From the Department of Internal Medicine and Comprehensive Cancer
Center, Ohio State University, Columbus, Ohio 43210
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INTRODUCTION |
A family of proteins that bind to a consensus DNA sequence CANNTG,
also known as E-box, has been identified. E-box-binding proteins share
a common amino acid sequence motif that is proposed to form two
amphipathic helices interrupted by a loop, designated the
helix-loop-helix (HLH)1
motif. The HLH domain of these proteins mediates the dimerization, and
a basic region located N-terminal of the HLH domain is responsible for
DNA binding (1). HLH proteins are mainly of two types, ubiquitous and
tissue-specific. The interplay of these HLH proteins is
particularly interesting because it plays a significant role in
the regulation of tissue-specific gene expression (1).
Fibroblast growth factor (FGF)-1 is a prototype member of the
structurally related FGF family, which comprises 14 proteins (2, 3). It
is found primarily in brain and retina, although low levels of its
mRNA have been demonstrated in other tissues (4-6). However, the
role of FGF-1 in brain is not yet well understood. By in
situ hybridization and immunohistochemical analysis, it has been
shown that the expression of this growth factor in brain is exclusively
in neuronal cells but not in glial cells (7-9). It was also shown that
the expression of FGF-1 mRNA in neurons is correlated with specific
developmental events (10) and that FGF-1 coexists with tyrosine
hydroxylase in neuronal cells (11). Importantly, transcription of
tyrosine hydroxylase, a key enzyme for catecholamine synthesis,
requires both FGF-1 and an activator (12). FGF-1 mRNA and protein
levels were also reported to elevate in lesioned rat brain (13, 14).
Interestingly, FGF-1 mRNA expression was reported to increase
significantly in glioblastoma, the major human intracranial tumor (5,
15). However, the regulatory mechanism of FGF-1 gene expression is only
beginning to be understood.
The study of tissue- and cell-specific distribution of FGF-1
transcripts has led to the identification of four different transcripts having the same protein coding exons but different 5'-untranslated exons (16-20). FGF-1.B is the predominant transcript in brain, gliomas, and some glioblastoma cell lines (e.g. U1240MG and
U251MG) (5, 21). We have identified a 23-bp cis-element
(
489 to
467) in FGF-1.B promoter that binds to a 37-kDa protein,
p37brn, and this binding is linked to the enhanced functional
activity of the promoter (22). Here we screened a human brain stem
cDNA expression library for DNA-binding proteins using a labeled
oligonucleotide probe made of four tandem repeats of the sequence
492
to
467. We have isolated and expressed three overlapping cDNA
clones. All three clones represent a splice variant of the E2-2 gene
product (23, 24), and the encoded protein binds to an imperfect E-box present in the probe. We further showed that overexpression of an E2-2
variant lacking RSRS represses the transcriptional activities of both
the FGF-1.B promoter and the E47 reporter containing a hexameric E-box
site.
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MATERIALS AND METHODS |
Screening of the cDNA Library--
A
gt11 expression
library, made from human brain stem as described previously (16), was
screened for expression of DNA-binding proteins using the protocol
described by Singh et al. (25). A
SpeI-BamHI fragment with four tandem repeats of
the sequence
492 to
467 (in the head-to-tail orientation) was
labeled with [
-32P]dATP by the Klenow reaction and
used as a probe. Three positive clones, VL1, VL2, and VL3, were
plaque-purified using the same DNA probe.
Sequencing Analysis--
Standard procedures (26) were used to
purify the phage DNA, and the cDNA inserts from the phage clones
were subcloned into the EcoRI site of pBluescript II KS(+).
The cDNA clones were sequenced using Sequenase (U.S.
Biochemical Corp.). DNA sequence alignment was performed using DNASTAR
software (Madison, WI).
Northern Blot Analysis--
The VL2 cDNA was used as a probe
to hybridize to human multiple-tissue Northern blot (BIOS
Laboratories). The Northern blot contained 20 µg of total RNA
extracted from different human tissues. The blot was subjected to
Northern blot analysis using standard procedures (26). Hybridizations
were performed in a buffer containing 50% formamide at 42 °C for
16 h. The final washes were done in 0.1× SSC at 50 °C for 30 min.
Production and Purification of GST-fusion Proteins and
Antibodies--
The cDNA fragments of 1272, 1037, and 700 bp
obtained by EcoRI digestion of VL2, VL3, and VL1 phage
clones, respectively, were blunt ended and inserted, in frame with the
glutathione S-transferase gene, into blunt ended
AvaI site of pGEX-2T (Amersham Pharmacia Biotech). The
proper orientation of the insert was confirmed by restriction
digestion. The GST-fusion proteins were expressed and purified from
Escherichia coli (BL21, pLysS) using glutathione-Sepharose beads (Amersham Pharmacia Biotech) as described by Smith and Johnson (27). The purified GST-VL2 protein was used as the antigen for antibody
production in rabbits (Cocalico Biologicals, Reamstown, PA). The
antibodies were purified by protein A-Sepharose (Amersham Pharmacia
Biotech) affinity column as described by Harlow and Lane (28).
Anti-E2-2 and anti-E2A antibodies were purchased from Pharmingen.
Western Blot Analysis--
Purified GST fusion proteins of the
spliced E2-2 gene products (GST-VL1, GST-VL2, and GST-VL3) and Nalm6
nuclear extract were subjected to electrophoresis in 10%
SDS-polyacrylamide gel. For immunodetection, proteins were transferred
to nitrocellulose membrane and blocked by 3% nonfat dry milk in
phosphate-buffered saline (pH 7.2) containing 0.1% Nonidet 40 (Nonidet
P-40). The membranes were probed with either monoclonal anti E2-2
antibody or polyclonal anti-GST-VL2 antibody at 1:1000 dilution and
washed three times (each for 15 min) with the blocking solution. The
blots were then incubated with either horseradish peroxidase-conjugated
anti-mouse IgG (for the monoclonal anti-E2-2 antibody) or horseradish
peroxidase-conjugated anti-rabbit IgG (for the polyclonal anti-GST-VL2
antibody) at a dilution of 1:1000. Finally, the immunodetection was
carried out by autoradiography using ECL kits (Amersham Pharmacia
Biotech).
Electrophoretic Mobility Shift Assay (EMSA)--
The 26-bp
(
492 to
467) double-stranded oligonucleotide was end-labeled
with [
-32P]dATP using the Klenow enzyme. The
sequence was described elsewhere (29). The binding reaction contained
the binding buffer (25 mM Hepes, pH 7.9, 1 mM
MgCl2, 0.5 mM dithiothreitol, 40 mM
KCl, 5% glycerol, and 0.1% Nonidet P-40) with radiolabeled DNA probe (30,000 cpm), 0.2 µg of poly(dI-dC), the GST fusion protein (0.3 µg), and a cold competitor, when desired, in a total volume of 20 µl. The brain nuclear extract (2 µg) was also assayed in EMSA in
the presence of 0.1 µg of GST or GST-VL2 under similar conditions except that 0.5 µg of poly(dI-dC) was used. Following 30 min of incubation at room temperature, the reaction mixture, when indicated, was further incubated with antibody for 1 h at 0 °C. The
reaction mix (12 µl) was analyzed in a 4% polyacrylamide gel in
0.25× TBE (22.25 mM Tris borate, 0.5 mM EDTA,
pH 8.0). Following electrophoresis, the gel was dried and
autoradiographed. The human brain nuclear extract was made in
accordance with the method described by Gorski et al.
(30).
Methylation Interference Assay--
The
HindIII-EcoRI fragment from plasmid pFGF-1.B
492/
467 (22) was 3'-end-labeled at HindIII site
(antisense strand), partially methylated with dimethyl sulfate, and
used as a probe in binding reactions containing GST-VL2. DNA-protein
complexes were separated from the free DNA by EMSA, eluted from the
gel, cleaved with piperidine, and then analyzed in 8% sequencing gel
as described (22).
Plasmid Constructs--
VL3-FL is a full-length E2-2 expression
plasmid that was constructed by replacing, in frame, the
EcoRV-EcoRI fragment of pCMV2961 (SEF2-1B) (24)
with the EcoRV-EcoRI fragment from VL3. This replacement allows the resulting construct to encode full-length E2-2/SEF2-1 with four additional amino acids (RSRS) as shown in Fig.
1. The constructs SEF2-1B, pCMV
CAT, and E47 luciferase reporter (31) were kindly provided by Drs. Brit Corneliussen and Thomas Grundström. SEF2-1B, previously designated as pCMV2961, was
cloned in the expression vector pCMV
CAT (24). E47/pCSA, an
expression vector for the bHLH protein E47 (32), was provided by Dr.
Andrew Lassar (Harvard Medical School, Boston). Plasmid pRL-tk
(Promega) contains the sea pansy Renilla luciferase gene
driven by the herpes simplex virus thymidine kinase promoter.
Transfection Assay--
The transfection protocols were as
described previously (22). Briefly, U1240MG cells were plated in 60-mm
Falcon tissue culture dishes (Becton Dickinson Labware) to achieve
60-80% confluence by day 2. On day 2, cells were transfected with the
reporter and expression plasmid DNA using the cationic lipid
transfection reagent, DOTAP (Boehringer Mannheim). Plasmid DNA was
prepared using the Qiagen plasmid kit (Qiagen Inc., Chatsworth, CA). In
each experiment, all constructs were transfected in triplicate. The
results show the mean of at least two separate experiments. The
luciferase activity was normalized by the protein contents as described
(22) or by the Renilla luciferase activity using the
Dual-luciferase Reporter Assay system (Promega) as described by the
manufacturer's protocol.
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RESULTS |
Cloning of VL1, VL2, and VL3 from Human Brain Stem cDNA
Library--
Using both functional and biochemical analyses, we
previously identified a 26-bp region (
492 to
467) within RR2 of the
brain-specific FGF-1.B promoter (22, 29). The 26-bp sequence was
concatenated, and a probe with four tandem repeats was used to screen a
human brain stem cDNA expression library constructed in
gt11
(16). Two positive clones (VL1 and VL2) were isolated during the first screening, and an additional clone (VL3) was isolated during the second
screening. The cDNA inserts from three phage clones were subcloned
into pBluescript II KS(+). Sequence analysis showed that the three
clones have an identical sequence with E2-2, also known as SEF2-1 and
ITF-2, a basic HLH (bHLH) protein which was previously isolated using
E-box probes (23, 24). Both E2-2 (23) and one of the four SEF2-1 (24)
cDNAs lack four amino acids (RSRS) just upstream of the bHLH
region. Using sequence analysis and restriction digestion, we showed
that all three of our cDNA clones contain the RSRS sequence (Fig.
1). The difference most likely resulted
from alternative splicing of the same E2-2/SEF2-1 gene (23, 24).

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Fig. 1.
Cloning of VL1, VL2, and VL3 and alignment of
each cDNA with human E2-2/SEF2-1 cDNA. A
schematic diagram of human E2-2 cDNA clone,
SEF2-1B (GenBankTM/EMBL accession number M74719) is shown.
The relative positions of VL1, VL2, and VL3 cDNA are delineated by
the vertical bars. The common restriction sites,
NcoI, EcoRV, and AvaI, in each
cDNA are aligned and are indicated by the arrows. The
open box represents the open reading frame, and
the hatched box represents the bHLH domain. The
four amino acids RSRS, which are absent in both E2-2 (23) and SEF2-1B
(24) cDNA clones, are indicated by open
triangles. The cDNA sizes and the predicted sizes of the
cDNA-encoded and the GST fusion proteins for VL1, VL2, and VL3 are
indicated next to the diagram.
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E2-2/SEF2-1 Is Abundantly Expressed in Human Brain--
To
understand the tissue distribution of E2-2 mRNA, we hybridized
Northern blot containing an equal amount of RNA from different human
tissues with VL2 cDNA probe. As shown in Fig.
2, the 7.5-kilobase pair human E2-2
transcript is detected in brain (lane 1), lung (lane 4), and to a lesser extent spleen
(lane 6). The expression of the 7.5-kilobase
transcript in other tissues (e.g. liver, heart, ovary,
skeletal muscle, and duodenum) was below the detectable level. It is
also noted that the expression of E2-2 mRNA is relatively higher
in the brain.

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Fig. 2.
Northern blot analysis of E2-2/SEF2-1
expression in human tissues. Each lane contains an
equal amount (20 µg) of total RNA from different human tissues as
indicated at the top. The 28 and 18 S RNA controls are shown
by the arrowheads in the left. The
arrow on the right indicates the 7.5-kilobase
transcript of E2-2/SEF2-1.
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The Protein Encoded by the VL cDNA Immunologically Reacts with
Anti-E2-2 Antibody--
In order to characterize the protein encoded
by our cDNA clones (VL1, VL2, and VL3), we expressed these
cDNAs as GST fusion proteins and raised antibody in rabbit against
one of the fusion proteins (GST-VL2) (Fig.
3). The fusion proteins were then tested for the immunoreactivity with the commercially available E2-2 antibody
or anti-GST-VL2 antibody. In the same assay, we also included nuclear
extract from Nalm6 (pre-B) cell line, which is known to express E2-2
protein (32). It is evident from the results that anti-E2-2 antibody
recognized GST-VL3 (lane 3) and GST-VL2 (lane 4), but it did not cross-react with GST
(lane 1) nor with GST-VL1 (lane
2). Multiple protein bands were observed to cross-react with
anti-E2-2 antibody in the case of GST-VL3 or GST-VL2. This could be
due to degradation of the fusion proteins or its premature termination
of translation. The sizes of the major GST fusion proteins that were
recognized by anti-E2-2 antibody for GST-VL2 and for GST-VL3 are
similar to the size predicted based on the nucleotide sequence
information (Fig. 1). Two major proteins of 90 and 70 kDa (marked in
Fig. 3 by the open arrowheads) in nuclear proteins from Nalm6 cells also cross-reacted with the same antibody (lane 5). The nucleotide sequence analyses showed
that the three clones reported in this paper are the splice variant of
the E2-2 gene. The fact that GST-VL1 was not recognized by anti-E2-2
is probably due to the lack of epitope in this protein that can be recognized by the monoclonal anti-E2-2 antibody. Hence, we
repeated the same experiment with anti-GST-VL2 antibody
(lanes 6-10). This antibody recognized all the
GST fusion proteins (lanes 7-9) and GST itself
(lane 6). The same two major polypeptides (90 and
70 kDa) were found to be present in Nalm6 cells that could cross-react with both anti-E2-2 (lane 5, open
arrowhead) and anti-GST-VL2 (lane 10, closed
arrowhead) antibodies. It is noteworthy to mention that Bain
et al. (32) detected a 90-kDa protein in Nalm6 cells cross-reacting with anti-E2-2 antibody. Therefore, it is most likely
that the antigenic determinant for anti-GST-VL2 antibody includes both
the GST and E2-2 portion of the fusion proteins. We do not know
whether the 70-kDa protein is a degradative E2-2 protein or a related
protein having the common epitope. These results suggest that the
epitope for the anti-E2-2 antibody lies in the 100-amino acid domain
encoded by nucleotides 1396-1720, and that VL1, VL2, and VL3 represent
the splice variant of E2-2 gene product.

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Fig. 3.
Western blot analysis of recombinant GST,
GST-VL1, GST-VL2, and GST-VL3 proteins. An equal amount (100 ng)
of the purified GST (lanes 1 and 6),
GST-VL1 (lanes 2 and 7), GST-VL3
(lanes 3 and 8), or GST-VL2
(lanes 4 and 9) recombinant protein or
10 µg of nuclear extract prepared from Nalm6 pre-B cells
(lanes 5 and 10) was separated in 10%
SDS-PAGE, electrotransferred onto a nitrocellulose membrane, and probed
with anti-E2-2 antibody (lanes 1-5) or anti-GST-VL2
antibody (lanes 6-10). Two major proteins (90 and 70 kDa)
in the Nalm6 nuclear extract that cross-react with anti-E2-2 antibody
and anti-GST-VL2 are indicated by open and closed
triangles, respectively.
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The Protein Product of VL1, VL2, and VL3 binds to an Imperfect
E-box (µE5) Site Present in RR2 of the FGF-1.B Promoter--
To
confirm the sequence-specific binding, we performed EMSA using the GST
fusion protein and labeled oligonucleotide probe containing the
sequence
492 to
467 in the absence or presence of a 50-fold molar
excess of cold competitor A (
492 to
467) or B (
484 to
467)
(Fig. 4). It is evident that GST-VL1
(lane 1), GST-VL3 (lane 6),
and GST-VL2 (lane 11) retarded the migration of
the labeled oligonucleotide probe, and the degree of retardation was
dependent on the size of the binding protein. It is also clear that
these retarded DNA-protein complexes are specific, since they could be
competed by the cold competitor A (lanes 2,
7, and 12). The lack of competition by the same
molar excess of cold competitor B (lanes 3,
8, and 13) suggests that the site where these
proteins bind resides within the seqence
492 to
484. Interestingly, a closer look of this binding site revealed a sequence
ACGACCTGC (29) that contains an imperfect E-box (µE5)
site, GACCTG (23). To further confirm the identity of these proteins,
we added anti-E2-2 or anti-E2A antibody following the DNA-protein
complex formation. As shown in Fig. 4, anti-E2-2 antibody supershifted
the DNA-protein complex formed either by GST-VL3 (lane
9) or GST-VL2 (lane 14). These
supershifts are also specific for the E2-2 antibody, since anti-E2A
was unable to supershift the retarded complex (lanes 10 and 15). However, the DNA-protein complex
formed by GST-VL1 was not supershifted in the same assay by anti-E2-2
antibody. This result conforms to the earlier data (Fig. 3), which
showed that GST-VL1 lacked the epitope to be recognized by anti-E2-2 antibody. However, anti-GST-VL2 could supershift the DNA-protein complex formed with GST-VL1 (Fig. 5,
lane 4). The same antibody was also shown to
supershift GST-VL3-DNA complex (lane 9). The control antibody (IgG from the same rabbit used to generate
anti-GST-VL2) failed to supershift GST-VL1-DNA (lane
5) or GST-VL3-DNA (lane 10)
complex.

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Fig. 4.
Sequence-specific binding of GST-VL1,
GST-VL2, or GST-VL3 with the 492 to 467 sequence of the FGF-1.B
promoter. Approximately 300 ng of purified GST-VL1 (lanes
1-5), GST-VL3 (lanes 6-10), or GST-VL2 (lanes
11-15) protein was incubated in the absence ( ) or presence (+)
of a 50-fold molar excess of cold competitor oligonucleotide A ( 492
to 467) (lanes 2, 7, and
12) or B ( 484 to 467) (lanes 3,
8, and 13). The identity of the protein in the
DNA-protein complex was ascertained by a supershift assay with
anti-E2-2 antibody (lanes 4, 9, and
14). The anti-E2A antibody (lanes 5,
10, and 15) was used as a negative control for
the supershift assay.
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Fig. 5.
Anti-GST-VL2 antibody supershifts the
DNA-protein complex formed with GST-VL1 or GST-VL3 in EMSA.
Approximately 300 ng of GST-VL1 (lanes 1-5) or GST-VL3
(lanes 6-10) protein was incubated with the labeled
oligonucleotide A in the absence (lanes 1 and 6)
or presence (lanes 2 and 7) of a
50-fold molar excess of cold competitor A as described in Fig. 4. The
identity of protein was tested by an antibody supershift assay using
anti-E2-2 antibody (lanes 3 and 8),
anti-GST-VL2 antibody (lanes 4 and 9),
or control antibody (lanes 5 and
10).
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To determine the exact contact point in the DNA where these fusion
proteins bind, we performed the methylation interference assay using
GST-VL2 and the methylated probe as described under "Materials and
Methods." Fig. 6A shows that
the guanine nucleotide in the antisense strand at
484 is important
for the binding of this GST fusion protein with the DNA. It is
noteworthy to mention that this guanine nucleotide at
484 is in close
proximity to the imperfect E-box (µE5) site. Fig. 6B
represents the quantitative levels by a comparison of the G-ladder
radioactivity of free versus bound. It is also obvious from
this analysis that the peak radioactivity at
484 in the bound is
significantly reduced (42%) in comparison with the same in the
free.

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Fig. 6.
Methylation interference showing binding of
E2-2 to the guanine nucleotide at position 484 of the FGF-1.B
promoter. A, the methylated
HindIII-EcoRI fragment containing nucleotides
492 to 467 with the antisense strand radioactively labeled was
incubated with GST-VL2 protein. The specific DNA-protein complex and
free DNA, localized following EMSA, were eluted, treated with
piperidine, and analyzed on an 8% sequencing gel. Lane
G, partial chemical degradation specific for guanine
residues; lane F, free DNA probe; lane
B, bound DNA probe. The sequence of the antisense strand
( 467 to 492) as well as the positions of the guanine nucleotides
are depicted on the left. The asterisk represents
the guanine nucleotide at position 484 critical for the specific
DNA-protein complex formation. B, quantitation of
methylation interference assay using a PhosphorImager (Molecular
Dynamics, Inc.). The amount of radioactivity in the free
(top) versus bound fraction (bottom)
is depicted. The peak value of nucleotide 484 (*) in the bound
fraction is 42% of that in the free fraction. The dagger
indicates that the cytosine nucleotide at position 485 is also the
DNA-protein contact site.
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To confirm the methylation interference data, we performed EMSA using
GST-VL2 and the 26-bp (
492 to
467) oligonucleotide probe having the
wild type sequence or having mutation C to A at
484,
478, and
472
(Fig. 7). The binding of GST-VL2 with the
mutant probe was reduced dramatically in comparison with that achieved
by the wild type probe. Since the binding site of GST-VL2 was
identified to be in the region
492 to
484 (Fig. 4), it is most
likely that the mutation at
484 caused this diminution of the
DNA-protein interaction.

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Fig. 7.
EMSA of the wild type (WT) and
mutant (mut) sequence 492 to 467 of FGF-1.B promoter
with E2-2. Equal counts (50,000 cpm) of wild type
(lanes 1 and 2) or mutant
(lanes 3 and 4) oligonucleotide were
incubated with 300 ng of GST-VL2 protein in the absence ( )
(lanes 1 and 3) or presence (+)
(lanes 2 and 4) of a 50-fold molar
excess of self-sequence. In the mutant oligonucleotide, cytosine
residues in the sense strand at positions 484, 478, and 472 were
replaced by adenine residues.
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E2-2 Forms a Heterodimer with Proteins Present in Brain Nuclear
Extract--
Since GST-VL2 binds to the sequence from
492 to
484, we looked for the endogenous E2-2 protein in crude brain
nuclear extract that can bind to that site (Fig.
8). Although we could detect one such
complex (shown by the open arrowhead,
lane 7), it was not recognized by anti-E2-2
antibody (lane 9) or by anti-E2A (lane 10). However, we could detect an additional complex (shown
by the solid arrowhead, lane
1) having mobility faster than GST-VL2 (lane
12) when the crude brain nuclear extract was preincubated with GST-VL2. This complex was not formed when the nuclear extract was
preincubated with GST under the same conditions (lanes
6-10). Importantly, this additional complex was
supershifted by anti-E2-2 antibody (lane 4) but
not by the anti-E2A antibody (lane 5). These results suggest that E2-2 forms a heterodimer with partner proteins present in brain and that the heterodimer binds to the site
492 to
484.

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Fig. 8.
GST-VL2 forms a heterodimer with cellular
protein(s) present in human brain nuclear extract. Two micrograms
of human brain nuclear extract was assayed in the presence of 100 ng of
GST-VL2 (lanes 1-5) or GST (lanes 6-10) in EMSA
with the oligonucleotide probe A ( 492 to 467) in the absence
(lanes 1 and 6) or presence of a 100-fold molar
excess of cold competitor A (lanes 3 and
8) or B ( 484 to 467) (lanes 2,
4, 5, 7, 9, and
10). An equal amount (100 ng) of GST-VL2 was also assayed in
the presence of 2 µg of bovine serum albumin (lane
11). Lanes 4 and 9, the
reaction carried out in the presence of anti-E2-2 antibody;
lanes 5 and 10, the reaction carried
out in the presence of anti-E2A antibody; lane
12, GST-VL2 assayed in the presence of a lower concentration
of poly(dI-dC) (0.2 µg/assay) as shown in Fig. 4. The
closed arrowhead indicates the heterodimer
complex formed in the presence of GST-VL2.
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In Vivo Effect of E2-2 Proteins in U1240MG Cells on the Activity
of Promoter Containing E-box (µE5) Site(s)--
Two spliced forms of
E2-2 proteins, with and without RSRS, are known to be expressed in
different cells (23, 24, 33, 34). The three cDNA clones (VL1, VL2,
and VL3) isolated from human brain stem cDNA library represent the
splice variant of E2-2 containing RSRS (Fig. 1). However, an E2-2
cDNA that lacks the sequence encoding RSRS was isolated from a
murine brain cDNA library (33). Hence, the potential presence of
both spliced forms of E2-2 in brain is possible. Considering these
facts, we tested the effect of transient overexpression of both
spliced forms (with and without RSRS) on the FGF-1.B promoter activity in U1240MG cells (Fig. 9). We observed
that SEF2-1B, which encodes full-length E2-2 protein without RSRS
(24), significantly reduced the promoter activity, while the other
splice variant with RSRS (VL3-FL) did not affect this promoter
activity. Under the same conditions, the empty vector pCMV
CAT and
another bHLH protein NeuroD (35) did not alter the activity of the
FGF-1.B promoter.

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Fig. 9.
The splice variant of E2-2 without RSRS
significantly reduced the FGF-1.B ( 540) promoter activity. One
microgram of expression plasmids, pCMV CAT, NeuroD, SEF2-1B, or
VL3-FL (which is SEF2-1B with four additional amino acids, RSRS) was
cotransfected into U1240MG cells with 10 µg of
FGF-1.B( 540)-luciferase reporter construct as described under
"Materials and Methods." Luciferase activity was measured using a
Lumat LB9501 luminometer and normalized by protein content. Each
pair of bars (closed and
hatched boxes) represents two independent
experiments. The S.E. values of the triplicates carried out in each
experiment are within 5-10%.
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To determine whether repression of FGF-1.B promoter activity is
specific, we tested E47 promoter reporter construct which contains a
hexameric E-box (µE5-µE2) site. The reporter activity of this
promoter construct was previously shown to increase upon expression of
E47 effector cDNA (23, 31, 32). Fig.
10A shows that the
expression plasmid E47/pCSA, encoding E47 protein (an E2A gene
product), activated the E47 reporter, and this activation attained a
peak level of 27-fold at a 0.5-µg dose in comparison with that
achieved by the same dose of the empty vector, pCMV
CAT. These
results suggest that the activation of the promoter through the
µE5-µE2 sites is also reproducible in U1240MG cells. On the other
hand, at the same dose level and under the similar experimental conditions, SEF2-1B, which encodes the full-length E2-2 protein without RSRS exerted a dramatic inhibition (7-fold) of the promoter activity. This inhibition of the reporter activity was dependent on the
dose of this E2-2 splice variant having the repression greater than
40-fold when 5 µg of the SEF2-1B effector plasmid was cotransfected
(Fig. 10A). In contrast, VL3-FL (E2-2 with RSRS) did not
confer any remarkable effect on this E47 reporter activity at any of
the doses tested (Fig. 10A). Interestingly, the activity of
the FGF-1.B(
540) promoter (containing an imperfect µE5 site at
490 to
485) was also repressed in SEF2-1B-co-transfected cells in
a dose-dependent manner (Fig. 10B). Although the
degree of reduction of FGF-1.B promoter activity was not as dramatic as
that of E47 reporter, it was reproducible in all five experiments carried out in our laboratory. The transfection of E47/pCSA, on the
other hand, did not affect the FGF-1.B promoter activity. These results
suggest that the µE5 site present in both promoters may be the
target of suppression by E2-2 protein lacking the RSRS domain.

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Fig. 10.
SEF2-1B (lacking RSRS) represses the
luciferase reporter activity mediated by a hexameric E-box
(µE5-µE2) linked to alkaline phosphatase TATA box (E47 reporter)
(A) or FGF-1.B promoter containing an imperfect E-box
(µE5) site (B). Effector plasmids SEF2-1B (lacking
RSRS) (closed circle), E47/pCSA (open
circle), VL3-FL (with RSRS) (closed
triangle), and pCMV CAT (open
triangle) (described under "Materials and Methods") at
different doses (0-5 µg) were cotransfected into U1240MG cells with
10 µg of E47 reporter plasmid (A) or FGF-1.B( 540) in
pGL2Basic (B). Plasmid pRL-tk (0.5 µg) was also
cotransfected in the same cell line in each experiment as an internal
control. Sonicated herring sperm DNA was supplemented to make up 15.5 µg of total DNA in each transfection. Luciferase activity was
normalized by the dual-luciferase reporter assay system (Promega). The
results of a representative experiment are shown here. The standard
errors of the triplicates carried out in each experiment are within
5-10%.
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DISCUSSION |
The present study was undertaken to identify the
trans-acting factor(s) that binds to RR2 of FGF-1.B
promoter. Earlier studies (22, 29) showed that the specific DNA-protein
interaction in a 23-bp sequence (
489 to
467) of RR2 is linked to
the enhanced functional activity of the FGF-1.B promoter in the
FGF-1.B-positive glioblastoma cell line, U1240MG. The present paper
reports the identification of three E2-2/SEF2-1 cDNA clones (VL1,
VL2, and VL3) from the brain stem cDNA library using four tandem
repeats of the sequence
492 to
467. Comparison of VL1, VL2, and VL3 with the full-length SEF2-1B cDNA (24) showed that our clones encode four additional amino acids (RSRS) N-terminal to the bHLH domain
(Fig. 1). The immunoreactivity of GST fusion proteins of VL1, VL2, and
VL3 to the monoclonal anti-E2-2 antibody in Western blot and in EMSA
further confirmed that these clones represent a splice variant of the
E2-2 gene product.
The localization of the binding site by using competitor
oligonucleotides of overlapping sequence as well as by methylation interference assay revealed that the protein product of these cDNA
clones binds to an imperfect E-box (µE5) site within the sequence
492 to
484. Recently, we showed that the 18-bp sequence (
484 to
467) of FGF-1.B is sufficient to activate the promoter in U1240MG
cells (29). Hence, the DNA-protein interaction at the µE5 site may
not be essential in the context of FGF-1.B promoter activity in U1240MG
cells. On the other hand, this study's findings are important because
(i) FGF-1.B is expressed in neuronal cells (7, 8, 10, 14); (ii) the HLH
proteins, which bind to E-box as homo- or heterodimers (1), are also
expressed in neuronal cells (35, 36); (iii) the expression of
these HLH factors in neurons also regulates the development of the
nervous system (35-40); and (iv) the expression of FGF-1 gene in brain
is also developmentally regulated (7, 10).
We have shown here that the specific binding of these E2-2 proteins
(GST-VL1, GST-VL2, and GST-VL3) with the oligonucleotide probe (
492
to
467) was through the imperfect E-box (µE5 site). However, we
were unable to detect, by EMSA, cellular E2-2 homodimer either in
U1240MG cells (data not shown) or in human brain nuclear extract (Fig.
8) that can bind to the imperfect µE5 site. The Northern blot
analysis showed that the steady state levels of SEF2-1 mRNA is
relatively higher in brain tissue than in other tissues studied.
Therefore, it is unlikely that the level of this protein in the brain
tissue is too low to be detectable. However, it is pertinent to mention
that protein binding to the µE5 site has not been detected in crude
nuclear extract (41, 42). We do not know at this point whether our
failure to detect the endogenous DNA-protein complex is due to the low
affinity of the E2-2 proteins for the imperfect µE5 site or to the
fact that the concentration of this protein in crude nuclear extract in
a suitable form to bind to this site is too low to be detectable. The
former possibility is unlikely, because the fusion of VL1 cDNA with
the GAL4 activation domain could activate, in the yeast one-hybrid
system (43), the minimal histidine promoter containing four tandem
repeats of the same DNA binding sequence (
492 to
467) (data not
shown). Moreover, the addition of GST-VL2 protein in the brain nuclear extract generated a unique retarded complex (most likely a
heterodimer), which can be recognized by the E2-2 antibody (Fig.
8).
Given the fact that the 18-bp (
484 to
467) cis-sequence
is sufficient to activate the FGF-1.B promoter in normally growing U1240MG cells (29), the regulation of FGF-1.B promoter activity in
other condition (e.g. in neuronal cells at different stages of development) awaits further exploration. In fact, the transfection experiment in U1240MG cells showed that the overexpression of the
full-length E2-2 protein lacking the RSRS domain can reduce the
activity of an heterologous promoter through µE5-µE2 sites or of
FGF-1.B promoter containing the µE5 site. The stretch of the four
amino acids, RSRS, is absent in SEF2-1B cDNA obtained from a human
thymocyte cDNA library (24) but is present in all three cDNA
clones isolated from a human brain cDNA library. This suggests that
the brain contains proportionally more E2-2 with RSRS than without
RSRS. Although E2-2 has been shown to repress muscle-specific cardiac
-actin promoter activity by heterodimerizing with MyoD (34), our
observation here represents the first report showing that a splice
variant of homodimeric E2-2 (without RSRS) has repressor activity.
The mechanism that could discriminate the two splice variants of E2-2
protein (i.e. with or without RSRS) in terms of their effect
on the promoter activity through the µE5 site is not known. The
possibility of squelching effect through overexpressed SEF2-1B (without RSRS) also has not been ruled out. However, it is interesting to note that RSRS is inside a highly negatively charged stretch of 50 amino acids. In fact, the activation domains of many transcriptional activators are highly negatively charged (44). Thus, the presence or
absence of RSRS inside of the highly negatively charged domain in the
E2-2/SEF2-1 protein (encoded by one of the splice variants) could be
very important, contributing to the different transcriptional properties of these proteins. It is likely that the relative abundance and dynamic equilibrium of the two splice variants in the brain could
be an important determinant for the expression of FGF-1.B. Finally, we
showed that in brain nuclear extract, a heterodimer comprising E2-2
binds to the imperfect µE5 site of the FGF-1.B promoter (Fig. 8). It
would be of interest to identify the brain-specific bHLH transcription
factor that partners with E2-2 or with other bHLH proteins to activate
the FGF-1.B gene expression in the brain.
We thank Drs. Brit Corneliussen and Thomas
Grundström for providing pCMV2961 (SEF2-1B), pCMV
CAT, and the
E47 reporter construct; Dr. Andrew Lassar for E47/pCSA expression
construct; and Dr. Jacky Lee for Xenopus NeuroD expression
construct.