinity. As a result, competition with up to a 25
fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in
the levels of C1 and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the
5′-gGGGAGGG-3′ sequence previously implicated in the response of the atrial natriuretic factor promoter to the α-adrenergic
agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the
37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically
significant. Furthermore, there was no difference in the gel mobility shift (C1–4) pattern obtained with the 37 bp A/G-rich
oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of
norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5′-flanking DNA, including the AGGGrepeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not
able to induce a significant phenylephrine response in neonatal rat cardiac myocytes. (Mol Cell Biochem 188: 169–176, 1998)
Key words: FGF-2, transcription, gene transfer, HSV-thymidine kinase promoter
Introduction
Fibroblast growth factor-2 (FGF-2) or basic fibroblast growth
factor (bFGF) and its high affinity receptor FGFR-1, a
member of the tyrosine kinase family, are essential for the
normal growth and development of the myocardium [1, 2].
With regard to the post natal heart, there is increasing evidence
that FGF-2, which is released with contraction [3, 4], is
Address for offprints: P.A. Cattini, Gene Technology Group and Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg,
Manitoba, Canada R3E 3J7
170
involved in the maintenance of a healthy myocardium
through its angiogenic [5–7] as well as cardioprotective
properties [8, 9]. Many of the studies regarding FGF-2 have
made use of exogenous addition of this factor, and focussed
on its interaction with its high affinity cell surface receptor,
which signals its mitogenic and angiogenic responses. In
contrast, there have been relatively few studies on the
control of FGF-2 synthesis, particularly at the transcriptional
level, and specifically in the heart. The human and rat FGF2 promoter sequences have been reported [10, 11], and
although they do not share extensive structural similarity,
short (~20 base pair) stretches of highly conserved sequences
were identified in the proximal promoter region [11]. Also,
both promoters lacked a TATA box but contained G/C rich
regions and Spl sites [10, 11]. The human FGF-2 promoter
was shown to be regulated by mutant and wild type p53 in
a positive and negative manner, respectively, in reporter
gene/transfection studies using glioblastoma and hepatocellular carcinoma cells [12]. In addition, neuropeptides
were shown to influence the human FGF-2 promoter via the
zinc finger transcription factor Egr-1 in glial cells [13]. A
consensus Egr-1 site is also contained in the rat FGF-2
promoter region [11].
Based on an alignment of reported human and rat FGF-2
genomic sequences [10, 11], the rat fragment appears to
extend further upstream [11]. Here we report the detection
of an A/G rich domain in this extended DNA region containing
a repeat of the sequence 5′-AGGGAGGG-3′. This sequence
shows some similarity to a previously reported phenylephrine
responsive element (5′-gGGGAGGG-3′) in the rat atrial
natriuretic factor promoter, which is functional in transfected
neonatal rat cardiac myocytes [14]. We have assessed these
sequences for activity using the firefly luciferase reporter
gene assay and transient gene transfer into neonatal cardiac
myocytes and non cardiac cells, as well as their ability to
bind nuclear protein. Our data indicate that these A/G rich
sequences can confer stimulatory activity on a promoter,
without tissue- or species-specificity, and make high affinity/
specificity interactions with nuclear protein. These sequences
did not, however, show a significant response to phenylephrine
in neonatal rat cardiac myocytes after gene transfer.
Materials and methods
Cell culture
Neonatal rat cardiac myocyte cultures were prepared
essentially as previously described [15]. Briefly, ventricles
from Sprague-Dawley rat pups (at 1–2 days after birth) were
dissected and the cells dissociated in a spinner flask using
a combination of trypsin and DNase I. Myocytes were
separated from non muscle cells on a discontinuous Percoll
gradient and plated on collagen coated plates at a density
of 1 × 106 cells per 35 mm dish. Cells were initially plated
in Ham’s F10 medium containing 10% fetal bovine serum
(FBS), 10% horse serum, antibiotic (1000 units/ml penicillin,
1 mg/ml streptomycin) and calcium chloride supplemented
to 1.05 mM. Rat glioma C6 and human astrocytoma U87MG cells were obtained from the American Type Culture
Collection and grown in monolayer culture in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
(v/v) FBS with antibiotic at 37°C in the presence of 5% CO2.
Plating densities for C6 and U87-MG cells were 0.5 × 106
and 1.0 × 106 per 100 mm dish, respectively. All cultureware
was purchased from Corning (Fisher Scientific, Nepean,
ON, Canada), and all media and reagents from Gibco-BRL
(Life Technologies, Burlington, ON, Canada).
Nuclear protein preparations
Heart tissue was dissected aseptically from neonatal (1–2
days) Sprague Dawley rats, and nuclear protein was prepared
essentially as described previously 116], with final dialysis
against 20 mM Hepes pH 7.9, 20% v/v glycerol, 0.1 M KCl,
0.2 mM EDTA, 0.5 mM DTT and 1 mM PMSF. The nuclear
extract was mixed with heparin-agarose and washed with
0.1M KCl before elution of protein with 0.6 M KCl.
Following determination of protein concentration using the
Bradford Assay (Bio-Rad Laboratories, Mississauga, ON,
Canada), nuclear extracts were aliquoted and maintained at
–70 °C. A modified, rapid extraction protocol [17] was used
for isolation of nuclear protein from cultured neonatal rat
cardiac ventricular myocytes grown in the presence or
absence of 0.01 mM norepinephrine for 6 hours under serumfree conditions.
Hybrid gene constructions
The plasmid TKp.luc (pT81.luc, 118]) contains a portion of
the herpes simplex virus thymidine kinase (TK) promoter
(positions –81 to +52) fused to the firefly luciferase gene.
A 37 bp A/G rich (double-stranded) oligonucleotide,
corresponding to nucleotide positions –785/–749 [11], and
containing the putative AGGG-repeat sequences, 5′GGGAAAGGGAGGGGGAAGGAAAGGAGGGAGGGAAGGA3′, was synthesised and inserted upstream of the TK promoter
in TKp.luc to generate A/G-TKp.luc. The 5 bp of sequence
upstream and downstream of the AGGG-repeat was included
in an attempt to limit any end effect that might interfere with
specific protein binding. The promoterless firefly luciferase
gene (–p.luc, contained in the vector pXPl) was described
previously [11]. The pRL-CMV vector, containing the Renilla
luciferase gene under the control of the cytomegalovirus
171
promoter was obtained from Promega Corporation (Fisher
Scientific).
Gene transfer
Cardiac myocytes and glial tumor cells were transfected by
the calcium phosphate-DNA precipitation method. Briefly,
30 µg of test plasmid (hybrid firefly luciferase gene) and 3
µg of control plasmid (pRL-CMV) was made up to a volume
of 0.5 ml in 252 mM CaCl2 and added gradually to an equal
volume of aerated 2 × HEBS buffer (280 mM NaCl, 50 mM
HEPES-KOH, pH 7.1, and 1.5 mM Na2PO4). Precipitate
was allowed to form at room temperature for 30 min, and
310 µl was added to each of 3 culture dishes of cardiac
myocytes (35 mm) or glial tumor cells (100 mm) containing
4 ml or 8 ml of DMEM/10% FBS, respectively. Cells were
transfected for 16 h and then washed thoroughly with
calcium- and magnesium-free phosphate-buffered saline.
Cells were refed with DMEM/10% FBS and maintained for
48 h before processing. For phenylephrine treatment,
transfected cardiac myocytes were refed with DMEM-F12
containing l × Redu-Ser II (Upstate Biotechnology, Lake
Placid, NY, U.S.A), 0.02 mg/ml ascorbic acid and antibiotics
with or without 0.1 mM phenylephrine, and maintained for
48 h before processing. Cells were also transfected with
–p.luc as a control for random transcription initiation. Cotransfection with pRL-CMV was used as a control for DNA
uptake and values were used subsequently to normalize the
firefly luciferase activity (firefly luciferase/Renilla luciferase)
from the ‘test’ genes. For experiments with phenylephrine
treatment, firefly luciferase activity was normalized
against lysate protein content (luciferase activity/ng
protein) which was determined using the Bradford Assay
(Bio-Rad Laboratories).
was 32P-end-labelled, in the presence of 2 µg of poly dI-dC.
Incubation of the reaction (in 10 mM Hepes-NaOH pH 7.9,
50 mM KCl, 0.5 mM EDTA, 10% glycerol, 1 mM DTT with
5 mM MgCl2) at room temperature for 30 min was followed
by electrophoresis in non-denaturing 4% polyacrylamide
gels. For competition, specific unlabelled 37 bp A/G-rich
oligonucleotide or non specific RF-1 element [21] (at a 1–
100 fold molar excess over radiolabelled oligonucleotide)
were added to nuclear extracts and incubated at room
temperature for 10 min prior to the addition of the radiolabelled DNA and incubation at room temperature for a
further 20 min.
Statistical analysis
Data presented in the text and figures represent the mean ±
S.E. from at least 2 independent experiments each done in
triplicate. Statistical analysis of the results was done using the
Mann-Whitney nonparametric test. Results were considered
significant if p was determined to be < 0.05.
Results
Sequence analysis reveals an A/G-rich domain in the rat
FGF-2 gene
We have reported the cloning of a fragment of the rat FGF2 gene, including a promoter region of 1058 bp based on a
primary transcription start site (+1) determined using brain
RNA [11]. Inspection of these sequences reveals a 74 bp
region which is 97% adenine/guanine (A/G-rich, located
between nucleotide positions –793 and –720 (Fig. 1). Indeed,
a 65 bp stretch within this region (–793/–729) contains only
A or G residues. Located at the core of this region are two
Reporter gene assays
Both firefly and Renilla luciferase activities were measured,
following ‘active lysis of cells by scraping’, using the ‘DualLuciferase™ Reporter Assay System’ (Promega) and a
luminometer (ILA900 Luminometer, Tropix Inc., Bedford,
MA, USA) according to the manufacturer’s instructions. The
protein content of cell Iysates generated in this manner was
determined by the bicinchoninic acid protein assay [19].
Gel mobility shift assay
The gel mobility shift assay was performed essentially as
described by Baldwin [20]. Nuclear extract (5 µg) was
incubated with the 37 bp A/G-rich oligonucleotide which
Fig. 1. Region containing A/G-rich sequences in the rat FGF-2 5-flanking
DNA. The A/G-rich region corresponds to nucleotides –793/–720, and its
position is based on the major transcription initiation site detected in rat
brain RNA [11]. The two copies of the AGGG-repeat sequence, 5′AGGGAGGG-3′ are boxed.
172
copies of the an AGGG-repeat sequence, 5′-AGGGAGGG3′. This sequence shows a high degree of similarity with a
sequence, 5′-gGGGAGGG-3′, found in the atrial natriuretic
factor (ANF) promoter and implicated in its response to α1specific adrenergic activation by phenylephrine [14]. A 37 bp
double-stranded A/G-rich oligonucleotide, corresponding to
nucleotides –785/–749 of the FGF-2 promoter and containing
both copies of 5′-AGGGAGGG-3′, was synthesized (Fig. 1,
boxed sequence).
An A/G-rich region containing an AGGG-repeat
stimulates heterologous promoter activity in a tissue- and
species-independent manner
To assess any effect of the AGGG-repeat sequences on
promoter activity, the 37 bp A/G-rich oligonucleotide was
inserted upstream of a minimal viral TK gene promoter,
which was fused to the firefly luciferase gene (TKp.luc) to
generate A/G-TKp.luc. Both TKp.luc and A/G-TKp.luc. were
co-transfected with the hybrid Renilla luciferase gene (pRL
CMV) into neonatal rat cardiac myocytes as well as rat C6
and human U87 glial tumor cells, and then tested for activity
after 48 h. The results (firefly luciferase/Renilla luciferase
activity) are shown in Fig. 2A. A significant increase in TK
promoter activity was observed in the presence of the A/Grich sequences in neonatal rat cardiac myocytes (p < 0.005,
n = 6), rat C6 glioma cells (p < 0.01, n = 5) and human U87MG astrocytoma cells (p < 0.05, n = 5). However, the level
of stimulation was greater in rat or human glial tumor cells
Fig. 2. (A) Effect of the 37 bp A/G-rich oligonucleotide on TK promoter
activity (firefly luciferase/Renilla luciferase) in neonatal rat cardiac
myocytes (CM) as well as rat C6 and human U87 glial tumor cells after
transient gene transfer. Results are expressed as the mean from at least two
independent experiments. The bars represent the standard error of the mean.
(B) The results from (A) are presented to show fold effect of the A/G-rich
sequences on TK promoter activity (A/G-TKp.luc/TKp.luc) in the various
cell types.
(~11 fold) versus primary cardiac myocytes (~3 fold; Fig.
2B).
Neonatal rat heart nuclear proteins make high affinity/
specificity interactions with the A/G-rich sequences
The gel mobility shift assay was used to investigate the
presence of specific neonatal rat heart nuclear protein
interactions with the 37 bp A/G-rich oligonucleotide. The
radiolabelled DNA (0.5 ng) was incubated with 5 µg of
neonatal rat heart nuclear protein in the absence or presence
of a 25, 50 or 100 fold molar excess of unlabelled A/G-rich
oligonucleotide. As a further control, an RF-1 DNA element,
containing an unrelated sequence, was also used at a 25, 50
or 100 fold molar excess as a non specific oligonucleotide
competitor. Four specific complexes (C1–4) were identified
(Fig. 3). Both C2 and C4 were competed completely with a
25 fold molar excess of specific (A/G-rich oligonucleotide)
but not non specific (RF-1 element) competitor. A slight
increase in the amount of C1 and C3 complexes was detected
corresponding to the complete competition of C2 and C4 with
a 25 (and to a lesser extent with a 50) fold molar excess of
specific competitor. The C1 and C3 complexes required a 100
fold molar excess of specific A/G-rich oligonucleotide to be
competed efficiently (Fig. 3).
Fig. 3. Gel mobility shift assay of neonatal rat heart nuclear proteins and
the 37 bp A/G-rich oligonucleotide. Specificity was determined by
competition with ‘specific’ unlabelled A/G-rich or ‘non specific’ RF-1
element oligonucleotide competitors. The radiolabelled A/G-rich fragment
(free probe, FP) was incubated in the (a) absence or (b-h) presence of (5
µg) nuclear protein. For competition, (b) none or (c) 25, (d) 50, or (e)100
fold molar excess of specific competitor, or (f) 25, (g) 50, or (h)100 fold
molar excess of non specific competitor was used. The positions/mobilities
of four specific complexes (C1-C4) are indicated.
173
Fig. 4. Detection of very high affinity interactions between neonatal rat
heart nuclear protein and the 37 bp A/G-rich DNA fragment. Affinity was
determined by gel mobility shift assay and competition with low amounts
of ‘specific’ unlabelled A/G-rich oligonucleotide competitor. The radiolabelled A/G-rich fragment (FP) was incubated in the (a) absence or (b-g)
presence of (5 µg) nuclear protein, (b) without or with a (c) 1, (d) 2, (e) 5,
(f) 10, or (g) 15 fold molar excess of specific competitor. The positions/
mobilities of the four specific complexes (C1-C4) are indicated.
Fig. 5. Effect of 0.1 mM phenylephrine treatment for 48 h on TK promoter
activity in the presence (A/G-TKp.luc) or absence (TKp.luc) of the 37 bp
A/G-rich oligonucleotide in transiently transfected neonatal rat cardiac
myocytes. Results are expressed as mean promoter activity (firefly
luciferase/ng protein). Basal levels for –p.luc in the presence and absence
of phenylephrine were 0.078 ± 0.007 and 0.030 ± 0.004, respectively. Bars
represent standard error of the mean.
To assess the relative affinity of nuclear protein for DNA
in the C2 vs. C4 complexes, gel mobility shift assays were
done using lesser amounts (1, 2, 5, 10, and 15 fold molar
excess) of 37 bp A/G-rich oligonucleotide for competition
(Fig. 4). Complex C2 represents a very high affinity/
specificity interaction since it was competed efficiently with
only a 2 fold molar excess of specific A/G-rich oligonucleotide. C4 was competed completely with a 10 fold
molar excess of specific competitor. The transient increase
in the amount of C1 and C3 with competition of C2 and C4
was also apparent.
sequences, respectively. However, although there was an
additional ~1.5 fold increase in activity observed in the
presence of the 5′-AGGGAGGG-3′ sequence with phenylephrine treatment, this was not considered statistically
significant (p = 0. 19).
To complement this study, we compared the gel mobility
shift assay patterns obtained using the 37 bp A/G-rich
oligonucleotide, containing two copies of the 5′-AGGGAGGG3′ sequence, with nuclear protein isolated from neonatal rat
cardiac myocytes grown in the absence versus presence of
0.01 mM norepinephrine. Four complexes (C1–4) were
observed (Fig. 6). This pattern was not altered by norepinephrine stimulation. There was also no difference in the
degree of competition with a 50 or 100 fold molar excess
of specific A/G-rich oligonucleotide, suggesting no change
in affinity of these complexes with adrenergic stimulation
(Fig. 6).
Effect of phenylephrine treatment on the pattern of
interaction between cardiac myocyte nuclear protein and
DNA containing the 5′-AGGGAGGG-3′ sequence
To assess the effect of phenylephrine on TK promoter
activity in the absence or presence of the 5′-AGGGAGGG3′ sequences, neonatal rat cardiac myocytes were transfected
with TKp.luc or A/G-TKp.luc. Transfected cells were treated
without or with 0.1 mM phenylephrine for 48 h, harvested
and firefly luciferase activity per ng protein was determined
(Fig. 5). Phenylephrine treatment increased TK promoter
activity 4.6 ± 0.8 (p < 0.0001, n = 17) and 6.7 ± 1.1 fold (p <
0.008, n = 5) in the absence and presence of the A/G-rich
Discussion
We have used both functional and structural approaches in
the form of transient gene transfer and gel mobility shift
assays to characterise the repeat elements 5′-AGGGAGGG3′ contained in a 37 bp DNA fragment comprised totally of
adenosine (A) or guanosine (G) monophosphates. These
174
Fig. 6. Comparison of gel mobility shift patterns seen with the 37 bp A/
G-rich oligonucleotide and nuclear protein from isolated cardiac myocytes
grown in the absence (b-f) or presence (e-k) of 0.01 mM norepinephrine
for 6 h. Affinity/specificity was assessed by competition with ‘specific’
unlabelled A/G-rich or ‘non specific’ RF-1 oligonucleodde competitors.
The radiolabelled A/G-rich fragment (FP) was incubated in the (a) absence
or (b, g) presence of 2.5 ±g or (c-f, h-k) 5 µg of nuclear protein, (c, h)
without or (d, i) with a 50, or (e, j) 100 fold molar excess of specific
competitor, or (f, k) 100 fold molar excess of non specific competitor.
The positions of four specific complexes (C 1-C4) are indicated.
sequences represent the core of a 74 bp region located at
nucleotide position –793/–720 that is 97% A/G residues,
and of which a 65 bp stretch contains only A or G nucleotides
(Fig. 1). A/G-rich regions have been linked to gene regulation
[14] as well as alterations in DNA structure, specifically
with regard to the human β-globin locus control region [22].
The ability of the A/G-rich oligonucleotide to increase the
activity of a minimal 81 bp viral thymidine kinase promoter
in neonatal rat cardiac myocytes as well as rat C6 and human
U87 glial tumor cells (Fig. 2) suggests that these sequences,
and more specifically the A/G-rich elements, can regulate
gene expression. Furthermore, the stimulation of promoter
activity in cells of different tissues (cardiac myocytes vs.
brain glial cells) or species (rat versus human glial cells)
origin correlates with the ubiquitous pattern of FGF-2
synthesis. FGF-2 has been found in every tissue examined
so far [23, 24] and its structure is highly conserved between
species, such that mouse and human FGF-2 share 94%
sequence similarity at the amino acid level [25]. However,
although the A/G oligonucleotide was able to stimulate
promoter activity in the context of a heterologous promoter
and reporter gene (Fig. 2), a deletion analysis of the rat FGF2 5′-flanking DNA does not support a major role for the
AGGG-repeats, alone, in the regulation of the FGF-2
promoter in cardiac myocytes or glial cells [Detillieux,
Meyers and Cattini, unpublished observations]. Of course,
this does not rule out the possibility that additional elements
and/or factors participate in common complexes with those
associated with the A/G-rich sequences, thereby modifying
their action and, thus, relative importance.
The data obtained from gel mobility shift assays indicate
the presence of multiple protein/DNA interactions. Four
complexes (C1–4) between neonatal rat heart nuclear protein
and the A/G-rich oligonucleotide were identified (Figs 3 and
4). The same pattern of complexes was also suggested when
nuclear protein isolated from neonatal rat cardiac myocytes
was used (Fig 5B), indicating that these interactions are not
only the product of non muscle cell proteins present in the
heart. All four complexes were specific as they were
competed by increasing amounts (25–100 fold molar
excess) of unlabelled A/G-rich oligonucleotide, but not by
equivalent doses of the RF-1 DNA element [21] containing
unrelated sequences (Fig. 3). The complete competition of
C2 and C4 with a 25 fold molar excess indicates that these
complexes possess a higher affinity/specificity than C1 and
C3, which required 100 fold molar excess of specific
oligonucleotide to see efficient competition. Interestingly,
an increase in C1 and C3 was suggested with the complete
competition of C2 and C4 in the presence of a 25 fold molar
excess of A/G-rich oligonucleotide (Fig. 3). The experiment
was repeated with a reduced level of specific competitor
DNA fragment (1–15 fold molar excess) to further dissect
this observation as well as determine the relative affinity of
the C2 and C4 complexes (Fig. 4). C2 and C4 were efficiently competed with a 2 and 10 fold molar excess, respectively, confirming that these are both very high affinity/
specificity interactions (C2 > C4). These results also show
that there is a transient increase in C1 and C3 binding which
corresponds to the competition of C2 and C4. This suggests
the possibility that the protein/DNA interactions reflected
by C2/C4 and C1/C3 are mutually exclusive with a preference,
under the experimental conditions used, for C2/C4 to form.
When the higher affinity C2/C4 events are made invisible by
competition (with a low molar excess of A/G-rich oligonucleotide), there is an increased opportunity for the
proteins associated with C1 and C3 to bind. The ability to
detect all four complexes (C14) simultaneously suggests
that the proteins associated with lower affinity C1 and C3
are present in excess in the neonatal rat heart. This scenario
offers a possible mechanism for regulation of nuclear protein
binding and hence function.
The AGGG-repeat sequence 5′-AGGGAGGG-3′ present
in the FGF-2 gene is related structurally to the sequence 5′gGGGAGGG-3′, which was reported previously to be the
phenylephrine responsive element in the ANF promoter
[14]. Indeed, the sequence 5′AGGGAGGG-3′ exists in the
175
promoter of human α-skeletal actin, and was suggested to
play a role in its phenylephrine responsiveness [14]. A
subsequent investigation demonstrated the requirement of at
least one additional element, resembling a serum response
element, to produce an efficient response of the ANF promoter
to phenylephrine treatment [26]. Although we detected an
additional ~1.5 fold increase in viral thymidine kinase
promoter activity that could be attributed to the presence of
AGGG-repeat sequences after phenylephrine treatment of
transfected neonatal rat cardiac myocytes, this effect was not
statistically significant (Fig. 5). Furthermore, there was no
change in the pattern of DNA interaction with nuclear
proteins (C1–4) isolated from neonatal rat cardiac myocytes
grown in the absence or presence of norepinephrine (Fig. 6).
Thus, it is possible that the duplicated 5′-AGGGAGGG-3′
sequences present in the FGF-2 gene constitute elements
that contribute to phenylephrine regulation of the FGF-2
promoter, but are themselves insufficient for a complete
response.
In summary, examination of sequences upstream of the rat
FGF-2 coding region revealed a 65 bp domain containing
only A or G nucleotides. Located at the core of this region
are two copies of the repeat sequence 5′-AGGGAGGG-3′,
which shows a strong similarity to a phenylephrine response
element. These A/G-rich sequences bind cardiac nuclear
protein with high affinity and are able to confer stimulatory
activity on a viral promoter in a tissue- and species-independent
manner, but do not respond significantly to phenylephrine in
transfected neonatal rat cardiac myocytes.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Acknowledgements
14.
15.
The authors would like to thank Rama Mohan Surabhi for his
careful review of the manuscript. This work was supported
by a grant from the Medical Research Council of Canada
(MT-13398). KAD is the recipient of a Heart and Stroke
Foundation Studentship and AFAM is the recipient of a
Medical Research Council Studentship Award.
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