(Received for publication, December 27, 1994; and in revised form, January 25, 1995)
From the
Expression of alternatively spliced human FGF-1 (or aFGF) transcripts is regulated in a tissue-specific manner via multiple promoters. To identify the cis-regulatory elements in the brain-specific FGF-1.B promoter, we constructed a series of promoter deletions fused to the luciferase reporter gene and transfected into an FGF-1.B positive glioblastoma cell line, U1240MG, and a 1.B negative cell line, U1242MG. Results of transient transfections indicate three elements that are involved in the positive regulation of FGF-1.B expression. The core promoter is located in a 40-base pair region (between -92 and -49), and two regulatory regions (RR-1 and RR-2) are located within the 540-base pair region 5` to the major transcription start site (defined as +1). Electrophoretic mobility shift assays and footprinting analysis have identified sequence-specific binding sites in RR-1 and RR-2. Mutants of RR-2 abolished binding to nuclear proteins and showed diminished luciferase reporter activity. The effects seen are specific for the U1240MG cell line, supporting a role for RR-2 in the tissue-specific regulation of FGF-1.B. Southwestern analysis using an oligonucleotide probe derived from RR-2 (nucleotides -489 to -467) further identified a 37-kDa protein that is present in nuclear extracts from U1240MG and brain but not from U1242MG.
Acidic fibroblast growth factor (or aFGF-1) ()is a
member of the fibroblast growth factor family of proteins. To date,
this family consists of nine members that are involved in proliferation
of a variety of cells of mesodermal and neuroectodermal origin and are
angiogenic (Basilico and Moscatelli, 1992; Burgess and Maciag, 1989;
Chiu et al., 1990a). The most recent additions to this family
include FGF-8, an androgen-induced growth factor isolated from the
conditioned medium of an androgen-dependent murine mammary carcinoma
cell line (Tanaka et al., 1992). FGF-9 (glial activating
factor), was purified from conditioned medium of a human glioma cell
line (Miyamoto et al., 1993). The gene organizations of the
members of this family are very similar, with the protein coding region
contained in a three exon, two intron structure. These growth factors
have a high affinity for heparin, heparan sulfate proteoglycans, and
glycosaminoglycans, and these interactions potentiate their effects
(Basilico and Moscatelli, 1992).
Three of the FGF family members were originally identified as oncogenes: Int-2 (FGF-3) (Dickson and Peters, 1987), K-fgf/hst (FGF-4) (Delli-Bovi et al., 1987; Yoshida et al., 1987), and FGF-5 (Zhan et al., 1988). Other evidence also suggests that the FGFs have oncogenic potential. Overexpression of FGF-1 protein in NIH3T3 cells results in a transformed phenotype, including the ability of these cells to form tumors when injected into nude mice (Bunnag et al., 1991). Therefore, the expression of FGF-1 protein may play a role in cellular transformation and in the proliferation of neoplastic cells. In addition, FGF-1 is also an angiogenic factor in vivo (Folkman and Klagsbrun, 1987) and may play an important role in tumor progression by promoting tumor vascularization.
FGF-1 is a mitogen for a variety of mesoderm- and neuro-ectoderm-derived cells (Basilico and Moscatelli, 1992; Chiu et al., 1990a; Gospodarowicz, 1987) and an inducer of mesoderm development in the Xenopus embryo (Whitman and Melton, 1989). In addition, expression of FGF-1 in smooth muscle cells of the vascular wall has been documented and FGF-1 is thought to be an important player in atherosclerosis (Brogi et al., 1993; Weich et al., 1990; Winkles et al., 1987). Because this family of growth factors is involved in a variety of physiological and pathological functions such as tissue growth, wound healing, neovascularization, mesodermal development, atherosclerosis, and oncogenesis, understanding the regulation of gene expression of these growth factors will provide valuable knowledge. Currently, little is known about the cis- and trans-regulatory elements of the FGF genes. The best studied to date is K-fgf (FGF-4). The FGF-4 gene has a canonical TATA box, and its physiological transcription depends on an enhancer element in the 3`-untranslated region (Curatola and Basilico, 1990). The Int-2 (FGF-3) gene contains three distinct promoters and two alternative polyadenylation sites, generating six different mRNAs that contain the same coding region (Grinberg et al., 1991; Smith et al., 1988a).
We have shown that the FGF-1 gene contains multiple polyadenylation sites (Chiu et al., 1990b) and have reported multiple FGF-1 transcripts, which are generated by alternative promoter usage and splicing and are distributed in a tissue-specific manner (Myers et al., 1993; Payson et al., 1993). The major transcript in human brain is different than that in human kidney (Myers et al., 1993). It is important to note that FGF-1 mRNA levels are elevated in lesioned rat brain (Logan, 1988). Furthermore, several glioblastoma cell lines have a different pattern of FGF-1 transcripts than seen in normal brain cortex (Myers et al., 1993). Here, we report our studies on the characterization of the brain specific FGF-1 promoter, FGF-1.B. Using luciferase reporter gene constructs and electrophoretic mobility shift analyses (EMSA), we have identified the core promoter sequences in a 40-bp region and two cis-regulatory elements in the FGF-1.B proximal promoter that are necessary for transcription. Mutation analyses have allowed us to identify a 23-bp region in the promoter that interacts specifically through contacts of three guanine residues to nuclear transcriptional factors. Using Southwestern analysis, we have also identified a brain-specific 37-kDa protein that binds this cis-acting sequence.
Figure 10: Sequence of the FGF-1.B promoter. The major transcription start site is designated by the closeddiamond at +1. The two minor start sites are indicated by the opendiamonds at -28 and -24. The splice donor site of the exon is denoted by the opentriangle at +110. cis-Regulatory regions RR-1 and RR-2 are boxed in gray. Sequence with 80% or higher sequence identity (with a window of 30 bp) to the murine FGF-1 gene are doubleunderlined. The NcoI and DraIII restriction sites used in the cloning of the -831 reporter construct are shown.
Figure 1: Promoter activity of the sequential 5`-deletions of FGF-1.B in U1240MG and U1242MG cells. The plasmid pGL2-Enhancer and the deletion mutants described under ``Materials and Methods'' were used to transfect the two glioblastoma cell lines. Luciferase activity was measured using a Lumat LB 9501 luminometer. The bars represent the mean of multiple experiments using at least two different plasmid preparations for each construct. The numberofexperiments done is indicated above each construct. The linediagrambelow summarizes the FGF-1.B proximal promoter region. FGF-1.B sequences are located between the NcoI(-831) and the DraIII (+31) sites. The thickenedlines indicate vector sequences; the SmaI and HindIII polylinker sites used to do cassette cloning between pGL2-Basic and pGL2-Enhancer vectors are shown. The luciferase data predicted two positive cis-regulatory elements, designated RR-1 and RR-2 for regulatory regions 1 and 2. RR-1 is localized to nucleotides -145 to -114, and RR-2 is localized to the sequences between -507 to -467. Enh represents pGL2-Enhancer, while (-196) represents the construct containing nucleotides -196 to +31 in the opposite orientation.
To generate
specific deletion mutants and introduce point mutations into the
FGF-1.B proximal promoter, we used mutagenic oligonucleotide primers as
described (Deng and Nickoloff, 1992). For each mutant clone, one primer
contained the desired mutation (mutagenic primer) while the second
primer (selection primer) mutated the unique AlwNI vector site
and introduced a unique SpeI site. This enabled selection
against the parent plasmid by AlwNI digestion and screening
for mutant clones with SpeI digestion. The sequence of the
selection primer was 5`-GCAGCCACTAGTAACAGGATT-3`, with the
underlined portion showing the SpeI site and the bold showing
the base changed during the mutagenesis. Point mutations were
introduced into the -540 wt luciferase construct at positions
-472, -478, and -484, and the clone was designated
-540 mut. The sequence of the mutant oligonucleotide was
5`-ACCTGATGTTTACCTGGAAACTC3-`, with the
nucleotides in bold showing the bases changed. These positions in the
wild type sequence were cytosine residues. Two deletion clones were
generated, each designed to eliminate small portions of the FGF-1.B
promoter and introduce a unique XhoI restriction site for
screening purposes. Del-1 represents the parent -540 wt, which
contains a deletion of nucleotides -512 to -485; Del-2
represents the parent -540 wt, which contains a deletion of
nucleotides -490 to -467. Oligonucleotide primers were
designed to contain sequences flanking the deletion areas as well as a
few additional bases to introduce the unique XhoI site. The
oligonucleotide sequence used to generate Del-1 was
5`-GCCTTCTGACTCGAGCTGTTTCCCTGG-3` and that for Del-2 was
5`-GTCTCCGAGCCACTCGAGGCCTCAAAAT-3`, with the bold nucleotides
showing the added bases. For each clone, the selection primer and the
mutation primer were phosphorylated prior to annealing to the
-540 wt plasmid. A 20-µl annealing reaction was set up
containing -540 wt plasmid DNA (0.025 pmol) and each of the
primers (5 pmol) in annealing buffer (20 mM Tris-HCl, pH 7.5,
10 mM MgCl, 50 mM NaCl). The reaction was
boiled for 3 min and chilled in an ice bath for 5 min. The annealed
mixture was then used in a 30-µl synthesis reaction containing 5
mM of each dNTP and 3 units of T4 DNA polymerase and T4 DNA
ligase in synthesis buffer (10 mM Tris-HCl, pH 7.5, 1 mM ATP, 2 mM DTT) at 37 °C for 2 h. The reaction was
then heated at 70 °C for 5 min and allowed to cool slowly to room
temperature. The DNA was then digested with AlwNI for 1.5 h.
Approximately 25 ng of digested DNA was used to transform BMH
71-18 mutS (Clontech), which is a bacterial strain defective in
mismatch repair. Transformants were grown in a pool, and plasmid DNA
was isolated from the mixed bacterial population. The isolated DNA was
again digested with AlwNI and transformed into DH5
bacteria. Colonies are grown, and plasmid DNA was isolated and screened
by digestion with either AlwNI and SpeI (all mutant
constructs) or XhoI (Del-1 and Del-2 mutants). The identities
of -540 mut, Del-1, and Del-2 clones were confirmed by
sequencing.
The results show that the core promoter is located in a 40-bp region from 49 to 92 bp upstream of the major transcription start site, with luciferase activity in U1240MG but not U1242MG. Additionally, deletion of sequences between -145 and -114 reduced the luciferase reporter activity by 34%. When sequences between -196 and +31 are cloned in the opposite orientation in the luciferase construct, reporter activity is lost (Fig. 1), demonstrating that this region functions as a promoter. Enhanced reporter activity is seen when sequences between -654 and -507 were included in the luciferase constructs, with levels being approximately 2-fold higher than those seen in the core promoter region. In all constructs, luciferase activity is much higher in the U1240MG cells as compared with the U1242MG cells reflecting the endogenous FGF-1.B mRNA levels (Fig. 1). Thus, the SV40 enhancer is not affecting the cell line specificity of the FGF-1.B promoter. RNase protection analysis conducted on transfected U1240MG cells confirmed that the reporter constructs were utilizing the same three start sites (namely -28, -24, and +1; data not shown) as we previously reported for the endogenous FGF-1 gene (Myers et al., 1993). We conclude that the FGF-1.B proximal promoter is relatively weak, and our data further suggest that some endogenous enhancer element(s) is necessary for optimal expression. The two positive regulatory regions identified by the functional analyses and subsequent electrophoretic mobility shift analysis (see below) are diagrammed and designated regulatory region 1 and 2 (RR-1 and RR-2) in Fig. 1.
Figure 2: U1240MG nuclear extract interacts with RR-1. The radiolabeled probes A and B were incubated with U1240MG nuclear extract (15 µg) without competitor(-) or with 40- and 100-fold excess unlabeled probes A, B, or C. The most prominent complex is labeled as 1. Complex 2 is also a specific complex, which is less predominant than factor 1. Complex 3 is observed only when probe A is competed with large excess of fragment B. The probelane represents the free probe (no incubation with nuclear extract).
There was an 86% increase in activity when sequences between -237 and -196 were deleted (Fig. 1), which may indicate a negative regulatory element in this region. Oligonucleotides containing sequence between -237 and -196 were synthesized, end labeled, and used in EMSA with nuclear extract from U1240MG. This region was chosen because of the decrease in luciferase activity seen in this region. One major complex was seen that occasionally resolved as a doublet and could be partially competed by a 50-200-fold molar excess of unlabeled probe but not by 200-fold excess of a nonspecific fragment of the same length (Fig. 3). Two other less prominent bands were observed that were not competed. These results indicate a sequence-specific DNA-protein interaction in sequence between -237 and -196, which may function as a negative regulatory element.
Figure 3: U1240MG nuclear extract interacts with a negative regulatory element. The radiolabeled oligonucleotide -237 to -196 was incubated with U1240MG nuclear extract (15 µg) without competitor or with 50-500-fold excess unlabeled oligonucleotide -237 to -196 or with oligonucleotide containing nonspecific sequence. The doublearrows denote a complex that can be competed with 50-200-fold excess competitor. The singlearrows indicate faint complexes that are not competed with up to 200-fold excess of unlabeled oligonucleotide.
To identify the region responsible for the enhanced reporter activity seen in constructs containing sequence 5` to -467 (RR-2 in Fig. 1), overlapping genomic fragments encompassing the region between -540 and -357 were used in EMSA with both U1240MG and U1242MG nuclear extracts. The results reveal a complex that appears as a tight doublet when sequence A or B is used as the probe (Fig. 4). The complex is readily competed with 50-fold molar excess of sequence A or B but not by sequence C, localizing the region of DNA-protein interaction between -507 and -467. Probe C could not be retarded in EMSA when used in similar analyses (data not shown). The same retarded complex was seen when brain and U1242MG nuclear extract was used (Fig. 4C), with U1242MG extract showing a less intensive band than the brain or U1240 extract.
Figure 4: Nuclear extracts interact with RR-2. The radiolabeled probes A and B were incubated with U1240MG, U1242MG, and brain nuclear extracts (15 µg) without competitor(-) or with 50-fold excess of unlabeled sequence A, B, or C. The complex denoted by the doublearrows is seen when both A (panelA) and B (panelsB and C) are used as the probe. The band is readily competed by A and B but not by sequences in C, suggesting that the sequences involved in the protein interaction reside between -507 and -467.
The similar band detected in U1242MG cells suggested that the protein-DNA interaction in the region between -507 and -467 might not be critical for the enhanced activity seen in U1240MG cells. Alternatively, similar bandshift observed in U1242MG cells could be due to nonspecific binding to the probe, which results in coincidental bandshift. If the latter is the case, a smaller probe is likely to differentiate the mobility differences. Therefore, oligonucleotides containing sequences between -507 and -467 were synthesized, end labeled, and used in EMSA, which included nuclear extract isolated from brain as well as the U1240MG and U1242MG extracts. The results shown in Fig. 5, confirmed the location of the sequences responsible for the protein binding to -507 to -467. In addition, a faster migrating doublet appeared in the binding reaction containing the nuclear extract isolated from human brain tissue. This doublet has been seen in other U1240MG extract preparations but inconsistently (data not shown). It is interesting that both complexes run as a doublet, implying multiple protein-DNA interactions in a small region. Importantly, the shifted protein-DNA complex in U1242MG cells have slightly different mobility than those observed in brain and U1240MG cells. This difference argues that the nature of the complex in U1242MG cells differs from those in FGF-1.B-producing cells. Further proof will be provided by Southwestern blotting analysis using the same probe (see Fig. 11).
Figure 5: RR-2 is localized to sequences -507 to -467. The radiolabeled oligonucleotide -507 to -467 was incubated with nuclear extract isolated from U1240MG, U1242MG, and brain (15 µg) without competitor or with 50-fold excess unlabeled oligonucleotide. The results confirm those seen in Fig. 4. The brain extract shows an additional doublet that migrates faster in the gel than the major complex.
Figure 11:
Southwestern analysis using
oligonucleotide probe E and mutE (-489 to -467). Partially
purified nuclear extract from U1240MG, U1242MG, and human brain tissue
were electrophoresed in a 12% SDS-polyacrylamide gel and transferred to
a nitrocellulose membrane. The blot was probed with P-labeled (1.5
10
cpm) oligonucleotide
probe E (panelA) or mutE (panelB)
and exposed to an x-ray film. The arrowhead in panelA indicates the 37-kDa protein that is present only in
nuclear extracts from U1240MG and brain but not from U1242MG. The openarrowhead in panelB points to
the position of the 37-kDa protein, which does not bind to
mutE.
Figure 6: Methylation interference footprinting analysis of RR-2. Oligonucleotide containing noncoding strand sequence between -507 to -467 was methylated with dimethyl sulfate prior to incubation with U1240MG nuclear extract. The complex and free DNA were localized following electrophoresis through a polyacrylamide gel, eluted, treated with piperidine, and analyzed on a 6% sequencing gel. LaneG, partial chemical degradation products of the probe cleaved at guanine nucleotides; laneB, bound probe DNA; laneF, free probe DNA. The sequence shown is the noncoding strand between -501 (top) to -472 (bottom) with stars denoting nucleotides in the sequence whose methylation interferes strongly with complex formation.
Figure 7: EMSA of FGF-1.B mutant oligonucleotides. Oligonucleotide probes D, E, and mutE were radiolabeled and incubated with U1240MG nuclear extract (15 µg) without competitor or with 50-fold excess competitor sequence as denoted. Probes D and E contain wild type sequence. Probe mutE is identical to probe E with the exception of three point mutations at positions -484, -478, and -472.
Figure 8: EMSA of FGF-1.B proximal promoter mutants. Genomic probes A and mutA were radiolabeled and incubated with U1240MG nuclear extract (15 µg) without competitor or with 50-fold excess competitor sequence as shown. Probes A-E are wild type sequence with the indicated boundaries. mutA and mutE contain point mutations at positions -484, -478, and -472.
Additionally, we shuttled appropriate 1B deletion fragments from the pGL2-Enhancer to pGL2-Basic vector. The reporter constructs were then transfected into U1240MG and U1242MG to test for luciferase activity. Although they have a much lower luciferase activity, the Basic constructs confirmed the functional significance of nucleotides between -489 and -467 in U1240MG cells (Fig. 9). Remarkably, the -540 mut in the basic construct diminishes its activity to the same level as the -467 clone. Potential loss of functionality of Del.1 could be due to the loss of nucleotides -489 to -485. In U1242MG cells, none of these constructs had luciferase activity greater than 50% above pGL2-Basic level (data not shown), reflecting that the activity observed in U1240MG is cell specific. Fig. 10summarizes the FGF-1.B proximal promoter sequences. Sequences for RR-1 and RR-2 are boxed. Areas having 80% or greater homology with murine FGF-1 gene are indicated.
Figure 9: Functionality of RR-2 in pGL2-Basic vector. The plasmid pGL2-Basic (pGL2-B) and the deletion mutants cloned into pGL2-Basic were used to transfect the U1240MG glioblastoma cells. Del-1 and Del-2 are derived from -540 wt with a deletion (indicated by a dashedline) of nucleotides -512 to -485 and -490 to -467, respectively. Asterisks indicate the mutations in -540 Mut at positions -484, -478, and -472. The bars represent results of three separate transfection experiments for each construct. The percentage of luciferase activity for each construct as compared with -540 wt in pGL2-Basic is indicated above each construct.
It is possible that some additional regulatory protein in U1240MG, which may not directly bind DNA, is necessary for the enhancer activity seen in RR-2. Alternatively, posttranslational modification, such as phosphorylation, may be required for activation of the regulatory molecule. A third possibility is that these cell lines contain proteins that share a DNA binding domain but differ in their activation domain, resulting in the difference in functional activity observed between the two cell lines.
We have previously shown that the expression of alternatively spliced human FGF-1 transcripts is regulated in a tissue-specific manner via multiple promoters (Myers et al., 1993; Payson et al., 1993). The brain-specific 1.B promoter is a non-TATA promoter and contains three transcription start sites clustered in a 30-bp region with the major start site designated as +1 (Myers et al., 1993). In this paper, we present our studies on the characterization of the FGF-1.B promoter. We have identified two cis-regulatory elements in addition to the core promoter, which are necessary for transcription of the FGF-1.B transcript, and a putative negative regulatory element.
The functional analyses of
sequential 5`-deletions identified that the basal promoter activity is
located in a 40-bp region between positions -92 and -49,
showing activity in the U1240MG but not in U1242MG cells. It is
important to note the two minor start sites at -28 and -24
conform to the initiator sequences (Javahery et al., 1994).
Since TATA box-binding protein binds to sequences 30 bp upstream of the
initiator (Zenzie-Gregory et al., 1992), it is not surprising
that deletion to -49 results in complete loss of the
transcriptional activity. When -196 to +31 was reverted in
the promoter construct, the luciferase activity was completely
abolished. These data are consistent with the function of initiators
being unidirectional (Smale and Baltimore, 1989). The luciferase data
also located two enhancing elements (RR-1 and RR-2 in Fig. 1) in
the proximal promoter between positions -145 to -114 and
-507 to -467. There was also a decrease in activity between
-237 and -196 (Fig. 1). We did repeated experiments
with multiple plasmid preparations for each construct and were able to
obtain consistent results. Initially, transfection efficiency was
normalized using cotransfection with a -galactosidase expression
vector. However, we found that these glioblastoma cell lines had a high
background of
-galactosidase activity, and we were not able to
normalize our luciferase activity using this approach. Also, these
human cell lines had a low transfection efficiency, and we were not
able to use the less sensitive chloramphenicol acetyltransferase assay
to normalize the luciferase data. However, multiple plasmid
preparations consistently supported our conclusions regarding the cis-regulatory elements in the proximal promoter region.
Promoter constructs containing sequences up to 3900 bp upstream from
the 1.B start site showed similar luciferase activities to the
-540 wt construct (data not shown), suggesting that the essential cis-acting sequences reside within the -540-bp region.
In addition, EMSA using DNA probes encompassing sequences in RR-1 and
RR-2 provided evidence of sequence-specific protein-DNA interaction in
these regions (Fig. 2Fig. 3Fig. 4Fig. 5),
supporting the results of the functional analyses. The reporter gene
levels were much lower in the U1242MG cell line as compared with the
U1240MG cell line (averaging 4% of U1240MG levels), reflecting the
natural levels of FGF-1.B in these cells (Myers et al., 1993).
Therefore, the use of the SV40 enhancer did not affect the
physiological expression pattern of this transcript. Similarly, the
SV40 enhancer did not interfere with studies of other promoters (Pech et al., 1989; Rao et al., 1988). The difference in
FGF-1.B promoter activity in the two cell lines is not due to different
transfection efficiency since the SV40 promoter strength in these two
cell lines is comparable. The reporter activity of the SV40 promoter in
U1242MG averages 70% of that in U1240MG (data not shown).
To
identify contact residues in RR-2, methylation interference analyses
were conducted on sequences between -507 and -467. Three
potential guanine residues were identified at positions -484,
-478, and -472, which, when methylated, interfered with
protein binding (Fig. 6). EMSA on mutant oligonucleotides (Fig. 7) as well as DNA fragments isolated from mutant FGF-1.B
reporter constructs (Fig. 8) confirmed that these residues are
essential for protein binding. Interestingly, human and mouse sequences
in this region are identical. ()The functional analyses of
-540 mut as well as two additional deletion clones (Del-1 and
Del-2) showed that mutations in this region resulted in a decrease in
the luciferase activity. Two mutant constructs in the Enhancer vector,
namely -540 mut and Del-2, showed reporter activity 60% of wild
type in U1240MG. The construct containing RR1 (-145 wt) confers
transcriptional activity, averaging 46% of the -540 wt activity (Fig. 1). Surprisingly, the effect was more dramatic with Del-1,
with transcription being only 45% of the wild type level. This was
unexpected, since the point mutations in -540 mut alone were so
effective in eliminating the specific protein binding. The activities
of promoter constructs in pGL2-Enhancer were confirmed using promoter
constructs in pGL2-Basic. Notably, Del-2 has higher diminishing effect
than Del-1 when the Basic constructs were used (Fig. 9). The
diminishing effect observed in Del-1 is mostly likely due to the
deletion of -489 to -485. Altogether, these results suggest
that the protein binding between -489 and -467 functions as
an architectural component necessary for the proper assembly of a
multiprotein complex that can interact with RR-1 and/or the initiation
complex at the start site. There is increasing evidence that the
formation of a stereospecific nucleoprotein complex may serve as a
mechanism for achieving a high level of specificity and gene activation
(Tjian and Maniatis, 1994). Studies of the human interferon
gene
have identified a protein, HMGI(Y), whose binding alters DNA structure
and increases the DNA binding affinity of NF-kB and ATF-2 (Du et
al., 1993). We are currently conducting a more detailed analyses
of RR-2 to delineate the boundaries and functionality of the binding
region.
None of the mutations had an effect in the FGF-1.B negative
cell line, U1242MG, supporting the role of RR-2 in the tissue-specific
regulation of FGF-1.B. EMSA shows that the protein(s) interacting with
RR-2 are present in both cell lines yet with slightly different
molecular weight (Fig. 5). Results of Southwestern analysis of
partially purified nuclear extracts from U1240MG, U1242MG, and human
brain tissue revealed a 37-kDa protein in U1240MG and brain tissue but
absent from U1242MG (Fig. 11). Most importantly, this 37-kDa
protein does not interact with the mutE probe. It is possible that this
protein, acting in conjunction with other proteins, is responsible for
the tissue-specific regulation of FGF-1.B. We have used a concatenated
probe derived from nucleotides -489 to -467 to screen brain
expression cDNA libraries and have identified two cDNA clones that are
capable to be bound to the oligonucleotide probe. ()Further
characterization of these two clones will allow us to explore the
nature of brain-specific transcription of the FGF-1 mRNA.
Fig. 10shows the sequence of the FGF-1.B promoter region.
Sequences with a 80% sequence identity with the murine FGF-1 gene ()are indicated. Interestingly, the sequences surrounding
RR-2 and the core promoter regions have significant homology with the
murine gene, suggesting biological significance of this region.
Sequence analysis of RR-2 shows an area of identity (-482 to
-476) to a cis-acting element in the nodulin-23 gene
(Mauro et al., 1985). Likewise, in RR-1, an area (-139
to -131) identical to a positive regulatory element in the human
apolipoprotein E gene is found (Smith et al., 1988b). The
significance of these sequences in the FGF-1.B promoter remains to be
determined.
In conclusion, we have characterized the FGF-1.B proximal promoter and have identified two positive regulatory elements in addition to the core promoter. We have identified a 41-bp enhancer region that binds to multiple proteins, including a tissue-specific protein of 37 kDa. It will be important to further identify these interactions and to characterize the protein(s) and ascertain the mechanism of the tissue-specific transcriptional activation of FGF-1.B. With its four promoters and alternative splicing to generate multiple transcripts, the regulation of FGF-1 is complex. Complex regulation may be indicative of a requirement for strict activation and inactivation during differentiation or development. Understanding the regulation of this gene will have important implications for the understanding of the role of FGF-1 in cell growth and differentiation.