From the Departament de Bioquímica i Biologia
Molecular, Facultat de Biologia, Diagonal 645, Universitat de
Barcelona, Barcelona 08028, Spain, the ¶ Molecular Cardiology
Unit, Laboratory of Cardiovascular Science, Gerontology Research
Center, NIA, National Institutes of Health,
Baltimore, Maryland 21224, and the
Cardiothoracic Surgery,
National Heart and Lung Institute, Imperial College School of Medicine,
Dovehouse St., London SW3 6LY, United Kingdom
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ABSTRACT |
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Glucose constitutes a major fuel for the heart,
and high glucose uptake during fetal development is coincident with the
highest level of expression of the glucose transporter GLUT-1 during
life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the
fetal stage, becomes the main glucose transporter in the adult. Here,
we show that the perinatal expression of GLUT-1 and GLUT-4 glucose
transporters in heart is controlled directly at the level of gene
transcription. Transient transfection assays show that the Facilitative glucose uptake in mammalian cells is mediated by a
family of glucose transporter proteins (GLUT-1 to GLUT-5) (1). The
pattern of expression of these proteins is very complex: GLUT-1 is
found in virtually all tissues and seems to be responsible for the
basal glucose uptake (2), whereas GLUT-4 is mainly expressed in the
peripheral insulin-sensitive tissues (skeletal and cardiac muscle and
brown and white adipose tissue) (1). In insulin-sensitive-tissues,
insulin is able to induce a rapid increase in glucose uptake, and this
effect is due to the recruitment of GLUT-4 glucose transporters from an
intracellular pool to the plasma membrane (3). Insulin-sensitive
tissues express both GLUT-4 and GLUT-1, although GLUT-4 is the major
glucose transporter isoform. Thus, in the rat adipose tissue, 90% of
glucose transporters expressed are GLUT-4 (4), and a similar percentage
has been observed in skeletal muscle (5). A higher relative expression of GLUT-1 has been observed in rat cardiomyocytes, where GLUT-1 expression accounts for 30% of the total glucose transporters (6).
There are several reports (7, 8) indicating that in the fetal heart,
and also in other muscles, glucose consumption is very high during the
late fetal stage and that the response to insulin increases postnatally
(7). Moreover, the glycogen content in fetal heart and other tissues is
higher in near-term fetuses than in the mature rat (9). This may
constitute an adaptive trait that would confer protection against
hypoxic stress in the fetus during delivery (10). Given that glucose
transport is a rate-limiting step for glycolysis (11), it is feasible that maintaining a high level of expression of GLUT-1 would be crucial
for the fetus, because a constitutive high glucose transport rate would
be ensured this way and would help to overcome any hypoxic events that
may arise during birth. Thus, anaerobic metabolism of glucose would
fulfill the ATP demand of the fetus during the hypoxic episode.
Furthermore, the relevance of maintaining appropriate expression levels
of GLUT-1 in vivo has been recently highlighted by Seidner
et al. (12); they attribute the cause of a severe human
brain disorder to the existence of mutations in GLUT-1 gene that reduce the expression of this transporter, which is specially detrimental to the energy metabolism of brain. It has also been pointed
out that an increase in glucose availability may be beneficial for
reducing the stress during cardiac ischemic episodes (13-15).
The expression of GLUT-1 and GLUT-4 glucose transporters is strongly
regulated during the perinatal development of rat heart, skeletal
muscle, and brown adipose tissue. We have described (16) that around
birth and during the first weeks of neonatal life, glucose transporter
expression is characterized by a dramatic change in the accumulations
of GLUT-1 and GLUT-4, both mRNA and protein, in rat heart, in
skeletal muscle, and in brown adipose tissue. Regarding the signals
regulating these processes, we have previously shown (17) that thyroid
hormones have an essential role in the maintenance of the postnatal
induction of GLUT-4 and the repression of GLUT-1 in rat heart. Whether
this effect is direct or not is still unknown. In further studies
performed in the L6E9 skeletal muscle cell line, we have observed that
the differentiation of myoblasts into myotubes is associated with the
repression of the expression of GLUT-1 and the induction of GLUT-4
(18). In these cells, the Materials--
[
Clones prGT3 (which contains the
2572-bp1 EcoRI rat
GLUT-1 cDNA insert) and pSM111 (containing the 2470-bp
EcoRI rat GLUT-4 cDNA insert) were kindly provided by
Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA) (19,
20).
Nuclear Run-on--
The protocol for the isolation of nuclei was
adapted from one previously described (21), although we introduced
several modifications, described below. Nuclei were isolated from
pooled hearts removed from several litters of anesthetized fetuses or after decapitation of neonates and washed in ice-cold saline. 0.25 g of rat ventricular muscle was homogenized in 25 ml of NA buffer (1 mM Tris-Cl, pH 8, 300 mM sucrose (Merck), 2.5 mM magnesium acetate, 3 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.25% (v/v) Triton
X-100, and 40 units of hrRNasin/ml of buffer), with 15 strokes in a
motor-driven Potter-Elvehjem homogenizer in the cold-room. The
homogenate was filtered through four layers of cheesecloth and mixed
with one volume of NB buffer (1 mM Tris-Cl, pH 8, 2.4 M sucrose, 2.5 mM acetate, 3 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1%
(v/v) Triton X-100, and 40 units of rRNasin/ml of buffer). This mixture
was layered onto 12-ml cushions of NB buffer and ultracentrifuged in a
SW28 Beckman rotor at 27,000 rpm for 60 min at 4 °C. The supernatant
and the interphase were discarded, and the nuclei pellet was dislodged with a spatula in 20 ml of NA buffer (without Triton X-100). The nuclear suspension was centrifuged in a SS34 Sorvall rotor for 10 min
at 2000 × g, and the nuclei resuspended in 1.5 ml of
NA buffer (without Triton X-100). Nuclei were counted in a
hemocytometer and pelleted at 4000 rpm for 5 min in a microcentrifuge.
The nuclei pellet was resuspended in Keller storage buffer (22)
containing 200 units of rRNasin/ml, frozen in liquid nitrogen, and
stored in 200-µl aliquots of approximately 2 × 107
nuclei each at
Nuclear run-on reactions were carried out by incubating approximately
2 × 107 heart nuclei in a mixture containing 0.625 mM ATP, 0.312 mM CTP, 0.312 mM GTP,
0.625 µM UTP, 0.5 mCi of 800 Ci/mmol
[
Detection of GLUT-4 and GLUT-1 newly transcribed RNA was carried out by
hybridization of the labeled RNA to membranes onto which the plasmids
containing the cDNAs for GLUT-1 and GLUT-4 (10 µg of each per
slot) had been previously slot-blotted. Hybridization and washes were
performed as described (21). Data are expressed in ppm as described
previously (23, 24)
GLUT-1 CAT Reporter Constructs, Transient Transfection, and CAT
Assays--
Preparation of enriched rat neonatal cardiomyocyte culture
has been described previously (25). Briefly, ventricular myocardium of
1-2-day Sprague-Dawley rat neonates was minced and digested with
collagenase and pancreatin. Isolated cells were collected by
centrifugation, pooled, and then preplated for 30 min on plastic dishes
(Primaria, Becton Dickinson) to remove non-myocytes, which attach
rapidly to plastic. The supernatant, which contains >95% cardiomyocytes as determined by immunohistochemistry with an
anti-sarcomeric actin antibody
(26)2 was used to seed
0.5 × 106 cells/well in 35-mm gelatin-coated plastic
tissue culture dishes in culture medium (10% horse serum, 5% fetal
bovine serum in 4:1 Dulbecco's modified Eagle's medium/199 medium
plus 1% (v/v) antibiotics (10,000 units/ml penicillin G and 10 mg/ml
streptomycin), 2 mM glutamine, 25 mM HEPES, pH
7.4) containing bromodeoxyuridine (100 µM), in order to
stop proliferation of non-myocytic cells.
A series of CAT reporter constructs containing different 5' deletions
of rat GLUT-1 promoter extending to a common 3'-end point at +134 (18)
were transfected into cardiomyocytes (10 µg each). Site-directed
mutagenesis of the
CAT activity was measured by incubating 75 µl of cytoplasmic extract
with 0.1 µCi of [14C]chloramphenicol, 1.3 mM acetyl-CoA, 200 mM Tris-HCl, pH 7.5, for
3.5 h at 37 °C. At the end of the incubation, extraction into ethyl acetate and thin layer chromatography (29) were performed. The
CAT activity was quantitated using an InstantImager (Packard Instrument
Co.). Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclei were
isolated from adult rat ventricular muscle for the preparation of
nuclear extracts essentially as described above (under "Nuclear
Run-on"), although a different homogenization procedure was used,
given the particular features of the tissue. Hearts were removed from
20 male Wistar rats after cervical dislocation and were washed in
ice-cold saline. Atria were removed from the ventricles, and three sets
of ventricular tissue of 4 g each were finely minced with scissors
in 35 ml of NA buffer. The tissue was then homogenized with three
strokes of 12 s each at 3000 rpm in a Polytron homogenizer
(Kynematica, Littau, Switzerland). The homogenate was centrifuged at
2600 × g for 10 min, and the pellets were resuspended
in another 35 ml of fresh NA buffer. The suspension was further
homogenized in a Potter-Elvehjelm homogenizer with eight strokes of the
pestle and then filtered through four layers of cheesecloth. The
filtered homogenate was centrifuged again at 2600 × g
for 10 min. 25 ml of NA buffer supplemented with Triton X-100 was used
in order to resuspend the pellet, and the suspension was centrifuged
again at 2600 × g for 10 min. After this last centrifugation, all three pellets were resuspended in the same 35 ml of
NB buffer (see the protocol of the nuclear run-on), and this volume was
ultracentrifuged as described under "Nuclear Run-on". The nuclei
pellet was saved and processed for the nuclear extraction.
Nuclei from fetal hind limb skeletal muscle were isolated as described
under "Nuclear Run-on." However, 2.2 M sucrose was used
for making NB buffer. As to adult rat skeletal muscle, nuclei were
isolated according to Zahradka et al. (31) and Neufer
et al. (32). Nuclei from L6E9 myoblasts and myotubes were
isolated as described (33).
Nuclear extract preparation and EMSAs were performed as described
previously (18). The Electrophoresis and Immunoblotting--
SDS-polyacrylamide gel
electrophoresis was performed in accordance with the method of Laemmli
(34). Proteins were transferred to Immobilon as reported (35). Transfer
was confirmed by Coomassie Blue staining of the gel after the
electroblot. An anti-Sp1 affinity-purified rabbit polyclonal antibody
(PEP-2, Santa Cruz Biotechnology) was used at a 5 mg/ml dilution in 1%
nonfat dry milk, 0.02% sodium azide in phosphate-buffered saline and
incubated overnight at 4 °C. Detection of the immune complexes with
the rabbit polyclonal antibody was accomplished using the ECL Western
blot detection system (Amersham Pharmacia Biotech). Immunoblots were
performed under conditions in which autoradiographic detection was in
the linear response range.
GLUT-1 and GLUT-4 Transcriptional Activity Is Regulated during
Development--
We have previously shown (16) that both GLUT-4 and
GLUT-1 glucose transporters are strongly regulated during perinatal
development in heart, skeletal muscle, and brown adipose tissue. This
results in changes to both mRNA and protein accumulation. The above
observations led us to consider that the induction of GLUT-4 mRNA
and repression of GLUT-1 during perinatal development may be due to
transcriptional control. To test this hypothesis, we carried out a
series of nuclear run-on experiments. These experiments were performed
on nuclei isolated from fetal and neonatal rat heart, and the
transcriptional rate of both GLUT-1 and GLUT-4
genes was analyzed over a period of time spanning from fetal day 19 to
postnatal day 20. The transcriptional run-on reactions were linear
throughout the experiment (Fig. 1), indicating that the incubation conditions were appropriate during the
reaction, and substrate was not limiting for the elongation of nascent
transcripts. The rate of incorporation of labeled UTP into RNA
inversely correlated with the age of the animals from which nuclei were
prepared. Thus, transcriptional activity was higher in nuclei from
fetal heart than in 15- or 20-day-old neonates. Such a postnatal
decrease of general transcription in rat ventricle agrees with previous
observations (36). Fig. 1B shows an autoradiograph of a
run-on reaction performed with nuclei isolated from 19-day fetal heart.
The signals corresponding to the hybridization of newly transcribed
GLUT-1 and GLUT-4 RNAs were well above that of background (pBluescript
and pGEM), thus indicating that transcription of both genes was active
at this developmental stage.
The data corresponding to the transcriptional activity of
GLUT-1 and GLUT-4 genes during rat heart
perinatal development are shown in Fig.
2. Transcription of GLUT-1 was
maximal in 19-day fetal heart and had decreased by 50% at birth; in
20-day-old neonates, GLUT-1 transcription was lowest and
accounted for <25% of the maximal values (Fig. 2). In contrast,
GLUT-4 transcription increased markedly between the late
fetal stage and 20-day-old neonates (a nearly 4-fold increase).
GLUT-4 transcription levels increased very rapidly after
birth, and in 5-day-old neonates were nearly 2-fold greater than those
in fetal heart nuclei. As a control, total RNA was prepared from hearts
saved from the same litters as the ones used in the nuclear run-on
experiments and used to detect the mRNA levels of GLUT-1 and GLUT-4
glucose transporters (data not shown). Thus, GLUT-1 mRNA levels in
heart were highest in the fetal stage (18-day-old fetuses) but steadily
decreased after birth (1- and 20-day-old rats showed 34 and 10% of the
level in 18-day-old fetal heart, respectively), which is in contrast to
the increase in GLUT-4 mRNA abundance observed over the same period
of time (in 17-day-old fetal heart, expression levels accounted for
only 35% of those observed in 20-day-old rat heart, whereas 5-day-old
rat heart contained mRNA levels similar to those observed in
20-day-old rat heart). Given the similarity observed between the
expression profiles of protein, mRNA, and transcriptional activity
of GLUT-1 and GLUT-4 transporters in rat ventricle during perinatal
development, the regulation of these two genes in development appears
to be regulated chiefly at the level of transcription.
The 99-bp Region Upstream of GLUT-1 Transcription Initiation Site
Is Sufficient to Drive Transcription in Isolated Cardiac
Myocytes--
Our next goal was to identify the
cis-elements that may regulate GLUT-1
transcription and to understand how GLUT-1 expression was repressed
postnatally in heart and skeletal muscle. In a previous report we
showed that the Sp1 Transcription Factor Is Present in Fetal Heart and Muscle
Nuclei Extracts and Binds to a Region Essential for the Function of the
GLUT-1 Promoter--
We have reported previously that Sp1 is able to
trans-activate a chimeric construct containing the rat
GLUT-1 promoter inserted upstream of the CAT reporter gene
in L6E9 cells (18), and it binds to a probe encompassing positions
In order to analyze the functional significance of the Sp1 Levels Are Down-regulated in Adult Cardiac and Skeletal
Muscle--
Additional data were obtained by Western blot carried out
with fetal rat heart, 21-day-old rat heart, and fetal and adult rat
skeletal muscle nuclear extracts. Sp1 protein was detected in these
samples with the same antibody used in the supershift experiments. This
antibody detected a band with an apparent molecular mass of 106 kDa,
which co-migrated with human recombinant Sp1 (Fig.
7A). Notably, both fetal heart
and muscle extracts contained greater levels of Sp1 protein than
20-day-old neonatal heart and adult muscle extracts. In comparison,
21-day-old rat heart extracts only contained 5% of the levels observed
in fetal muscle, and Sp1 protein was barely detected in the adult
skeletal muscle extract under these conditions. These results confirm
that Sp1 levels decrease in both skeletal muscle and heart during
perinatal development. Indeed, strong variations in Sp1 protein
expression in nuclear extracts along with perinatal development have
been previously reported in heart (37), and aortic smooth muscle cells
(38). Moreover, Saffer et al. (39) have reported a wide
variation in Sp1 mRNA levels between tissues in adult mice (in
which they detected extreme differences in Sp1 mRNA levels of up to
100-fold), as well as a decrease in Sp1 mRNA abundance during
postnatal development in most of the tissues examined. We also measured
Sp3 protein levels in fetal and adult heart nuclear extracts with the
antiserum against Sp3. Fetal heart extracts contained high levels of
the two reported Sp3 polypeptides (40), whereas Sp3 expression was barely detectable in adult heart extracts, paralleling the pattern of
expression observed for Sp1(data not shown).
The expression of GLUT-1 and GLUT-4 mRNA levels is regulated
during rat heart perinatal development. Furthermore, GLUT-1 and GLUT-4
mRNA expression in heart correlates with the pattern of protein
expression during perinatal development, which had been reported
previously (16). We show here that these changes in expression
represent direct alterations in the transcriptional activity of
GLUT-1 and GLUT-4 genes, as deduced from nuclear
run-on experiments. We have analyzed the DNA region upstream of the
GLUT-1 transcription initiation site in order to look for
regions responsible for the control of GLUT-1 transcription in cardiac
muscle. To this end, we have performed transient transfection
experiments in rat neonatal cardiomyocytes, with a series of
GLUT-1 promoter-CAT chimeric constructs spanning positions
Using nuclear run on assays, we were able to observe a decrease in the
transcriptional activity of GLUT-1 during the perinatal development, which explains the decrease in the GLUT-1 mRNA and protein levels observed in the same period (16). The changes in GLUT-1
mRNA expression in rat heart are probably due to the regulation of
the expression of this transporter in cardiomyocytes and not in other
cell types, because in situ hybridization experiments reveal
that the expression of GLUT-1 mRNA in the late fetal heart is
higher in the ventricular cardiac muscle than in any of the surrounding
tissue.3 We do not know,
however, whether the levels of transcriptional activity of this gene
were higher in earlier stages of rat heart development prior to
embryonic day 17, because isolation of fetal nuclei from earlier time
represented a technically unaffordable task. However, we cannot discard
this possibility because the fetal period is characterized by a high
glucose consumption rate, which is typical of cells in an active
proliferative state.
Although both GLUT-1 protein and mRNA are still detectable in the
adult rat heart (16), we found that the postnatal transcriptional activity of GLUT-1 was very low. These data suggest
potential posttranscriptional regulatory mechanisms. Such a mechanism
has already been postulated for the Na+/Ca2+
exchanger (24) and SR Ca2+-ATPase (41). The
Na+/Ca2+ exchanger in rat heart is also
down-regulated perinatally, at the levels of both protein and mRNA.
However, even though the protein is still detectable in adult rat heart
samples, the transcriptional rate and mRNA expression of this gene
fall below detection levels soon after birth, suggesting a long
half-life for the Na+/Ca2+ exchanger protein in
the adult rat heart (24). It is unclear whether the half-life of GLUT-1
is also developmentally regulated in rat heart. Nevertheless, the
stability of the GLUT-1 mRNA in L6 myoblasts (42), or of the GLUT-1
protein in 3T3-L1 adipocytes (43), is up-regulated by oxidative stress
and glucose deprivation, respectively. Therefore, it is likely that
posttranscriptional and/or translational regulatory mechanisms are
occurring in addition to the transcriptional regulation we have described.
Heart growth is characterized by a fetal and early neonatal
hyperplastic phase (cell division), which is followed by the withdrawal of rat cardiomyocytes from the cell cycle (a fact that is particularly relevant because in adulthood, dead cardiac myocytes cannot be replaced), and the onset of a hypertrophic phase of growth (increase in
cell mass). Thus, the repression of GLUT-1 occurs when myocardial cells
are still involved in a period of active replication but are starting
to withdraw from the cell cycle. Although cardiac fibers are fully
functional, cardiomyocytes retain the ability to divide during the
hyperplastic growth phase. This is in contrast to skeletal muscle,
where myoblasts withdraw irreversibly form the cell cycle prior to
differentiation (44). In fact, the incorporation of
[3H]thymidine into DNA in heart is much higher in the
late fetal stage than later on (45), and it decreases rapidly after
birth (46, 47), ceasing completely by the 17th day of neonatal life. From this moment on, the growth of heart depends exclusively on the
hypertrophy of preexisting cardiomyocytes (48). It is possible that the
signal regulating the perinatal repression of GLUT-1 and induction of
GLUT-4 is in some way related to the withdrawal from the cell cycle. In
fact, the expression of GLUT-1 is rapidly induced by proliferative
stimuli, such as growth factors (49), and is high in transformed cells
(50). Moreover, in the L6E9 muscle cell line, cells exit the cell cycle
prior to differentiating into myotubes (44), and this is accompanied by
the parallel repression of GLUT-1 and the induction of GLUT-4 (18).
GLUT-4 transcription is mainly induced postnatally, as
deduced from our transcriptional activity data. This transporter is characteristically expressed in differentiated skeletal muscle and
heart. However, the rat heart is already pumping blood as early as in
the ninth day of gestation, and therefore is an organ with a precocious
differentiation. Nevertheless, GLUT-4 transcriptional activation does not occur until the late fetal or early postnatal phase. This fact indicates that, although the differentiation of
cardiac fibers is necessary for the induction of GLUT-4 expression, this may not be sufficient, and other factors may be required in order
to achieve high GLUT-4 expression levels. One candidate may be thyroid
hormone, because the responsiveness to this hormone is elicited at the
end of the fetal stage. We have previously shown (17) the pivotal role
of this hormone in the perinatal induction of GLUT-4. A recent report
(51) presents some evidence for the existence of a low-affinity binding
site in the GLUT-4 promoter, in a region that had been shown
as functionally important for the thyroid hormone-dependent
regulation of GLUT-4 in the skeletal muscle cell line C2C12 (52).
Nevertheless, we cannot exclude the importance of other transcription
factors, such as those facilitating the establishment of the
differentiated muscle-fiber phenotype, in the onset of
GLUT-4 transcription. In keeping with this, it has been
shown that MEF2A and/or C transcription factors are able to bind to the
human GLUT-4 promoter (53), to a site that is functionally
important for the muscle-specific expression of this transporter in the
C2C12 skeletal muscle cell line (54).
As a first step to characterize the transcription factors regulating
the transcriptional activity of GLUT-1, we looked for cis-acting regions that may account for the transcriptional
activity of GLUT-1 in cardiac cells. Our experiments (Fig.
3) show that the 99 bp upstream of the GLUT-1 transcription
initiation site were essential for basal transcriptional activity in
primary cultures of rat neonatal cardiomyocytes. Next, we tested
whether Sp1 transcription factor, which regulates, to a certain extent,
GLUT-1 transcription in the skeletal muscle cell line L6E9
(18), might be responsible for the high level of expression of this
glucose transporter in fetal heart. Results from EMSA experiments
indicate that Sp1 binds to a region in the GLUT-1 promoter
that we have shown by mutational analysis to be essential for
GLUT-1 transcription in neonatal cardiomyocytes. Although
Sp1 factor was initially considered to have a function related to the
maintenance of the basal transcription of many genes, an increasing
number of studies, including ours, demonstrate that Sp1 regulates the
transcription of some genes by changes in binding activity or
expression levels (55, 56), and also through the interaction with
transcription factors, such as Egr-1 (57), MEF2C (58), and Smad family
members (59). In the case of GLUT-1, Sp1 protein levels in fetal heart
and muscle nuclear extracts, together with its binding activity,
correlate with the high levels of expression observed for GLUT-1
glucose transporter in the late fetal stage in both tissues. In a
recent report (60), an indispensable role for Sp1 in the development of
the mouse embryo has been shown, given that Sp1 Our results show that Sp1 expression in heart nuclear extracts is
down-regulated around birth. Little is known about the regulation of
the expression of Sp1. However, in our previous study (24), we showed
that MyoD may be a regulator of Sp1 expression because in cells
overexpressing this myogenic factor, Sp1 levels are dramatically reduced. This suggests the participation of a myogenic factor in the
regulation of the expression of a gene that may, in turn, be
responsible for the down-regulation of GLUT-1 transcription during terminal differentiation of muscle that takes place during perinatal development. However, MyoD cannot be exerting this effect on
Sp1 during heart development, because neither this myogenic factor nor
its relatives myogenin, myf-5, and MRF4 are expressed in this organ,
but only in developing skeletal muscle. We therefore postulate that
another unidentified factor might be partially mediating the GLUT-1
repression in cardiac muscle, through the down-regulation of the levels
of expression of Sp1. Given the parallel between the postnatal
reduction of Sp1 levels of expression in heart and the progressive cell
cycle withdrawal experienced by cardiac myocytes along the same period,
it is intriguing to think that cell cycle regulatory proteins may have
a role in the perinatal down-regulation of Sp1. In keeping with this, a
tight control of the Sp1-mediated activation of transcription by Rb and
other cell cycle regulatory proteins has been shown (61-63). However,
whether Rb or any other protein involved in cell cycle control is able
to regulate the expression of Sp1 itself remains unknown. Sp3, another
member of the Sp family of zinc-finger transcription factors, has been
shown to have opposite roles in the regulation of the transcription of
a number of genes. Thus, Sp3 has been attributed a role as an activator
for the transcription of genes such as SIS/platelet-derived growth
factor-B (64), the neuronal nicotinic acetylcholine receptor 4 subunit
(65), and GLUT-3 (66), but it has also been well
characterized as an inhibitor of Sp1-mediated transcativation in other
genes (40, 67). We have found that Sp3 is more abundant in extracts
from fetal heart than in adult heart and that the Sp3 transcription
factor present in fetal extracts is able to bind to the 99/
33
fragment of the GLUT-1 gene is sufficient to drive
transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the
transcription factor Sp1, a trans-activator of
GLUT-1 promoter, binds to the
102/
82 region of
GLUT-1 promoter during the fetal state but not during
adulthood. Mutation of the Sp1 site in this region demonstrates that
Sp1 is essential for maintaining a high transcriptional activity in
cardiac myocytes. Sp1 is markedly down-regulated both in heart and in
skeletal muscle during neonatal life, suggesting an active role for Sp1
in the regulation of GLUT-1 transcription. In all, these
results indicate that the expression of GLUT-1 and GLUT-4 in heart
during perinatal development is largely controlled at a transcriptional
level by mechanisms that might be related to hyperplasia and that are
independent from the signals that trigger cell hypertrophy in the
developing heart. Furthermore, our results provide the first functional
insight into the mechanisms regulating muscle GLUT-1 gene
expression in a live animal.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
99/
33 region of the GLUT-1 proximal promoter drives transcriptional activity of GLUT-1
and participates in the reduced transcription after muscle
differentiation. Furthermore, we have shown that the Sp1 zinc-finger
transcription factor is able to bind to a putative binding site in
91/
86 of the GLUT-1 promoter. Sp1 is able to
trans-activate the GLUT-1 promoter in L6E9 cells,
and its own expression undergoes down-regulation during muscle cell
differentiation and in response to overexpression of the myogenic basic
helix-loop-helix factor MyoD (18). Here we show that the changes in
GLUT-1 and GLUT-4 expression in rat heart are due, at least in part, to
alterations in the transcriptional rate of both genes. Moreover, the
99/
33 region of the GLUT-1 promoter is also essential
for the transcription of GLUT-1 in rat neonatal
cardiomyocytes in primary culture and that mutation of the Sp1 site at
91/
86 region compromises the transcriptional activity. Furthermore,
binding of Sp1 to this site can be detected in fetal heart and skeletal
muscle nuclear extracts but not in extracts from adult heart and
muscle. We also show that this reduced binding is due to a reduced Sp1
protein abundance in nuclear extracts from heart and muscle in the
adult. Together, these findings suggest that Sp1 contributes to the
high level of expression of GLUT-1 in the fetal heart.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP,
[
-32P]UTP, and [
-32P]ATP were
purchased from ICN, NEN Life Science Products, and Amersham Pharmacia
Biotech, respectively. Hybond N+ was from Amersham Pharmacia Biotech,
and random primed DNA labeling kit was from Roche Molecular
Biochemicals. Immobilon was obtained from Millipore. Most commonly used
chemicals were from Sigma. Dulbecco's modified Eagle's medium, fetal
bovine serum, glutamine, and antibiotics were obtained from Whittaker
(Walkersville, MD). Human recombinant Sp1, a double-stranded
oligonucleotide containing an Sp1 consensus binding site, and rRNasin
were obtained from Promega (Madison, WI).
80 °C until used.
-32P]UTP, 300 units of hrRNasin/ml, 40 mM
Tris-Cl, pH 8.3, 150 mM NH4Cl, and 12.5 mM MgCl2, in a final volume of 400 µl, for 20 min at 27 °C. 2-µl samples were removed from the reactions at 0, 5, 10, and 20 min in order to calculate the incorporation of [
-32P]UTP into RNA. DNA was digested after the
addition of an additional 80 units of hrRNasin to each reaction, with
75 µl of RQ1 RNase-free DNase (Promega) for 20 min at 27 °C, prior
to isolation of RNA. This was isolated by pelleting the nuclei at room
temperature for 2 min at low speed. The supernatant was discarded, and
the nuclei were resuspended in 500 µl of GuSCN solution (4 M guanidinium thiocyanate, 25 mM sodium
citrate, 0.5% N-laurylsarcosyne, 0.1 M
-mercaptoethanol). Next, 50 µl of 2 M sodium acetate
were added to the nuclei, prior to extracting RNA with 500 µl of
water-equilibrated acid phenol and 100 µl of chloroform:isoamyl
alcohol (49:1) solution. RNA was precipitated with 1 volume of
isopropanol and centrifuged for 5 min at full speed. The RNA pellet was
washed in 70% ethanol and resuspended in GuSCN solution, followed by
another precipitation in 100 µl of isopropanol. The final RNA pellet
was resuspended in 100 µl of TE, pH 8, and 2 µl was counted by
liquid scintillation, in order to calculate the activity of the RNA solution.
99/+134 GLUT-1 CAT construct was performed with
the QuickChangeTM kit from Stratagene, according to the manufacturer's
instructions. The oligonucleotide used for generating the mutant
construct was 5'-CCTCAGGCCCCGTACCCCGGCCCACC-3', which
contains a two-nucleotide substitution (underlined) in the core of the
Sp1 site (see below). The test plasmids were co-transfected with 7.5 µg of
-galactosidase expression plasmid pON239 (27) to normalize
for the efficiency of transfection. Cardiomyocytes were transfected on
the day following isolation by a calcium phosphate protocol (25, 28)
and harvested 2-3 days later for CAT assay (29). For the harvesting of
cardiomyocytes and preparation of cytoplasmic extracts, cells were
washed twice in phosphate-buffered saline and then lysed in 300 µl of
reporter lysis buffer (Promega) according to the manufacturer's
instructions. After centrifugation in a microcentrifuge for 5 min at
4 °C, the supernatants were stored at
80 °C.
-Galactosidase activity was measured as described (30). Data
are expressed as percentage of maximum expressing construct and
represent the average of at least three independent rounds of transfection.
102/
37 probe was obtained by digesting with
AvaII the
201/+134 CAT construct, purifying the 66-bp
fragment, and filling in the cohesive ends with the Klenow fragment of
the DNA polymerase and [
32P]dATP. Both a wild type
(wtG1Sp1, 5'-CCTCAGGCCCCGCCCCCCG-3') and a mutated (mutG1Sp1,
5'-CCTCAGGCCCCGTACCCCG-3'; mutation underlined) oligonucleotide encompassing positions
100 to
82 in the rat GLUT-1
proximal promoter were used as competitors in EMSA. When oligonucleotide wtG1Sp1 was used as a probe, 20 pmol of double stranded
oligonucleotide was end-labeled with [
-32P]ATP by
using T4 polynucleotide kinase (Promega), and 10,000 cpm of the probe
was incubated with 5 µg of the corresponding nuclear extracts as
described by Viñals et al. (18). All competitor oligonucleotides were used at 100-fold molar excess. Supershift experiments were performed as described (18).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The transcriptional activity of nuclei
isolated from rat ventricle decreases with development. Nuclei
isolated from pooled hearts of between 18-day-old fetuses and 15- and
20-day-old neonates were used in nuclear run-on reactions. Reactions
were carried out as described under "Experimental Procedures," and
the amount of [32P]UTP incorporated into total RNA was
measured during the course of each reaction. Data are represented as
cpm incorporated per nucleus (A) (F, fetal;
N, neonatal). B shows a representative
autoradiograph of a single run-on experiment, corresponding to a
reaction in which 2.2 × 107 nuclei isolated from
heart of 19-day-old fetuses were used. 2.3 × 106 cpm
of total 32P-labeled RNA were added to the hybridization
solution. The slots corresponding to pGEM4Z and pBluescript plasmids
are included as negative controls and show nonspecific hybridization.
This membrane was exposed to an X-OMAT AR Kodak film for 1 week.
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Fig. 2.
The transcriptional activity of
GLUT-1 and GLUT-4 genes is regulated
during rat heart perinatal development. Run-on reactions were
performed, as described under "Experimental Procedures," on nuclei
isolated from pooled hearts from animals with ages in a range between
embryonic day 19 to neonatal day 20. Results are expressed in ppm
(background value previously subtracted) of the RNA species of interest
relative to newly transcribed total RNA. Data are represented as
mean ± S.E. of 3-8 observations of each sample.
99/
33 fragment in GLUT-1 promoter drives
transcriptional activity of the CAT reporter gene in the L6E9 rat
muscle cell line (18). These findings prompted us to consider that
maybe this region would be also important for the transcription of
GLUT-1 in rat cardiomyocytes. This hypothesis was tested through the
transfection of a series of 5' deletion constructs of GLUT-1
promoter containing various lengths of the proximal and 5' upstream
sequence through to +134 fused to the CAT gene, into primary cultures
of rat neonatal cardiomyocytes prepared from 1-2-day-old neonates
(Fig. 3). These experiments showed that
transcription was maintained to a high level in the constructs
containing deletions from
812 to position
99 of the GLUT-1
promoter, but deletion of the region between
99 and
33 relative to
GLUT-1 transcription initiation site resulted in an 80%
decrease of CAT transcriptional activity relative to the
99/
33 CAT
construct. As observed for the skeletal muscle cell lines, this region,
which contains putative binding sites for known transcription factors,
such as Sp1, AP2, and a CAAT box, (18) appears to be crucial for
maintaining basal transcription in rat neonatal cardiomyocytes.
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Fig. 3.
Expression of GLUT-1
promoter 5' deletion constructs in rat cardiac myocytes. A
series of 5' deletions of the upstream region of the GLUT-1
gene from 812 to
33 bp relative to the transcription start site
were generated as indicated under "Experimental Procedures." Rat
neonatal cardiomyocytes were transiently transfected with the indicated
constructs and cells were harvested 72 h after transfection. The
data are expressed as relative CAT activity/
-galactosidase activity
±S.E. from three experiments, performed in duplicate, using at least
two preparations of DNA, with the
812/+134 CAT construct being set to
a value of 100.
102 to
37 of GLUT-1 promoter. Given the drop in basal
transcription of the GLUT-1 promoter in cardiomyocytes when
5' sequence is deleted from
99 to
33, we used the
102/
37
fragment of GLUT-1 promoter as a probe to determine whether
Sp1 protein was present in nuclear extracts prepared from fetal or
adult rat heart and skeletal muscle and able to bind to the
91/
86
Sp1 binding site in the GLUT-1 promoter, previously shown to
be functionally relevant in skeletal muscle cells (18). EMSA
experiments showed that both fetal heart and muscle nuclei extracts
generated a series of protein-DNA complexes when incubated with the
102/
37 probe (Fig. 4A).
Most of these bands are not present in adult heart and skeletal muscle
nuclear extracts. The arrows in Fig. 4A indicate
a band with a slow mobility, which is specific to fetal heart and
skeletal muscle, and also present in L6E9 myoblast nuclear extracts.
This band in the L6E9 myoblast nuclei extracts was shown in our
previous report to be due to the binding of Sp1 factor (18) and
disappears with differentiation of L6E9 myoblasts into myotubes.
Therefore, fetal heart and muscle extracts, but not their adult
counterparts, may exhibit binding of Sp1 to the GLUT-1 core
promoter. To ascertain whether that band in fetal heart and skeletal
muscle extracts corresponds to the Sp1 transcription factor, we
performed competition EMSA experiments by using a commercial
oligonucleotide containing a consensus binding site for Sp1. This
oligonucleotide was able to compete with the
102/
37 probe for the
binding of the protein producing the slow-migrating band, indicating
that Sp1 protein may be responsible for this DNA-protein complex (Fig.
4B). In order to provide more direct evidence that the Sp1
transcription factor present in fetal heart and skeletal muscle nuclear
extracts binds to this site in GLUT-1 promoter, we used a
polyclonal antibody against human Sp1 in supershift EMSA (according to
the supplier, this antibody cross-reacts with rat Sp1 and was therefore
appropriate for these studies). When an oligonucleotide encompassing
positions
102 to
82 in GLUT-1 promoter was radiolabeled and used as
a probe, it proved to be able to bind human recombinant Sp1 protein
(hrSp1). This protein was recognized specifically by the anti-Sp1
antibody, which produced a decrease in the apparent mobility of the
complex formed by this factor bound to the probe (Fig.
5, left panel). This
supershift was specific, because an irrelevant antibody (IgG) had no
effect on the mobility of the complex (Fig. 5, left panel).
Fetal heart and L6E9 myoblast nuclear extracts were incubated with the
same probe and produced several bands (arrows), one of which
had the same relative mobility as the one produced by hrSp1 (Fig. 5,
right panel). This band could also be supershifted by the
anti-Sp1 antibody, and this supershift was observed both in fetal heart
and in L6E9 myoblasts and was specific because the irrelevant antibody
was not able to produce them. These data show that Sp1 is present in
fetal heart nuclear extracts and can bind to the Sp1 site located at
91/
86 in the GLUT-1 promoter. We performed other supershift experiments with an antiserum against Sp3 (gift of Dr. Guntram Suske,
Phillips-Universität, Marburg, Germany) that showed this other
member of the Sp family of transcription factors was present in the
fetal heart nuclear extracts and able to bind to the same site in
GLUT-1 promoter (data not shown).
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Fig. 4.
The 102/
37 probe and a Sp1 consensus
oligonucleotide compete for the binding of a specific protein present
in fetal heart and muscle nuclear extracts. A, 5 µg
of protein nuclear extract from fetal (F) and adult
(A) heart or skeletal muscle, as well as from L6E9 myoblast
(Mb) and myotube (Mt) nuclear extracts, were
incubated as described under "Experimental Procedures" with 10,000 cpm of the radiolabeled
102/
37 fragment of GLUT-1
promoter. Samples were loaded on a nondenaturing 7% polyacrylamide gel
and run at 4 °C and 325 V for 90 min. The gel was then dried and
exposed to an AGFA CURIX film. Arrows point to a specific
band obtained after incubation with nuclear extracts from fetal heart,
fetal muscle, and L6E9 myoblasts, which was attributed to the binding
of Sp1 in L6E9 myoblasts extracts. B, EMSA experiments were
performed by incubating 5 µg of protein from fetal heart or muscle
nuclear extracts with 10,000 cpm of the radiolabeled GLUT-1 promoter
102/
37 probe. Arrows point to a specific band obtained
after incubation with fetal heart or fetal muscle extracts that was
prevented when the Sp1 consensus oligonucleotide was included as a
competitor in some of the reactions (100-fold molar excess).
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Fig. 5.
The Sp1 transcription factor present in
the nuclear extracts from fetal heart binds to the Sp1 site.
Supershift experiments were carried out by incubating 5 µg of protein
of fetal heart nuclear extract or L6E9 myoblast (Mb) extract
with 10,000 cpm of the radiolabeled oligonucleotide wtG1Sp1 ( 102/
82
fragment of GLUT-1 promoter), which contains the putative
Sp1 site present in this region (right panel). One µl of a
commercial solution of human recombinant Sp1 transcription factor
(hrSp1) was also used in the supershift experiments as a control
(left panel). Sp1 and IgG antibodies
stand for a polyclonal antibody raised against human Sp1 and MANRX, a
polyclonal antibody raised against rat rBAT amino acid transporter,
respectively. The latter was used as an irrelevant antibody in these
experiments (negative control). Arrows point to the bands
specifically displaced by the addition of the antibody against
Sp1.
91/
86 Sp1
site in GLUT-1 promoter, we tested the effect that a point mutation introduced into the
91/
86 Sp1 site would have in
(a) the binding of Sp1 to the GLUT-1 promoter,
and (b) the transcriptional activity of the
99/+134
GLUT-1 CAT construct transfected into rat neonatal
cardiomyocytes. First, a mutant version of the oligonucleotide encompassing positions
102 to
82 in GLUT-1 promoter was used in
competition EMSA experiments. The ability of this oligonucleotide to
compete for the binding of Sp1 to the
102/
37 probe was compared with that of the wild type oligonucleotide (Fig.
6A). Thus, whereas the wild
type competitor (wtG1Sp1) was able to compete with the probe, a
two-nucleotide substitution in the
91/
86 site (GC
TA) abolished
the ability of the oligonucleotide to compete in EMSA (Fig. 6A,
mutG1Sp1). The same two-nucleotide substitution was introduced
into the core of the Sp1 site in the
99/+134 GLUT-1 CAT
construct, and the transcriptional activity of this DNA construct was
then compared with that of the wild type after transfection into rat
neonatal cardiomyocytes. Fig. 6B shows that the mutation induced a reduction of transcriptional activity of the
99/+134 CAT
construct to a value that was 38% of the activity observed in the wild
type. Interestingly, this reduced level of activity is similar to that
of the
33/+134 CAT construct when compared with the activity of the
99/+134 CAT construct (the activity of the mutant
99/+134 CAT
construct accounts for 75% of the drop observed when the sequence
between
99 and
33 is deleted). These data indicate the functional
significance of this site in the maintenance of the transcriptional
activity of GLUT-1 in neonatal cardiomyocytes.
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Fig. 6.
Disruption of the Sp1 binding site in
GLUT-1 promoter compromises the binding of Sp1 and the
transcription of the 99/+134 GLUT-1 CAT construct in cardiac
myocytes. A, 5 µg of protein nuclear extract from
fetal heart were incubated as described under "Experimental
Procedures" with 10,000 cpm of the radiolabeled
102/
37 fragment
of GLUT-1 promoter. A wild type (wtG1Sp1) and a mutated
version (mutG1Sp1) of an oligonucleotide containing the
91/
86 Sp1
site in GLUT-1 promoter were included as competitors
(100-fold molar excess). The arrow points to a band that was
competed only by the wtG1Sp1 oligonucleotide and not by the mutant
version, in which the consensus Sp1 site was disrupted. B,
either a wild type or a mutated version of
99/+134 GLUT-1 CAT were
transiently transfected into rat neonatal cardiomyocytes as described
in Fig. 3 and under "Experimental Procedures." The sequence at the
Sp1 site is shown for both the wild type and the mutant construct. Note
that the same point mutation was included in the mutant competitor
oligonucleotide (mutG1Sp1) shown in A. The data are
expressed as relative CAT activity/
-galactosidase activity ± S.E. from four experiments, with the wild type
99/+134 CAT construct
being set to a value of 100.
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Fig. 7.
High abundance of Sp1 transcription factor in
fetal heart and muscle nuclear extracts. Twenty-five µg of
protein from nuclear extracts obtained from fetal heart and muscle,
20-day-old neonatal heart, and adult skeletal muscle were loaded
together with a 1:5 dilution of hrSp1 (human recombinant Sp1) on a
7.5% acrylamide minigel, and after electrotransfer onto an Immobilon
membrane, Sp1 protein was detected by using PEP2 polyclonal antibody
against human Sp1 factor (see under "Experimental Procedures").
Immunoblots were autoradiographed and subjected to scanning
densitometry in order to quantify the relative amount of Sp1 in these
samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
812 to
33 relative to GLUT-1 transcripition initiation
site. These experiments showed that the 99-bp region upstream of
GLUT-1 transcription site are essential for the
transcription of GLUT-1 in rat cardiomyocytes. We also
showed by electrophoretic mobility shift analysis that the zinc finger
transcription factor Sp1 is present in fetal heart and skeletal muscle
nuclear extracts and can bind to a site present in the
102/
37
fragment of GLUT-1 promoter. This was further demonstrated
by supershift experiments, using an oligonucleotide encompassing the
Sp1 site located at
91/
86 and an antibody raised against rat Sp1.
Furthermore, disruption of the Sp1 site produced a remarkable decrease
in the transcriptional activity of a GLUT-1 promoter-driven
CAT construct in neonatal cardiomyocytes, showing a functionally
essential role for Sp1 in the transcription of GLUT-1 in
those cells. Finally, we have observed that the levels of expression of
Sp1 in nuclear extracts from heart or muscle in the fetus are higher
than those in the adulthood. Together, these data indicate that the
expression of GLUT-1 and GLUT-4 in the developing heart is largely
regulated at a transcriptional level and that Sp1 plays a pivotal role
in the regulation of the expression of GLUT-1 in heart and muscle.
/
homozygous embryos die in utero at about day 11 of
gestation. These embryos show a delayed development although the
absence of Sp1 did not impair the early steps on the formation of
heart, neither of other organs. However, the early death of these
embryos prevents further study about the effects of such a mutation in the perinatal expression of GLUT-1 in heart.
91/
86 Sp1
site in GLUT-1 promoter. Therefore, Sp3, which is subject to a strong
regulation during heart development, may have a role in the regulation
of GLUT-1 transcription in heart. In keeping with this, unpublished data from our laboratory,4
show that, in the L6E9 myoblasts, Sp3 acts as a negative regulator of
GLUT-1 transcription. Moreover, despite both Sp1 and Sp3 being down-regulated in L6E9 myoblasts along differentiation, alterations in
the relative Sp1/Sp3 ratio at early stages of differentiation (through
a temporary increase in Sp3 abundance) may trigger the down-regulation
of GLUT-1 observed in those conditions. However, investigating whether
a similar mechanism operates during the down-regulation of GLUT-1
expression in heart perinatal development and in parallel to the exit
of the cardiac myocytes from the cell cycle lies beyond the scope of
the present study. The identification of the nature of the signals
responsible for the triggering of the perinatal repression of Sp1 and
Sp3 in rat heart constitutes an important issue and will be addressed
in future studies.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Penny S. Thomas and Paul Burton from the National Heart and Lung Institute (London, United Kingdom) for sharing unpublished observations.
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FOOTNOTES |
---|
* This work was supported in part by Research Project PB95/0971 from the Dirección General de Investigación Científica y Técnica (to A. Z.), Grant GRQ94-1040 from Generalitat de Catalunya, (to A. Z.) Spain, The British Council-Acciones Integradas Grants 1398, HB94-117, and HB96-0138 (to A. Z., K. R. B., and N. J. B.), and British Heart Foundation PG/93148 (to K. R. B.) and by professor Magdi H. Yacoub.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.
§ Recipients of predoctoral fellowships from the Ministerio de Educación y Ciencia, Spain.
** Recipient of a predoctoral fellowship from the Generalitat de Catalunya.
To whom correspondence should be addressed. Tel.: 34-934021519;
Fax: 34-934021559; E-mail: azorzano{at}porthos.bio.ub.es.
2 P. Burton, personal communication.
3 P. Thomas, personal communication.
4 C. Fandos, M. Sánchez-Feutrie, T. Santalucía, F. Viñals, P. Kaliman, J. Canicio, M. Palacín, and A. Zorzano, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); hrSp1, human recombinant Sp1; EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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