(Received for publication, October 22, 1996, and in revised form, January 10, 1997)
From the Department of Internal Medicine and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
Four different transcripts encoding fibroblast
growth factor 1 (FGF-1, also known as aFGF) have been previously
identified in our laboratory. Among them, FGF-1.B is the major
transcript expressed specifically in the neuronal cells in brain
tissue. Using the transient transfection experiment in a glioblastoma cell line, U1240MG, that expresses 1.B, we previously identified two
regulatory regions (RR1 and RR2) in the brain-specific promoter, FGF-1.B. In the present study, we showed that the minimal region required for the DNA-protein interaction in RR2 resides in an 18-base
pair (484 to
467) sequence, by using DNase I footprinting and
methylation interference studies and electrophoretic mobility shift
assays. This minimal cis-acting element was found to be sufficient in enhancing the reporter activity driven by the
heterologous herpes simplex virus thymidine kinase promoter in the
1.B-positive U1240MG cell line. This enhancing effect, however, was not
detected in a glioblastoma cell line, U1242MG, which is negative for
1.B expression. By electrophoretic mobility shift assays, we also identified a specific DNA-protein complex, namely complex I, which is
specific for 1.B-positive cell lines and human brain tissue. By
in situ UV cross-linking experiment, we further showed that complex I contains two major DNA-binding proteins of apparent molecular
masses of 37 and 98 kDa. Our results suggest that the formation of
complex I, resulting from the heterodimerization of a 37-kDa protein
(1.B-specific) and a 98-kDa protein (ubiquitous) may likely be a
prerequisite for the enhanced expression of 1.B transcript in neuronal
cells.
The FGF1 family consists of 14 structurally related polypeptide growth factors (1, 2). FGF-1, a prototype member, was originally identified as a mitogen for endothelial cells (3) and subsequently for a variety of mesoderm- and neuroectoderm-derived cells (4, 5). This growth factor is involved in a variety of physiological and pathological function such as tissue growth, wound healing, neovascularization, mesoderm development, and angiogenesis (6-10). FGF-1 confers tumorigenicity when introduced via expression vectors into normal cells (11-13). Unlike FGF-2, FGF-1 is found primarily in brain and retina, although low levels of its mRNA have been demonstrated in other tissues (14-16). FGF-1 may function in establishment of retinal fate and ganglion cell differentiation from the invaginated optic vesicle (17). 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 neural cells but not in glial cells (18, 19). It was also shown that the expression of FGF-1 mRNA in neurons is correlated with specific developmental events (20). FGF-1 was reported to coexist with tyrosine hydroxylase, a key enzyme for catecholamine synthesis, in neuronal cells (21) and is involved in the expression of this gene (22). FGF-1 mRNA (23) and the protein (24) levels were also reported to elevate in lesioned rat brain. Interestingly, in gliomas, a major human intracranial tumor (25), FGF-1 mRNA expression was reported to increase significantly (7, 26). However, the regulatory mechanism of FGF-1 gene expression is only beginning to be understood.
The human FGF-1 gene spans over 120 kilobase pairs containing three protein coding exons and at least four upstream untranslated exons, namely 1A, 1B, 1C, and 1D (27-31). Splicing of each of these untranslated exons to the first protein coding exon generates four different mRNA transcripts designated as 1.A, 1.B, 1.C, and 1.D. These multiple transcripts generated by alternate promotor usage and splicing are distributed in a tissue-specific manner. The major transcript in human brain, 1.B, is different from that in human kidney or prostate (31, 32). By RNase protection assay, it was shown that 1.B transcripts are highly expressed in glioblastoma tissues, as well as in some of the glioblastoma cell lines (26, 32). By in situ hybridization with cRNA probe specific for the murine FGF-1.B transcript, our laboratory has recently shown that 1.B in mouse brain are restricted largely to sensory and motor nuclei in the brainstem, and to the ventral spinal cord and cerebellum (33).
We have previously identified a 41-bp DNA sequence (507 to
467) in
the brain-specific FGF-1.B promoter, designated as regulatory region 2 (RR2), through luciferase reporter assays (34). RR2 binds nuclear
factors through a 23-bp (
489 to
467) region and this binding is
linked to the enhanced functional activity of the promoter (34). In the
present communication, we have further characterized the functional
importance of this region. We have narrowed down the minimal
cis-acting sequence to an 18-bp region (
484 to
467)
which can form more than one DNA-protein complex including the
1.B-specific complex, complex I. This 18-bp sequence is sufficient to
enhance the luciferase reporter gene expression driven by a
heterologous thymidine kinase (tk) promoter in a cell line-specific
manner. UV cross-linking analysis of this 1.B-specific complex reveals
that it contains two major DNA-binding proteins of 37 and 98 kDa.
U1240MG, U1242MG, U343MG, and U251MG glioblastoma cells (human glioblastoma cell lines originally established from surgical specimens of malignant gliomas) and the normal fetal glial cell line (CHII) were grown in minimal essential media containing 10% calf serum and antibiotics (Life Technologies, Inc.) as described (26, 34).
Transfection and Analysis of Promoter ActivityU1240MG cells were plated in 60-mm Falcon tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) to achieve 60-80% confluence by day 2. On day 2, cells were transfected with 10 µg of 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 duplicate. The results show the mean and standard error (S.E.) of at least two experiments. The transfection protocols were the same as described previously (34). The luciferase activity was normalized as described and was expressed as total light units per milligram of protein (34). The Bio-Rad DC protein assay system was used to determine the amount of protein in the cell lysates.
Construction of the FGF-1.B Insertion ClonesA pair of
complementary oligonucleotides containing nucleotides 492 to
467 of
FGF-1.B promoter were synthesized. The sequence of the sense
oligonucleotide is 5
-
ACGACCTGCTGTTTCCCTGGCAACTCG-3
with the underlined portion showing the BglII site, and the
antisense 3
-ATGCTGGACGACAAAGGGACCGTTGAG
-5
with the
underlined portion showing the BamHI site. The two
oligonucleotides were annealed and ligated into the BamHI
site of either pBluescriptII KS(+) or pUC19. A clone with four
repeating units in the same orientation was selected, double-digested
with SpeI and BamHI, resulting in the 104-bp
fragment. The 104-bp tetramer fragment was blunt-ended using Klenow
enzyme and subcloned into blunt-ended XhoI site of Del-2.
Del-2 contains a deletion of nucleotides
490 to
467 in the FGF-1.B
540 promoter reporter construct as described previously (34). The
540 construct contains nucleotides
540 to +31 of FGF-1.B promoter
sequence in the pGL2-Basic luciferase vector. The resulting tetramer
inserted clones were designated as 4U(+) with the tetramer in the sense
orientation and 4U(
) in antisense orientation. A clone containing one
repeat unit in the direct orientation cloned into the BamHI
site of pUC19 was designated as pFGF-1.B
492/
467. The reporter
plasmid containing the full-length tk promoter was constructed by
inserting nucleotides
200 to +50 of ptk-LUC (provided by Dr.
Ghyselinck; Ref. 35) into the SmaI-SacI sites of
pGL2-Basic. This plasmid was designated as tk(
200). Similarly, a
minimal tk promoter fragment (from
80 to +50) was cloned into
pGL2-Basic and was designated as tk. The heterologous promoter-reporter
plasmids 26bp/tk and 18bp/tk contain the 26-bp (
492 to
467) and
18-bp (
484 to
467) of FGF-1.B promoter in the tk, respectively.
Four pairs of oligonucleotides with SmaI and KpnI
sites representing the 18-bp wild type (WT) sequence (
484 to
467)
in either orientation (+ or
) and two mutant (mut) sequences (mut 456 and mut 56, Table I) were synthesized, annealed and subsequently cloned
upstream of tk plasmid at SmaI-KpnI site. The
resulting constructs are designated as 18bp(+)/tk, 18bp(
)/tk, mut
456/tk, and mut 56/tk. The copy number and orientation of each insert
were verified by sequencing.
|
NE was prepared from
the above cell lines as described before (34) with slight modification.
The hypotonically swollen cells were homogenized with a pestle (20 strokes) in a Dounce homogenizer and then treated with Nonidet P-40
(Nonidet P-40) to a final concentration of 0.5%. The nuclei were
pelleted by centrifugation for 15 min at 3,300 × g,
and the cytoplasmic fraction was removed. The nuclei were resuspended
with one packed nuclei volume of low salt buffer, buffer B (20 mM Hepes (pH 7.9), 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and
0.9 unit/ml aprotinin) plus 0.02 M KCl. To this suspension
one packed nuclei volume of high salt buffer, buffer B plus 1 M KCl, was added dropwise with slow mixing and rocked at
4 °C for 30 min. The extract was centrifuged for 30 min at 4 °C
at 25,000 × g. The supernatant was dialyzed against 50 volumes of dialysis buffer, buffer B plus 0.1 M KCl for
1.5 h at 4 °C. The NE was stored in aliquots at 80 °C
following centrifugation for 20 min at 4 °C at 25,000 × g. Protein concentration, measured by the Bio-Rad assay
using bovine serum albumin (BSA) as standard, showed 4-6 mg of
protein/ml of the NE. NE from human brain tissue and placenta was
prepared as described by Gorski et al. (36).
The WT
oligonucleotide representing the sequences 489 to
467 or
484 to
467 of FGF-1.B promoter and the mutants are described in Table I. To
localize the minimal region of DNA-protein interaction by
electrophoretic mobility shift assays (EMSA), we also synthesized the
following oligonucleotides, which upon annealing and end-filling represent the sequences
484 to
467,
484 to
473, and
480 to
467. The sequences of the pairs of oligonucleotides are,
respectively, as follows 5
-GATCTCTGTTTCCCTGGCAACTCG and
5
-GATCCGAGTTGCCAGGGAAACAGA, 5
-GATCTTCCCTGGCAACTCG and
5
-GATCCGAGTTGCCAGGGAA, and 5
-GATCTGTTTCCCTG and
5
-GATCCAGGGAAACA.
The pGL2-Basic reporter clones containing 540
to +31 or
594 to +31 of the FGF-1.B promoter were digested with
BbsI. The BbsI fragment containing
540 to
357
or
594 to
357 was labeled with [
-32P]dCTP by
end-filling following gel purification and digested with
SmaI. Finally, the SmaI (
540 or
594)-BbsI (
357) fragment was gel-purified. The labeled
HindIII-EcoRI fragment containing
492 to
467
sequences was obtained by digesting the plasmid pFGF-1.B
492/
467
with HindIII and EcoRI, followed by labeling with
Klenow enzyme in the presence of [
-32P]dATP. The
oligonucleotide probes were labeled with [
- 32P]ATP
using polynucleotide kinase following annealing of the complementary strand and purified by passing through spin columns (Sephadex G25).
The binding reaction contained the binding buffer (25 mM Hepes, pH 7.9, 1 mM MgCl2, 0.5 mM DTT, 40 mM KCl, and 5% glycerol) with radiolabeled DNA probe (30,000 cpm), 2 µg of poly(dI-dC), 5 µg of nuclear protein, and cold competitor when desired in a total volume of 20 µl. Each reaction was initiated by addition of protein and incubated for 30 min at room temperature, and then 12 µl was analyzed in a 4% polyacrylamide gel in 0.25 × TBE buffer (22.25 mM Tris borate, 0.5 mM EDTA, pH 8.0). Following electrophoresis, the gel was dried under vacuum and autoradiographed.
Sodium Deoxycholate (DOC) Treatment of U1240MG NETo exclude the possibility that formation of additional complexes was due to the interactions occurring between the DNA-binding protein and non-DNA-binding proteins, the NE was treated with DOC as described by Chellappan et al. (37). U1240MG NE (50 µg) was incubated in ice in a total volume of 10 µl with 0.7% DOC (final concentration) for 20 min, and then Nonidet P-40 was added to a final concentration of 1%. One µl of this reaction mix was assayed in EMSA in a total volume of 20 µl. An equivalent amount of untreated NE was also tested for comparison of the binding activity in the presence of 0.05% Nonidet P-40.
DNase I Footprinting AssayAn aliquot containing 0.3 ng
(30,000 cpm) of 540 to
357 or
594 to
357
SmaI-BbsI fragment of FGF-1.B promoter, labeled at the 3
-end at the BbsI site (sense strand), was incubated
with increasing amount of U1240MG nuclear extract (12-60 µg of
protein) or 20 µg of acetylated BSA in a total volume of 30 µl
containing 25 mM Hepes (pH 7.9), 1 mM
MgCl2, 0.5 mM DTT, 40-60 mM KCl,
5% glycerol, and 4 µg of poly(dI-dC) (Pharmacia). After 30 min of incubation, an equal volume (30 µl) of a solution containing 8 mM MgCl2 and 4 mM CaCl2
was added, followed by the addition of 10-25 ng (depending on the
amount of nuclear extract used) of freshly diluted DNase I (Boehringer
Mannheim). The digestions were carried out for 60 s. The reaction
was stopped by adding 140 µl of stop buffer (TE plus 0.1 M NaCl, 0.2% SDS), 200 µl of phenol, and 200 µl of
CHCl3. The aqueous layer was extracted further with 200 of
µl CHCl3 and ethanol-precipitated in the presence of 5 µg of tRNA. The precipitates were denatured, and equal counts were
analyzed in a 6% acrylamide sequencing gel.
The
HindIII-EcoRI fragment from plasmid pFGF-1.B
492/
467 was 3
-end-labeled at EcoRI site (sense strand),
partially methylated with dimethyl sulfate, and used as a probe in
binding reactions containing U1240MG NE. DNA-protein complexes were
separated from the free DNA by EMSA, eluted from the gel and cleaved
with piperidine, and then analyzed in an 8% sequencing gel as
described (34).
Analysis of the EMSA complexes
was done by in situ UV cross-linking according to the method
of Wu et al. (38) with a slight modification. The
32P-labeled photoaffinity probe used in this experiment was
made by annealing two complementary strands (5-GATCTGTTTCCCTG-3
and 5
-GATCCGAGTTGCCAGGGAAACAGA-3
) followed by end-filling with dGTP, 5-bromo-dUTP, [
-32P] dATP, and
[
-32P]dCTP by Klenow reaction. The gel shift binding
reaction, carried out using the labeled photoaffinity probe
(representing
484 to
467), was scaled up in the absence or presence
of a specific cold competitor. The reaction mixture was run in 1% low
melting agarose gel in 0.5 × TAE buffer (20 mM Tris
acetate, 1 mM EDTA, pH 8.0) at 4 °C. The gel was covered
with plastic wrap and exposed to UV light (254 nm) for 20 min at
4 °C. The DNA-protein complexes I, II, and III were localized
following autoradiography, and the gel slices containing each of these
complexes were melted. The labeled DNA cross-linked with the specific
DNA-binding protein was concentrated on glassfog beads (Mermaid Kits),
eluted by boiling with SDS-gel loading dye. The proteins thus labeled
were separated in SDS-polyacrylamide gel electrophoresis and visualized
following autoradiography of the dried gel.
Functional Rescue of FGF-1.B Del-2 Luciferase Reporter
Construct with Four Tandem Repeats of 492 to
467 in Sense
Orientation
To strengthen the functional link between the DNA-protein
interaction and the FGF-1.B promoter activity, we inserted four tandem
repeats of the DNA sequences 492 to
467 in the sense (4U(+)) or
antisense (4U(
)) orientation into the Del-2 construct (
540 to +31
with the deletion of nucleotides
490 to
467). These constructs were
transfected into U1240MG cells and assayed for the luciferase reporter
activity. Fig. 1 showed that the insertion of the four
repeats in the sense orientation significantly increased the reporter
activity (4-fold) in comparison to that with Del-2 construct. The 4U(+)
construct had a 20% increase in luciferase activity in comparison to
the wild type construct (
540 to +31). In contrast, the insertion of
the four repeats in the antisense orientation had no effect in
comparison to that observed with Del-2 construct. These results suggest
that the enhancer activity of the nucleotide sequences
492 to
467,
unlike the conventional enhancer, is
orientation-dependent.
Minimal Sequence Requirement for the DNA-Protein Interaction
To determine the minimal sequence in RR2 required for the specific
DNA-protein interaction, we performed (i) DNase I footprinting assay,
(ii) methylation interference assay, and (iii) EMSA with overlapping
sequences between 492 and
467.
To characterize the minimal
cis-acting element for the specific DNA-protein interaction,
we used end-labeled (sense strand labeled at its 3-end) DNA from
540
to
357 (Fig. 2A) or
594 to
357 (Fig.
2B) in DNase I footprinting assay with increasing amount of
U1240MG NE. The results showed a single footprint in the same position
(
485 to
467) when either probe was used and indicated that the
minimal sequence required for the DNA-protein interaction resides in
the sequence from
485 to
467.
Methylation of Guanine Nucleotide at
To identify the precise location of the nucleotides in
the sense strand contacting with the nuclear factors, methylation
interference analysis using a probe labeled at the 3-end of the sense
strand was performed. The previous study could not detect the contact sites in the sense strand (34); this may be due to the full methylation
of the genomic fragment (
507 to
467) having only 10 guanine
nucleotides in the same strand. This problem was overcome by using the
DNA fragment (
492 to
467) flanked by vector sequences at both ends.
It is evident from the results (Fig. 3) that the methylation of guanine nucleotides in the sense strand at positions
482,
474, and
473 strongly affects the specific DNA-protein interaction.
The Minimal cis-Acting Element Required for the Specific DNA-Protein Interaction Is from
The DNase I
footprinting assay (Fig. 2) showed that the site for the DNA-protein
interaction lies in the sequences from 485 to
467. We used
oligonucleotides of different sizes between
492 and
467 having
overlapping sequence either as cold competitors (Fig.
4A) or as probes (Fig. 4B) in EMSA
to determine the minimal sequence requirement for the DNA-protein
complex formation with U1240MG NE. In EMSA, we detected three distinct
complexes (I, II, and III) with the above NE (Fig. 4A). It
is evident from this figure that the sequence
484 to
467 is equally
effective to compete these complexes as the wild type oligonucleotide
(
489 to
467) does. The oligonucleotide
480 to
467 only
partially competes for these complexes, even at 100 molar excess, while
484 to
473 does not compete at all. Likewise, when these
oligonucleotides were used as probes (Fig. 4B), only
484
to
467 as well as
492 to
467 could specifically bind to the
nuclear factors. The oligonucleotide
480 to
467 showed very poor
binding, and no specific binding was detected with the
484 to
473
probe. These three experiments together determine the minimal
cis-acting element for the specific DNA-protein
interaction lies from
484 to
467.
The sequence 484 to
467 of FGF-1.B
promoter is sufficient to form the specific DNA-protein complexes.
Panel A, oligonucleotides of different sizes between
492
and
467 (oligonucleotide A) having overlapping sequences
(oligonucleotide B, C, or D) were synthesized and were used as cold
competitor at different molar excess levels in the DNA-protein binding assay. A
HindIII-EcoRI fragment containing the sequence
492 to
467 was end-labeled and used as a probe. I,
II, and III represent the three specific
complexes formed with U1240MG NE. Panel B, the
oligonucleotide (A, B, C, or
D) was labeled by end-filling reaction with Klenow enzyme in
the presence of [
-32p]dATP. Equal count (30,000 cpm)
of each oligonucleotide was incubated with 5 µg of U1240MG NE in the
absence (
) or presence (+) of 50-fold molar excess of cold
oligonucleotide A.
The Minimal 18-bp cis-Acting element (
To
address the functional consequence of the DNA-protein interaction with
the minimal 18-bp (484 to
467) region and the cell line specificity
of such interaction, we tested the activity of the minimal tk promoter
in absence or presence of the 18-bp or the 26-bp (
492 to
467) DNA
sequence of FGF-1.B in U1240MG or U1242MG cell lines. The results
showed that the insertion of the 18-bp sequence upstream of the tk
promoter enhanced the reporter activity by 6-fold relative to tk (Fig.
5A, left panel). It is also clear
that the 18-bp DNA sequence is sufficient to enhance the activity of
the heterologous tk promoter, since the 26-bp construct has no further
effect on the enhancement of the promoter activity. This enhancement is
cell-specific, since neither 18-bp nor 26-bp DNA sequence enhanced the
tk promoter activity in U1242MG cells (Fig. 5A, right
panel). It is important to note that a comparable level of
reporter activity (10-fold) was scored by both U1240MG and U1242MG
cells when tk(
200) was used in transient transfection assay (Fig.
5A). We also assayed the reporter activity of tk promoter in
the absence or presence of the 18-bp oligonucleotide in either orientation (Fig. 5B). The results showed that the 18-bp
sequence, when placed upstream of the tk promoter in the sense
orientation, activated it by 6.5-fold. When placed in the antisense
orientation, the activity was reduced to 1.7-fold relative to tk. This
result is consistent with the data shown in Fig. 1, demonstrating that the regulatory effect of the 18-bp sequence is
orientation-dependent. The insertion of mutant
oligonucleotide, mut 456 and mut 56, upstream of the tk (mut 456/tk or
mut 56/tk) resulted in a 71% and 57% reduction of the promoter
activity respectively relative to 18-bp(+)/tk (Fig. 5B).
Correlation of DNA-Protein Complex I in EMSA with the Expression of 1.B Transcript
Since the activation of the tk promoter through the 18-bp or 26-bp
DNA sequence was demonstrated to be cell-specific, we were interested
to know whether there is any cell-specific DNA-protein interaction with
the same DNA sequence and, if so, whether that correlates with the
expression of 1.B. We assayed the NE prepared from five different cell
lines (U1240MG, U1242MG, U251MG, U343MG, and CHII) of glial origin for
the specific binding of the nuclear protein(s) with the DNA probe
having the sequence 492 to
467 (Fig. 6). By RNase
protection, it has been shown that U1240MG and U251MG are positive for
1.B transcript, whereas the remaining three cell lines are negative
(26, 32). Fig. 6A showed that three distinct complexes (I,
II, and III) were formed with U1240MG NE (lane 1) and these
complexes were competed by a specific cold competitor (
489 to
467)
(lane 2). Notably, the fastest mobility complex, complex I,
is exclusively present in the 1.B-positive cell lines (U1240MG and
U251MG) but not in the 1.B-negative cell lines (CHII, U343MG, and
U1242MG) (Fig. 6, A and B). To understand whether
there is any tissue-specific expression of similar factors, we also
performed EMSA with the NE prepared from human brain and placenta (Fig.
6B). Interestingly, three specific DNA-protein complexes
including complex I were also formed with the NE from the human brain
(Fig. 6B, lane 5), but the nuclear proteins in the human placenta failed to form any of these complexes (lane 7). These results are consistent with the previous RNase
protection studies, which showed the human brain tissue is positive for
1.B (26, 32) whereas placenta is negative for FGF-1 mRNA (5).
The DNA-Protein Complexes I, II, and III Share a Common DNA Binding Site
Since the methylation of guanine (G) residues at 482,
474, and
473 in the sense strand affected the formation of three complexes (I,
II, and III) (Fig. 3), we were interested to know whether these
complexes result from the binding of nuclear factors with the DNA
sequence
492 to
467 at distinctly different site or an overlapping
site. We tested each of these complexes in methylation interference
assay and looked for the methylation of particular G residues that
affect individual complex. Fig. 7 showed that each of
these complexes preferably bound to the labeled and methylated DNA,
which had no methylation of the G residues at
474 and
473 in the
sense strand. In addition, methylation of the G residue at
482 was
also shown to interfere with complex II formation and to some degree
with complex I formation. These results indicate that these three
complexes share a common binding site including the nucleotides
474
and
473. We also made several mutant oligonucleotides of the sequence
484 to
467 or
489 to
467 considering the G residues important
for DNA-protein interaction in the sense strand (Fig. 3) and antisense
strand (34) and used them in EMSA (Fig. 8). The
sequences of these mutants are described in Table I. We
used these mutant oligonucleotides as well as the WT oligonucleotide in
EMSA as cold competitors (Fig. 8, A and C) or as
probes (Fig. 8B) to compare their abilities to affect the
formation of these three complexes I, II, and III. Fig. 8A
showed that the WT oligonucleotide as well as mut 1, mut 3, or mut 13 could compete the three complexes I, II, and III (lane 1) to
the same extent. The competition was drastically reduced by mutants
that contain mutation at nucleotide
478 including mut 2, mut 12, mut
23, or mut 123. Lane 10 showed the inability of an
irrelevant (IR) oligonucleotide to compete these complexes.
A comparison of the abilities of these oligonucleotides (WT and
mutants) to form specific DNA-protein complexes (Fig. 8B)
reveals that the binding ability of the single point mutant mut 1 or
mut 3 is virtually the same as that of the WT. However, the complex
formation is significantly reduced for the single point mutant mut 2. These results showed that a single mutation at
478 (mut 2) affects
the formation of each of these three complexes to the same proportion.
Similarly, mutations at
482,
474, and
473 (mut 456) or at
474
and
473 (mut 56) resulted in the loss of ability to compete these
complexes (Fig. 8C, Table I). When used as probes, both mut
456 and mut 56 failed to form these specific complexes (Table I). These
results suggest that all these complexes may share a DNA binding site
including the sequence from
478 to
473. The nearby G residues in
the sense strand at
482 and antisense strand at
484 and
472 may
be involved in the stabilization of these complexes.
DOC Treatment Has Little Effect on the Number of the Complexes Formed with U1240MG NE
Since complexes I, II, and III share a common DNA binding site, we
addressed the question whether these three complexes result from
protein-protein association of a common DNA-binding protein with
non-DNA-binding proteins. For example, the DNA-binding protein E2F
forms DNA-protein complexes of different mobilities in EMSA by
associating with other proteins that are not directly associated with
the DNA. In such a case, these complexes can be dissociated to a single
complex in the same assay by prior treatment with DOC (37). We treated
the NE with DOC to dissociate the protein-protein association of
similar nature (if any) and assayed in EMSA to detect the number of
specific DNA-protein complexes. Our results showed that DOC treatment
did not abolish any of the three complexes, although their intensities
were reduced in general (Fig. 9, lane 4).
This observation implies that each of these three complexes results
from the binding of DNA directly and that there are more than one
specific DNA-binding protein recognizing the sequence 484 to
467.
Complex I Contains Two DNA-binding Proteins of 37 and 98 kDa
We used U1240MG NE and a photo affinity probe (as described under
"Materials and Methods") to analyze the DNA-protein complexes I, II
and III by in situ UV cross-linking (Fig.
10). The bromo-dUTP-labeled photoaffinity probe also
formed the same three specific complexes I, II, and III (data not
shown) as formed by the regular probe 492 to
467 (Fig. 6). We
analyzed each of these complexes in denaturing polyacrylamide gels.
Fig. 10 showed that complex I contains two DNA-binding proteins of
apparent molecular masses of 37 and 98 kDa. Complex II contains a
single protein of 98 kDa, and complex III has two proteins of 98 and
145 kDa. Based on the protein profile (Fig. 10) and the mobility of
each complex in native gels (Fig. 6), it appears that complex I results
from the heterodimerization of p37 and p98. Complex II may likely be a
homodimer of p98 and complex III a heterodimer of p98 and p145 (Fig.
11).
The study of the tissue- or 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 (27-31). FGF-1.B is the predominant transcript in brain,
gliomas, and some glioblastoma cell lines (e.g. U1240MG and
U251MG) (26, 32). Using U1240MG cell line, we have recently identified
two regulatory regions, RR1 (
145 to
114) and RR2 (
507 to
467),
in the promoter of FGF-1 gene, which are important for FGF-1.B
expression (34). In the same study, we have also shown that the
deletion of
490 to
467 sequence (Del-2) significantly reduced the
reporter activity relative to the wild type promoter (
540 to +31). In
this study, we addressed the question whether the insertion of a tandem
repeats of the same sequence into Del-2 in either orientation can
functionally rescue the reporter activity above the wild type level. As
expected, the insertion of four tandem repeats in the sense orientation
(4U(+)) restored the reporter activity above the wild type level.
However, the insertion of the same tandem repeats in the antisense
orientation (4(
)) had little effect on the reporter activity relative
to Del-2. This result suggests that the sequence
492 to
467
positively regulates the FGF-1.B promoter in an
orientation-dependent manner. It is noted that other
regulatory elements, such as the one for the platelet-derived growth
factor B gene, are orientation-specific (38).
The region 540 to
467 has been shown previously (34) to be
important for the enhanced activity of FGF-1.B promoter. The DNase I
footprinting experiment carried out in the present study with two
overlapping genomic fragments
540 to
357, and
594 to
357
revealed that the nuclear protein(s) in U1240MG cells protects a common
region
485 to
467 in both fragments. The methylation interference
study as well as EMSA using overlapping oligonucleotides also
established that the minimal region for the DNA-protein interaction resides at
484 to
467. It is noted that mutation at
484,
478, and
472 (mut 123), which disrupts the formation of DNA-protein complexes, also reduces the transcriptional activity of the reporter activity by 76% in the context of
540 promoter of FGF-1.B (34). These results indicate that
484 to
467 is an essential element contributing to the promoter activation through the DNA-protein interaction.
To further test the above prediction, we determined the activity of the
minimal tk promoter in the absence or presence of the 18-bp (484 to
467) or 26-bp (
492 to
467) sequence in U1240MG or U1242MG cells.
It was demonstrated that both sequences enhanced the reporter activity
driven by the heterologous tk promoter to a similar extent in U1240MG.
The data indicated that the 18-bp DNA sequence, which is sufficient for
DNA-protein interaction, is also sufficient to enhance activity of the
tk promoter. This enhancer activity is cell-specific, since none of
these two chimeric promoter-reporter constructs enhanced the tk
promoter activity when transfected into U1242MG cells. By contrast, the
full-length tk construct (tk(
200)) activated the reporter activity to
the similar extent in both cell lines. The activation of tk promoter in
U1240MG cells through the 18-bp element is also site-specific because
the mutant oligonucleotides (mut 456 or mut 56), which are unable to
form specific DNA-protein complex, failed to enhance the activity of tk
promoter. Thus, the 18-bp cis-acting sequence functions not
only in the context of the native FGF-1.B promoter (34) but also in the
context of the heterologous tk promoter (Fig. 5). The orientation
dependence of this 18-bp sequence is, however, unconventional but
consistent with the data shown in Fig. 1. Moreover, these results also
indicate the presence of cell-specific factor(s) interacting with the
18-bp sequence is important for the enhancement of the promoter
activity.
EMSA revealed that complex I is unique when using the NE of U1240MG and U251MG cells; both cell lines are known to express 1.B (26, 32). The absence of complex I when using the NE of U1242MG or other 1.B-negative cells supports the view that nuclear factor(s) allowing the formation of complex I may be important for the activation of FGF-1.B promoter. Interestingly, the NE from the human brain tissue also formed three specific DNA-protein complexes including complex I, and this tissue predominantly expresses 1.B (26, 32). The human placenta is known to be negative for FGF-1 mRNA (5), and the NE prepared from this tissue failed to show any of these complexes. These data suggest tissue- or cell-specific expression of nuclear factor(s) may be important for the tissue- or cell-specific expression of 1.B.
Among the three complexes (I, II, and III), since complex I appears to
be linked with 1.B expression, we attempted to precisely locate the
binding site that is crucial for this complex formation. The
methylation interference assay using the bound probes eluted from each
individual complex revealed that complexes I, II, and III resulted from
the contact of nuclear factor(s) with G residues at 474 and
473 in
the sense strand. In addition, complex II has also been found to
contact with the G residue at
482 in the same strand. EMSA using
oligonucleotide with mutation at
482,
474 and
473 (mut 456) or at
474 and
473 (mut 56) also shows that the formation of each of these
complexes is affected to a similar extent in both cases. Furthermore,
EMSA using the oligonucleotide (
489 to
467) with mutation at
different sites (
484,
478, and
472) also failed to differentiate
these three complexes in terms of their specific binding sites and
showed that the G residue at
478 is crucial for the formation of
these complexes. These results suggest that these complexes share a
common DNA binding site including the sequence
478 to
473.
DNA-protein complexes with different mobilities may be formed by the
interaction of multiple DNA-binding proteins of different sizes to a
common binding site (39) or by the association of DNA-binding protein
with other proteins that do not directly bind the DNA (37). Our results
showed that more than one DNA-binding protein binds to the sequence
484 to
467. In situ UV cross-linking study of these
three complexes reveals that complex I contains two DNA-binding
proteins of 37 and 98 kDa, complex II contains a single DNA-binding
protein of 98 kDa, and complex III contains two DNA-binding proteins of
98 and 145 kDa. Based on the protein profile and the mobility of each
complex in native gels, it is most likely that complex I results from
heterodimerization of p37 and p98. Complexes II and III may likely be a
homodimer of p98 and a heterodimer of p98 and p145, respectively. These
results suggest that formation of the activating complex in the FGF-1.B promoter requires both p37 and p98. Remarkably, a DNA-binding protein
of 37 kDa has been shown to be present in human brain tissue and
U1240MG cells (but not in U1242MG cells) that can specifically bind to
the sequence
489 to
467 in Southwestern analysis (34). In EMSA,
however, we could not detect the specific binding of a 37-kDa protein
in U1240MG NE that can alone bind the same DNA sequence. This
discrepancy may be due to the variation of the assay conditions used to
study the DNA-protein interaction. It is pertinent to mention that a
specific faster mobility complex (faster than complex I) was
demonstrated in EMSA in the same cell line when 1 µg or less than 1 µg of poly(dI-dC) was used as nonspecific competitor (data not
shown). Moreover, it is significant that a DNA-binding protein of the
same size (i.e. 37 kDa) from the same source
(i.e. U1240MG cells) and the same binding specificity (34)
is present in complex I, which is also unique for 1.B-positive cells or
tissues.
Our data, taken together, allow us to propose a testable model (Fig.
11) for the DNA-protein interactions at the RR2 (precisely at positions
484 to
467) that may explain the cell line-specific activation of
FGF-1.B. Based on the present study, it is likely that a 98-kDa protein
binds to a minimal DNA sequence
484 to
467 as a homodimer. Two
other DNA-binding proteins of 37 and 145 kDa bind to the 98-kDa
protein, forming two heterodimers of different mobilities. Based on the
finding that complex I is present exclusively in 1.B-positive cells
(U1240MG, U251MG, and in human brain), we hypothesize that the
expression of the p37 followed by its heterodimerization with p98
resulting in the formation of complex I is a prerequisite for the
enhancer activity of RR2. Whether the FGF-1.B promoter commits itself
to activation in a given cell may depend on the stoichiometric amounts
of p37, p98, and p145. It is likely that the availability of p37 is
crucial for the expression of 1.B. Since the identified
cis-acting sequence shows no similarity with sequences
recognized by any other known transcription factors (GenBank, October
1996 release), it is most likely that p37 represents a novel
transcriptional factor, which we tentatively designated as
p37brn. Whether p98·p98 homodimer and p98·p145 heterodimer
compete with p37·p98 heterodimer for the common binding site and
thereby neutralizing the positive trans-acting effect of the
latter remains to be determined. As a corroboration to the model
proposed here, we have cloned the mouse FGF-1.B promoter (33) and
showed that the sequences most crucial for binding to
p37brn (between nucleotides
483 and
473) are identical
and positioned in the same context relative to the transcription start
sites between the two species (33). Isolation of the cDNA
expressing these DNA-binding proteins including p37brn, either
by screening the expression library using the minimal binding sequence
or by protein purification, will help us verify the above model and
study the regulation of 1.B expression in brain as well as in
glioblastoma.
We thank Drs. René Myers and Vera Leybman for helpful discussions.