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INTRODUCTION |
Fibroblast growth factors
(FGFs)1 are an important
family of intercellular signaling molecules involved in early embryonic development and organogenesis (reviewed in Refs. 1 and 2). In
vitro, the FGFs demonstrate a plethora of properties including the
modulation of cell proliferation, differentiation, and cell motility
(reviewed in Refs. 3 and 4). The principle route of signal transduction
is through a secreted FGF ligand that interacts with a high affinity
cell surface receptor causing activation of its cytoplasmic tyrosine
kinase (reviewed in Refs. 5 and 6). Four high affinity FGF receptor
genes (FGFR1-4) have been identified, although alternative
splicing by three of these FGFRs generates a more diverse set of
signaling receptor. Moreover, signal transduction requires the
presentation of FGFs by a second type of receptor, identified as a
heparan sulfate-containing proteoglycan.
We have been particularly interested in FGF3, a ligand first identified
as a proto-oncogene in virally induced mouse mammary cancer but later
shown to be expressed in a complex spatial-temporal pattern during
mouse embryogenesis and fetal development (7-9). In the
pre-implantation conceptus, fgf-3 expression is detected in the parietal endoderm and subsequently in the embryo at a number of
sites, including the primitive streak mesoderm, early hindbrain and
forebrain, cerebellum, sensory cells of the inner ear, pharyngeal pouches, retina, tooth mesenchyme, and tail bud. Although these multiple locations suggest an involvement in several developmental processes, mice deficient for fgf-3 only show skeletal
and inner ear abnormalities reflecting a subset of the known expression sites (10). However, the expression pattern of fgf-3
overlaps with several other FGF family members, which suggests a
potential for functional compensation and a possible masking of shared
roles (reviewed in Ref. 2).
To understand how the complex pattern of fgf-3
transcription is regulated, we have been analyzing the promoter to
identify the transcriptional regulators that control its activity. We
have previously demonstrated that a 1.7-kilobase fragment of genomic DNA, encompassing both the multiple transcription start sites and
sequences upstream of the transcribed region, can act as an inducible
promoter in F9 embryonal carcinoma cells but not in a number of other
common laboratory cell lines tested (11). F9 cells do not normally
express fgf-3 at a significant level until they are
induced to differentiate by the addition of retinoic acid and dibutyryl
cyclic AMP (Bt2cAMP) (12, 13). Treatment with retinoic acid
and Bt2cAMP causes F9 cultures to differentiate into cells
expressing markers of parietal endoderm, the cell lineage in which
fgf-3 is first detected in the conceptus (8).
A number of potential regulatory elements within the 5' proximal region
of the fgf-3 promoter were identified by DNase-1
protection assays using nuclear extracts from F9 cells (11). Functional studies using site-directed mutagenesis showed that three of the DNase-1 protected regions were necessary for full transcriptional activity, whereas two others were suppressors of transcription. Of the
three regions showing enhancer activity, one designated PS4A was found
to be absolutely required for promoter activity. In this paper, we
characterize the PS4A region, demonstrating that it can be divided into
three subregions. The centrally located subregion is essential for
enhancer activity and binds a protein that aids the binding of adjacent
5' and 3' transcription factors. We also demonstrate that within PS4A
the 3' binding protein is Gata-4 and that this transcription factor
confers retinoic acid inducibility of transcription upon the
fgf-3 promoter.
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EXPERIMENTAL PROCEDURES |
Plasmid DNA and Transfections--
The construction of
pfgf-3/CAT was described previously (11). Mutations in
the PS4A region of pfgf-3/CAT were introduced using a
two-step PCR procedure with primers containing a selected deletion or
base substitutions as described (14). pTKLuc was constructed by
inserting 136 nucleotides (
85 to +51) of the HSV TK promoter from
pBLCAT2 (15) upstream of the luciferase gene in pGV-B2 (Nippon Gene,
Tokyo). For derivatives of pTKLuc containing three copies in tandem of
individual or combined elements identified in PS4A, complimentary
synthetic oligonucleotides were designed with 5' blunt and 3'
EcoRI cohesive ends. After annealing the complimentary
oligonucleotides, the resulting duplex DNA was ligated into pTKLuc at
the SmaI and EcoRI sites located upstream of the TK promoter. Sequences of a single copy of each plasmid insert are
presented in Fig. 8. pfgf-3/Luc was constructed by
inserting the 1.7-kilobase fgf-3 fragment of
pfgf-3/CAT into pGV-B2 at the KpnI/XhoI site. A gata-4
expression plasmid, pG4S1, was constructed by cloning a PCR fragment
from a gata-4 mouse cDNA (16) into pCR3 (Invitrogen)
at the EcoRI site. DNA transfection experiments were carried
out in 6-cm dishes using either pG4S1 (6 µg) or pCR3 (6 µg) with
the reporter pfgf-3/Luc (3 µg) and with pRL-CMV (1 µg) (Nippon Gene, Tokyo) as internal control. Procedures for F9 cell
culture, transfections of F9 cells with plasmid DNA, isolation of
stably transfected cells, preparation of cell extracts, and CAT and
luciferase assays have been described previously (11, 12).
Preparation of Nuclear Extracts and Recombinant Gata-4
Proteins--
Nuclear extracts were prepared from undifferentiated F9
cells or differentiated F9 cells after 3 days of treatment with
retinoic acid (10
6 M) and dibutyryl
cyclic-AMP (10
4 M) by the method described
previously (17). Recombinant Gata-4 protein carrying a histidine tag at
the C terminus (HT-GATA-4) was obtained using Bac-to-Bac Baculovirus
Expression Systems (Life Technologies, Inc.). In brief, a
gata-4 mouse cDNA (16) was inserted in the genome of
AcNPV, and the resulting recombinant virus was used to infect
Sf9 cells. HT-GATA-4 was purified from infected cell extracts
using a Ni-nitrilotriacetic acid resin.
Electrophoretic Mobility Shift Assays--
Oligonucleotide
probes were labeled with [
-32P]ATP (5000 Ci/mM, Amersham Pharmacia Biotech) using T4 polynucleotide
kinase (New England BioLabs) and annealed by heating at 80 °C for 10 min followed by slow cooling. Nuclear extract (7 µg) and/or
recombinant HT-GATA-4 protein (about 10 pg) were preincubated with 1 µg of nonspecific competitor poly(dI)·poly(dC) (Amersham Pharmacia
Biotech) in 20-µl reaction mixtures for 15 min at 4 °C. The
reaction buffer consisted of 20 mM HEPES/KOH (pH 7.9), 50 mM KCl, 1 mM dithiothreitol, 2 mM
MgCl2, 1 mM EDTA, 0.1% Triton X-100, and 20%
glycerol. When indicated, double-stranded competitor oligonucleotides
were included. After addition of 25 fmol of 32P-labeled
probe, the reaction was incubated for 15 min at room temperature.
Finally, for supershift experiments, 2 µl of antiserum was added and
the reaction incubated for a further 30 min at room temperature.
Antisera to GATA-4 and GATA-6 were purchased from Santa Cruz
Biotechnology. In some initial experiments (not shown), we used an
antiserum to GATA-4, kindly provided by Dr. David B. Wilson (Washington
School of Medicine, St. Louis, MO) (16). Samples were loaded on 6%
polyacrylamide, 0.5× Tris borate-EDTA gels and run at 200 V for 2 h at 4 °C. Gels were dried and exposed to x-ray films.
Northern Blot Analysis--
Total RNA was isolated from
undifferentiated or differentiated F9 cells using an RNA Extraction Kit
(Amersham Pharmacia Biotech), and poly(A)+ RNA was selected
using mRNA Purification Kit (Amersham Pharmacia Biotech).
Procedures for Northern hybridization were described previously (18). 1 µg of poly(A)+ RNA for each sample was electrophoresed in
1.2% agarose gels and transferred to GeneScreen membrane (NEN Life
Science Products). gata-4 or gata-6
transcripts were detected using appropriately labeled PCR fragments.
These were prepared by RT-PCR using differentiated F9 cell RNA
as template, and the following primers: gata-4,
AACGGAAGCCCAAGAACCTG and CAGGCAGGTGGAGAATAAGG; gata-6,
TTGTCCAGCAGTCCAGATGG and AACACTGATTGCTGCAACGC.
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RESULTS |
PS4A Is a Critical Enhancer Element That Can Bind Multiple
Factors--
A schematic depiction of the proximal
fgf-3 promoter with the previously identified DNase-1
protected sequences (PS) is shown in Fig.
1A. Targeted deletion of these
DNase-1 protected sites showed that the region designated PS4A (located
approximately 60 nucleotides upstream of P1) was essential for
fgf-3 expression in F9 cells (11). To analyze the PS4A
enhancer in more detail, a series of six smaller nonoverlapping
deletions across this element were introduced into the expression
plasmid pfgf-3/CAT, and their effect on CAT expression
tested (Fig. 1, B and C). As transient transfection assays do not satisfactorily reproduce the retinoic acid
induction of the fgf-3 promoter, the effects of the
deletions within PS4A were assessed in stably transfected F9 cells. For these assays, cultures containing more than 100 transfected clones were
pooled to minimize positional effects caused by integration at random
sites within the genome. Two of the deletions designated 4A
c and
4A
d, abolished detectable CAT activity, whereas two others 4A
b
and 4A
f showed substantial reductions. Deletion mutants 4A
a and
4A
e gave values similar to the parental fgf-3
expression vector, indicating that these sequences are not a crucial
part of the enhancer element.

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Fig. 1.
Schematic depiction of DNase-1 protected
regions and the effect of PS4A deletions on fgf-3
promoter activity in undifferentiated and differentiated F9 cells.
A, schematic representation of previously identified DNase-1
protected regions within the proximal fgf-3 promoter.
The striped blocks represent the 5' exons with the position
of the three RNA start site clusters marked as P1,
P2, and P3 (13, 41). DNase-1 protected regions
that have shown enhancer activity in F9 cells are stippled
(PS2, PS4A, and PS5). Regions that
have a negative effect on transcription are shown as black
boxes. B, depiction of expression plasmid
pfgf-3/CAT with the PS4A sequence and position of
specific deletion mutants indicated. C, histogram showing
the CAT activity measured in pools of stably transfected clones
containing the different deletion mutations shown in panel
B. Solid and open bars represent
fgf-3 promoter activity in undifferentiated and
differentiated F9 cells, respectively. CAT activity was corrected for
protein content and average number of integrated plasmid copies.
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To delineate the sequences within these subdomains of PS4A that are
necessary for enhancer activity, base substitutions of one, two, or
three closely located bases were introduced into the expression plasmid
pfgf-3/CAT (Fig.
2A). The CAT activity estimated in pools of F9 cells stably transfected with these base substitution mutants confirmed that regions encompassed by 4A
b, c,
d, and f were necessary to maintain enhancer activity (Fig. 2B). Moreover, the point mutations demonstrated three types
of behavior; (i) mutations 4AxM0 and 4AxM1 showed a reduced activity in
both differentiated and undifferentiated F9 cells, (ii) 4AxM2 and the
mutations of 4Ay completely abolished CAT activity, (iii) whereas 4AzM1
and 4AzM2 showed primarily a loss of induction by retinoic
acid/Bt2cAMP. A consideration of these three types of behavior together with the general principle that 6-8 nucleotides normally define the core binding site of a transcription factor, suggested that three DNA binding proteins might each interact with the
PS4A enhancer. A comparison of the PS4A sequence with known
transcription factor binding sites, however, only revealed a GATA motif
(consensus (A/T)GATA(A/G)) within the 4A
f region.
Although perfect matches to consensus sites of other known transcription factors were not found, matches with a single nucleotide mismatch were identified, and these included AP1 (TGACTCt), and C/EBP
(ATTGt).

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Fig. 2.
The effect of base substitutions in the PS4A
enhancer on fgf-3 promoter activity. A,
schematic depiction of the PS4A base substitutions introduced into
pfgf-3/CAT. B, histogram showing CAT activity
associated with the base substitution shown in panel A. CAT
activity is corrected to cpm/100 µg of protein/single plasmid copy.
Solid and open bars represent
fgf-3 promoter activity in undifferentiated and
differentiated F9 cells, respectively.
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DNA-binding Proteins That Associate with PS4A--
To determine
the number and nature of DNA binding proteins that can specifically
associate with the PS4A enhancer, electrophoretic mobility shift assays
(EMSAs) were employed with extracts prepared from the nuclei of
undifferentiated (uF9NE) and differentiated (dF9NE) F9 cells. Several
retarded bands were clearly resolved with a full-length 4A probe (Fig.
3). The number of bands and the relative
amounts of the retarded bands depended on whether the nuclear extract
was derived from undifferentiated or differentiated F9 cells. The bands
showing the greatest amount of retardation, and designated 1, 2, and 3, were found to be common for nuclear extracts from both cell states and
bound the 4A probe to a similar level. In addition, undifferentiated F9
nuclear extract formed a number of minor complexes (bands 6 and 7). By
contrast, differentiated F9 nuclear extract yielded a major complex(es)
running as a strong, broad band (often resolving as a doublet) and
designated as bands 5 and 6 (Fig. 3). To localize the binding sites of
the different complexes, three overlapping probes designated 4Ax, 4Ay,
and 4Az were used in place of PS4A (Fig. 3). This approach showed that the complexes common to uF9NE and dF9NE (bands 1-3 and two minor undesignated bands below 3) were clearly associated with the 4Ax probe,
whereas the major complexes formed using dF9NE on the full-length probe
bound to 4Ay only at a significantly reduced level. Using probe 4Az, no
bands were observed with uF9NE and only a weak complex (band 4)
migrating at a novel position was observed with dF9NE. These latter
findings suggest that neither 4Ay or 4Az probes could support efficient
formation of the main complex(es) (bands 5 and 6) identified with the
full-length 4A probe and dF9NE.

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Fig. 3.
Analysis of DNA-protein complexes formed on
PS4A. 32P-labeled DNA probes 4A, 4Ax, 4Ay, and 4Az as
indicated were used with nuclear extracts from undifferentiated
(uF9NE) and differentiated (dF9NE) F9 cells. The
major retarded complexes are numbered.
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Identification of Core Binding Sites in PS4A--
To correlate
PS4A enhancer activity with transcription factor binding, an extensive
set of base substituted probes was generated for EMSA that also
included those mutations used for the functional analysis (Figs. 2, 4,
and 5B). Base substitutions in the 4Ax region of PS4A
resulted in a complete or partial loss of bands 1, 2, and 3, which
correlated with reduced promoter activity observed in both
undifferentiated and differentiated F9 cells (compare Figs. 2 and
4). Thus, from the combined point
mutational analyses, the sequence GTGACT(C) was deduced as the binding
domain for the three complexes of the 4Ax region (Figs. 4 and
5B). The crucial nucleotides
of the motif are GTGA because their substitution (probes 4AxM12, M12.1,
M12.2, and M0) resulted in loss of the three complexes. Changing the 3'
T to a G (4AxM13) resulted in a site with an apparent increased binding
of these complexes. In contrast, substitution of the C for A in
4AxM12.3 caused a loss of the triplet complex and the acquisition of a
single more retarded band, which also appeared with probe 4AxM14 but in
this case without loss of the 4Ax associated triplet.

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Fig. 4.
Correlation of DNA-protein complex formation
with specific base mutations in PS4A. Nuclear extracts from
undifferentiated (uF9NE) and differentiated
(dF9NE) F9 cells were used in an EMSA with
32P-labeled probes containing base substitutions identical
to those introduced into the expression plasmid
pfgf-3/CAT (Fig. 2A).
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Fig. 5.
Determination of the sequence motifs
essential for DNA binding proteins associated with the subregions 4Ax
and 4Ay. 32P-labeled PS4A probes containing single
base substitutions (A) were used for EMSA with dF9NE
(B) or both dF9NE and uF9NE (C). Probes
containing the base substitutions that altered protein binding to the
4Ax and 4Ay regions are boxed. Boxes with
solid lines show probe substitutions that are crucial for
complex formation. Base substitutions that only alter complex formation
are boxed with interrupted lines.
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Probes with base substitutions in the subregions 4Ay and 4Az, resulted
in a loss or severe reduction of the major complexes (bands 5 and 6)
unique to dF9NE (with the exception of 4AyM3; Fig. 4). It was
surprising that mutations across two subregions caused the loss of the
same complex(es) because the deletion analysis suggested the presence
of two distinct sites: one located in 4Ay (4A
c and 4A
d deletions)
and the other associated with retinoic acid/Bt2cAMP
inducibility in 4Az (4A
f deletion; Fig. 1). To identify the core
nucleotides in 4Ay necessary for formation of this major complex, a set
of probes containing single base substitutions at alternate nucleotides
across the region was used in a further EMSA (Fig. 5C). From
this experiment, the sequence ATTGT was deduced as the motif necessary
for efficient binding of protein to this region. For example, single
base changes at the first T (4AyM6) or at G (4AyM7) virtually abolished
complexes 5 and 6. Moreover, these base substitutions also reduced
formation of complexes associated with the 4Ax region, suggesting
cooperativity between proteins binding at 4Ax and 4Ay. Probes
containing point mutations immediately 3' of this sequence (4AyM8, M9
and M10) did not affect formation of the major complexes (bands 5 and
6), whereas substitution of the T residue 5' of the motif (4AyM5)
diminished their formation. Mutations affecting the ATTGT motif also
abolish activity of the fgf-3 promoter (compare Figs. 2
and 4).
Complexes associated with the ATTGT motif were also found to be
partially dependent on the integrity of the 3' GATA motif in the 4Az
subregion (Fig. 4). This suggested a cooperativity of binding between
the proteins associated with 4Ay and 4Az, in addition to that found
between 4Ax and 4Ay associated proteins. To establish the GATA motif as
the protein binding site in 4Az, a probe encompassing the 4Ay and 4Az
subregions was used as a target in an EMSA with unlabeled competitor
probes containing single point mutations in and proximal to the GATA
site (Fig. 6A). Competitor
probes in 10-fold excess, containing base substitutions in the G, A, or
T of the GATA-motif (TATC in sense strand), failed to compete for
binding with labeled target probe, whereas an A to C mutation in the
fourth position competed only poorly. However, base substitutions in
the 5' or 3' sequences flanking the four nucleotides TATC were able to
compete for binding as efficiently as a wild-type competitor,
confirming GATA as a crucial element in PS4A.

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Fig. 6.
Confirmation of a GATA binding site in
PS4A. A, EMSA using dF9NE with a
32P-labeled 4A probe in competition with a 10-fold excess
of unlabeled probes containing single base substitutions
(4AGM2-4AGM9). B, Northern blot analysis using
poly(A)+ RNA isolated from undifferentiated
(und) and differentiated (diff) F9 cells
hybridized with probes to fgf-3, gata-4
and gata-6, respectively. fgf-3 RNA is
shown as a marker for the induction of differentiation by retinoic acid
and Bt2cAMP.
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Identification of Gata-4 as a Binding Protein for 4Az--
To
determine whether F9 cells express known GATA proteins that could be
candidates for binding to PS4A, RNA from undifferentiated and
differentiated F9 cells was analyzed for expression of the GATA family
of transcription factors. An initial screen by RT/PCR using primers
unique to six different GATA genes (data not shown), revealed a low
level of gata-1 expression in undifferentiated F9 cells that
was diminished upon retinoic acid/Bt2cAMP differentiation. gata-2 and gata-3 RNA were both present at very
low levels in undifferentiated F9 cells and were slightly induced upon
differentiation. gata-5 was not detected, but
gata-4 and gata-6 were strongly induced in F9 cells following addition of retinoic acid/Bt2cAMP.
The induction of gata-4 and gata-6 mRNA
by retinoic acid/Bt2cAMP was confirmed by Northern analysis
(Fig. 6B). To determine whether Gata-4 and/or Gata-6
contribute to complex formation on PS4A, EMSA analysis was performed in
the presence of an antiserum directed to either GATA-4 or GATA-6 (Fig.
7A). The antiserum to GATA-4
caused a substantial loss of the major complex and the appearance of a
new band with a more severely retarded position. In contrast, the
antiserum to GATA-6 was unable to cause a similar supershift of the
major complex although this antiserum was less efficient at
super-shifting Gata-6 on a probe containing two consensus GATA sites
(Fig. 7A). These results provide strong evidence that the
major complexes formed on PS4A in the presence of dF9NE contain
Gata-4.

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Fig. 7.
Gata-4 forms a complex on PS4A but requires
both an intact GATA and ATTGT site. A, EMSA with a PS4A
probe and uF9NE or dF9NE in the presence of antiserum to GATA-4 or
GATA-6. Arrows indicate complexes formed in the presence of
dF9NE which are further retarded by antiserum to GATA-4 (left of
panel). Shown are control tracks using a probe with two consensus
GATA sites (right of panel). B, EMSA using either
probe 4A or probes with base substitutions that affect binding at one
of the three defined sites (see Figs. 2, 4, and 5) and incubated with
uF9NE and recombinant HT-GATA-4. The position of the complex containing
HT-GATA-4 is marked by an arrow.
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Gata-4 Binding Is Dependent on Another Factor Binding at
ATTGT--
The dependence of Gata-4 binding on the presence of an
unknown factor at the ATTGT motif was further investigated using
purified recombinant Gata-4 protein (HT-GATA-4) in an EMSA (Fig.
7B). Alone, HT-GATA-4 protein was not able to bind the 4A
probe, but in the presence of uF9NE it formed a clear complex with the
expected retardation characteristics. Moreover, probes containing base substitutions in either the ATTGT motif (4AyM7) or GATA site (4AzM1) failed to form a complex, even in the presence of uF9NE. These results
support the idea that in the context of PS4A, Gata-4 binding is
dependent both on the presence of the GATA site and an unknown second
protein that binds at the ATTGT motif.
Both the ATTGT and GATA-binding Proteins Are Necessary for Enhancer
Activity on a Heterologous Promoter--
The function of the three
component binding motifs within the PS4A enhancer were tested
individually and in combination for their ability to activate a minimal
TK promoter (Fig. 8A). For all
constructs, three copies of the elements were appended to the TK
promoter, driving expression of a luciferase marker gene. The TK
promoter alone gave no significant activity in pools of stably
transfected F9 cell clones. However, the complete PS4A enhancer
conferred a 100-fold increase in activity to the promoter in
undifferentiated cells and nearly a 2000-fold enhancement following treatment with retinoic acid/Bt2cAMP, thereby mimicking the
transcription pattern of the endogenous fgf-3 gene.
Individual elements containing binding sites GTGACT(C), ATTGT, or GATA
were not able to activate the promoter, and the combination of the
GTGACT(C) and ATTGT binding sites was also inactive. By contrast, the
ATTGT and GATA motifs together conferred a substantial inducible
activity, which was further increased when the two binding sites were
placed in their normal context. These results are entirely consistent
with the EMSA data showing the pivotal role of the ATTGT binding site
for the formation of a multimeric protein complex on the PS4A enhancer. They also show that the GATA site is necessary for the induction of
transcription by retinoic acid and Bt2cAMP and that the
GTGACTC motif contributes substantially to the full activity of the
complete enhancer.

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Fig. 8.
PS4A acts as a retinoic
acid/BT2cAMP regulated enhancer in conjunction
with a minimal TK promoter. A, activity of the TK
promoter containing individual or pairwise combinations of elements (in
triplicate) identified in the PS4A enhancer. B, activity of
pfgf-3/Luc in undifferentiated and differentiated F9
cells in the presence or absence of a gata-4 expression
plasmid.
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To determine whether the induction of gata-4 expression
in F9 cells is sufficient to account for the stimulation of
fgf-3 transcription upon F9 cell differentiation, the
undifferentiated cells were transfected with a gata-4
expression vector or an empty vector control, and the level of
fgf-3 promoter activity measured (Fig. 8B).
The results show that expression of gata-4 in
undifferentiated F9 cells resulted in a dramatic increase in
fgf-3 promoter activity, whereas it had little effect in
differentiated cells. This would be expected because endogenous Gata-4
is already expressed at high levels in differentiated F9 cells.
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DISCUSSION |
The results presented here clearly demonstrate that the PS4A
enhancer is essential for the transcription of fgf-3 in
F9 cells. Functional and in vitro analyses of PS4A have
delineated three distinct regions in the enhancer, each associated with
a different transcription factor binding site.
Base substitutions in the most 5' motif GTGACT(C) resulted in a
complete or partial loss of the most highly retarded complexes formed
on a PS4A probe in EMSA and a concomitant reduction of enhancer
activity when assayed in both undifferentiated and differentiated F9
cells (Figs. 2, 4, and 5B). However, none of these base
substitutions completely abolished enhancer activity, showing that the
GTGACT(C) binding site is not essential for enhancer function but
nevertheless contributes to its overall activity. The deduced binding
site is only one base different from a consensus AP1 site
(GTGACTCA); however, in vitro prepared AP1
composed of c-Fos and c-Jun did not bind to a 4A probe (data not
shown). The complexes associated with the GTGACT(C) site were not
supershifted by antisera that recognize members of the Jun or Fos
families of proteins.2 The
evidence to date suggests that the protein(s) binding at the GTGACT(C)
site is/are not related to the AP1 family of transcription factors.
Functional and DNA binding analyses revealed ATTGT as the essential
motif for PS4A enhancer activity (Figs. 2, 4, and 5C). Interestingly, probes used in EMSA that encompassed the ATTGT site
alone did not bind complexes associated with this motif as efficiently
as longer probes that included the 3' GATA motif (Fig. 3). Thus a
substantial proportion of the complexes that appeared to be associated
with the ATTGT site were in fact located at the GATA site because
Gata-4 binding depended on an intact ATTGT site (see below).
Furthermore, integrity of the ATTGT motif was also shown to be
necessary for efficient binding at the GTGACT(C) site (bands 1, 2, and 3 in Figs. 4 and 5C). These findings
demonstrate the interdependence of protein binding at the three
sequence motifs. Enhancer reconstitution experiments using a minimal TK
promoter also demonstrated a critical interdependence between the ATTGT and GATA sites for enhancer activity and confirmed the GTGACT(C) site
as contributing to, but not essential for, activity (Fig. 8A). Furthermore, together the ATTGT and GATA motifs were
sufficient to confer retinoic acid- and Bt2cAMP-regulated
activity to the TK promoter.
A strong interaction has been described between Gata-4 and the cardiac
transcription factor Nkx2-5, which are co-expressed in precardiac
cells and show a synergy in activating the ANF promoter (19). However,
there is little similarity between the Nkx2-5 core binding site
(GCAAGTG) and ATTGT (19). Interestingly, in these experiments, a
physical interaction was shown between Gata-4 and Nkx2-5, as well as a
strong specificity for Gata-4 when it was co-expressed with Gata-6 (see below).
In vertebrates, the GATA family presently constitutes six members that,
on the basis of sequence similarity and distribution patterns, can be
divided into two subgroups. gata-1,
gata-2, and gata-3 form one subgroup with
expression restricted primarily to the hematopoietic cell lineages
(20-26). The second subgroup of gata-4,
gata-5, and gata-6 are expressed in an
overlapping pattern in the precardiac mesoderm, heart, gut, and several
other endoderm tissues (16, 19, 27-34). Moreover, the generation of
embryonal stem cells that are Gata-4
/
suggests that Gata-4 and Gata-6 are involved in the early development of the extraembryonic tissues, visceral and parietal endoderm (35, 36).
Mutational analysis of PS4A in the expression vector pfgf-3/CAT showed that the loss of retinoic acid
inducibility was associated with the GATA site (Figs. 1 and 2).
Furthermore, the differentiation of F9 cells by retinoic
acid/Bt2cAMP into parietal endoderm-like cells correlates
with the concomitant induction of gata-4 and
gata-6 RNA as well as fgf-3 ( Refs. 13,
16, and 37, and Fig. 6B). Antiserum to GATA-4, but not
GATA-6, supershifted complexes associated with the GATA/ATTGT sites,
providing good evidence that Gata-4 is the main GATA family member
responsible for the retinoic acid inducibility. However, at present we
cannot exclude a similar role for Gata-6 because the antiserum to
Gata-6 was less effective at supershifting it even on a consensus
binding site (Fig. 7A). Nevertheless, the ability of Gata-4
to bind to the GATA site, and its dependence on an intact ATTGT site,
was confirmed using recombinant Gata-4 protein (Fig. 7B). In
addition, the expression of gata-4 in undifferentiated F9
cells from an introduced cDNA greatly stimulates
fgf-3 promoter activity, whereas there was no similar
effect in differentiated F9 cells that already express endogenous
gata-4 and gata-6 (Fig. 8B).
The increase in gata-4 transcription in differentiating F9
cells mediated by retinoic acid and Bt2cAMP has also been
implicated in the transcriptional regulation of platelet-derived growth
factor
receptor and the J6 serpin genes (38-40). Thus, there is
now good evidence to suggest that the induction of gene expression by
retinoic acid can be mediated directly through the activation of
retinoic acid receptors or indirectly by its ability to induce GATA proteins.