©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Characterization of the Brain-specific FGF-1 Promoter, FGF-1.B (*)

(Received for publication, December 27, 1994; and in revised form, January 25, 1995)

René L. Myers (§) Subir K. Ray Rebecca Eldridge Maqsood A. Chotani Ing-Ming Chiu (¶)

From the Department of Internal Medicine and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Acidic fibroblast growth factor (or aFGF-1) (^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.


MATERIALS AND METHODS

Construction of Deleted and Site-directed FGF-1.B Mutants

An 891-bp HindIII-DraIII fragment isolated from clone pKL111-7 (Myers et al., 1993) was blunt ended using T4 DNA polymerase and ligated with pGL2-Basic luciferase vector (Promega), which had been linearized with BglII and blunt ended. The insert contains the FGF-1.B proximal promoter sequences between the NcoI and DraIII sites (nucleotides -831 to +31 in Fig. 10). The resulting clone was designated pRM56-3. In addition, pRM56-3 polylinker sites XhoI and HindIII were used to subclone this same FGF-1.B region (-831 to +31) into the XhoI/HindIII sites of the pGL2-Enhancer luciferase vector (Promega), which contains the SV40-derived enhancer element. The enhancer clone was designated pRMenh-1. The two parent clones, pRM56-3 and pRMenh-1, were then used to generate sequential 5`-deletions using exonuclease III and mung bean nuclease (Stratagene) (Henikoff, 1984). Plasmids were double digested with KpnI and XhoI, unique 3`- and 5`-overhang restriction sites, respectively. The double-digested DNA was treated with exonuclease III to make a portion of the insert single stranded. A single exonuclease III reaction was set up (50 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 10 mM beta-mercaptoethanol, 10 µg/ml tRNA, 250 units of exonuclease III), and aliquots were removed at 15-s intervals for 2 min. Each time point was treated with mung bean nuclease (30 mM NaOAc, pH 5.0, 50 mM NaCl, 1 mM ZnCl(2), 5% glycerol, 15 units of mung bean nuclease) and ligated with T4 DNA ligase to recircularize the plasmid. The DNA was transformed into DH5alpha bacteria using 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and blue/white colony selection. Plasmid DNA from white colonies was analyzed by digestion with SmaI and compared with the parent plasmid to determine the amount of DNA deleted. A panel of deletions was selected, and their exact 5`-ends were determined by dideoxy sequencing using Sequenase (U. S. Biochemicals/Amersham Life Science). To generate identical FGF-1.B promoter construct in both the pGL2-Basic and the pGL2-Enhancer vectors for analyses, selected inserts were released by digestion with SmaI and HindIII and subcloned into the SmaI-HindIII sites of the alternate vector (see diagram in Fig. 1).


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(2), 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 DH5alpha 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.

Transfection and Analysis of Promoter Activity

U1240MG or U1242MG cells (human glioblastoma cell lines originally established from surgical specimens of malignant gliomas) were plated in 60-mm Falcon tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) to achieve 60-80% confluency 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. Briefly, 30 µl of the transfection reagent was diluted to 100 µl with HBS (20 mM Hepes, pH 7.4, 150 mM NaCl) in a polystyrene vial. Plasmid DNA was diluted separately to 100 µl with HBS in a polystyrene vial. Both solutions were mixed and incubated at room temperature for 10 min. 2 ml of culture medium was added to the DNA mixture and pipetted on the cells following aspiration of the old tissue culture medium. Cells were incubated at 37 °C, 5% CO(2) for 24 h. The medium was then aspirated and replaced by 5 ml of fresh medium and incubated for an additional 24 h. The cultures were then prepared for analysis of luciferase activity using the luciferase assay system from Promega. Briefly, medium was aspirated, and monolayers were washed twice with phosphate-buffered saline. Lysis buffer (250 µl, Promega) was added to the cells and swirled gently. Plates were incubated at room temperature for 10 min, and contents were scraped and transferred to a microfuge tube. Tubes were centrifuged briefly (15 s) to pellet large debris; 20 µl were transferred to a polystyrene reaction tube and placed in a Lumat LB 9501 luminometer (EG& Berthold, Berthold Systems Inc., Pittsburgh, PA). Luciferase assay reagent (100 µl, Promega) was injected, and the light produced was measured for 20 s. Protein determinations were made on 25 µl of the cell lysate using the Bio-Rad DC protein assay system. Data were expressed as total light units per milligram of protein.

Preparation of Nuclear Extracts

Nuclear extracts were prepared based on the method of Dignam et al.(1983) as outlined in Ausubel et al.(1989) with minor modifications. Cells were grown in T175 Falcon tissue culture flasks or roller bottles (Becton Dickinson). At confluency, cells (3-6 times 10^8) were washed with phosphate-buffered saline, scraped, and collected in conical tubes. Cells were centrifuged 10 min at 1,850 times g at 4 °C. The cell pellet was resuspended in 5 packed cell volumes of cold hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl(2), 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM DTT, 1 µg/ml leupeptin, 0.9 unit/ml aprotinin) and immediately centrifuged for 5 min at 1,850 times g. The pellet was resuspended in hypotonic buffer to 3 packed cell volumes and allowed to swell on ice for 10 min. Nonidet P-40 was added to a final concentration of 0.85%, and cells were homogenized 20 strokes in a Dounce homogenizer with pestle A and then vortexed for 20 s. The nuclei were pelleted 15 min at 3,300 times g, and the cytoplasmic extract was removed. Packed nuclei volume was determined, and nuclei were resuspended in 0.5 packed nuclei volume low salt buffer (20 mM Hepes, pH 7.9, 1.5 mM MgCl(2), 0.2 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, 0.2 mM PMSF, 1 µg/ml leupeptin, 0.9 unit/ml aprotinin). High salt buffer (0.9 M KCl) was slowly added, with constant mixing, to a final concentration of 0.3 M KCl. The nuclei were extracted at 4 °C for 30 min. The extract was centrifuged 30 min at 4 °C at 25,000 times g. The supernatant was dialyzed in micro-colloidion bags (Sartorius, Haywood, CA) against 50 volumes of dialysis buffer (20 mM Hepes, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.2 mM PMSF, 1 µg/ml leupeptin, 0.9 unit/ml aprotinin) for 1 h. The extract was removed from the dialysis bag and centrifuged for 20 min at 4 °C at 25,000 times g. Aliquots (20 µl) were frozen in liquid N(2) and stored at -80 °C. Protein concentrations were measured using the Bio-Rad protein assay. The extracts typically contained 5-7 mg of protein/ml and remained stable at -80 °C for several months. Nuclear extracts from human brain tissue were prepared as described by Gorski et al.(1986).

EMSA

DNA probes were prepared from various FGF-1.B reporter clones following digestion with the appropriate restriction enzymes, agarose gel electrophoresis, and band isolation using the Geneclean system (Bio 101, Inc., La Jolla, CA). DNA fragments were labeled by a fill-in reaction using Klenow enzyme (Boehringer Mannheim) and appropriate [alpha-P]dNTPs. Alternatively, oligonucleotides were labeled with [-P]ATP and polynucleotide kinase, annealed to their complementary strand by heating at 85 °C for 5 min and allowing the reaction to slowly cool to room temperature. Radiolabeled probes were then used in binding reactions combining 30,000 cpm of probe, 3 µg of poly(dI-dC), 15 µg of crude nuclear extract, 10% glycerol, and cold competitor when desired in a final reaction volume of 20 µl in incubation buffer (25 mM Hepes, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 1.5 mM DTT). The binding reactions were incubated at room temperature for 30 min. 2 µl of 0.25% bromphenol blue and xylene cyanol dye solution was added to each reaction prior to loading onto a 4% polyacrylamide gel. The gel was electrophoresed with 0.5 times TBE (45 mM Tris borate, 1 mM EDTA, pH 8.0) at a constant current of 20 mA. Following electrophoreses, the gel was dried under vacuum and exposed to an x-ray film.

Methylation Interference Footprinting

Methylation interference analyses were performed as outlined in Ausubel et al.(1989) with minor modifications. The noncoding strand FGF-1.B sequence between -507 and -467 was end labeled using [-P]ATP and polynucleotide kinase and methylated with dimethyl sulfate for 1 min at 20 °C. Binding reactions and electrophoresis were as described for the EMSA, except eight reactions were pooled and loaded across four wells to augment the signal. After electrophoresis, the DNA was localized by autoradiography of the wet gel for 6-12 h, and the bound and free bands were excised from the gel. The DNA was electroeluted from the gel in 0.5 times TBE and purified through a 0.22-µm cellulose acetate spin filter unit (Costar, Cambridge, MA). The DNA was then treated with 1 M piperidine at 90 °C for 30 min and lyophilized to dryness using a Speed-Vac concentrator. The sample was redissolved in 50 µl of H(2)O 2-3 times and lyophilized to dryness. The DNA (3,000 cpm) was denatured and electrophoresed on an 8% sequencing gel.

Partial Purification of the Nuclear Extract and Southwestern Analysis

3 mg of nuclear extract (U1240MG or U1242MG cells) was passed through a 0.5-ml heparin-agarose column at 4 °C pre-equilibrated with ice-cold buffer A (20 mM Hepes, pH 7.6, 0.1 mM EDTA, 0.02 mM DTT, 0.2 mM PMSF, and 10% glycerol) containing 0.1 M KCl. The column was washed with four bed volumes of buffer A containing 0.1 M KCl. The bound proteins were eluted at 4 °C with three bed volumes of ice-cold buffer A containing 0.75 M KCl and dialyzed against buffer containing 0.1 M KCl for 2.5 h at 4 °C. The proteins from the partially purified nuclear extracts (9.5 µg) of U1240MG and U1242MG cells as well as from brain nuclear extract (15 µg) were separated in a 12% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The proteins in the blot were denatured and renatured at 4 °C by sequential addition of 6.0, 3.0, 1.5, 0.75, 0.375, 0.1875, and 0.0938 M guanidine HCl in 1 times binding buffer (25 mM Hepes, pH 7.9, 3 mM MgCl(2), 40 mM KCl, 1 mM DTT, and 0.2% Nonidet P-40). In each step, the blot was gently shaken for 5 min. The blot was then blocked with 5% Blotto in 1 times binding buffer at room temperature for 2 h and prehybridized for 30 min at room temperature in 1 times binding buffer containing 0.25% milk, 10 µg/ml poly[d(I-C)], and 50 µg/ml tRNA. The blot was hybridized for 2 h with P-labeled (1.5 times 10^6 cpm) oligonucleotide E (-489 to -467) in 1 ml of prehybridization solution at room temperature. The blot was washed (three times, each time for 5 min) with 0.25% milk in 1 times binding buffer, rinsed quickly in 1 times binding buffer, and autoradiographed.


RESULTS

Luciferase Activity of FGF-1.B 5`-Deletion Clones

To identify cis-regulatory elements in the FGF-1.B proximal promoter, we constructed a series of sequential 5`-deletions fused to the luciferase reporter gene. Several reporter constructs containing up to -831 bp of FGF-1.B promoter were transfected into the FGF-1.B positive human glioblastoma cell line, U1240MG. Relatively low transcriptional activity was detected in these constructs. To assess this region for promoter activity and to identify cis-regulatory elements, the SV40 enhancer was included in the luciferase reporter constructs. The transcriptional activity of these recombinants was determined following transfection into U1240MG and U1242MG, a 1.B negative cell line (Myers et al., 1993). The results of transfection experiments using constructs containing the SV40 enhancer are summarized in Fig. 1. The data are expressed as total light units/mg of protein and represent the mean of at least two separate DNA preparations. The numbers above the bars indicate the number of experiments conducted.

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.

EMSA of the FGF-1.B Proximal Promoter

EMSA using radiolabeled probes spanning -540 to +35 from the 1.B promoter region have identified sequence-specific binding regions for nuclear protein in RR-1 and RR-2. Fig. 2shows the results of EMSA using overlapping genomic fragments containing FGF-1.B sequences between -196 and +35. A major complex (marked as 1) is seen with U1240MG nuclear extract when either fragment A or B was used as the probe, narrowing the region of protein interaction to sequences between -146 and -111. The complex is easily competed by 40-fold molar excess of fragment A and B but not by fragment C. This -146 to -111 DNA region corresponds to RR-1 in Fig. 1, and its deletion resulted in diminished reporter gene activity. There is also a second complex with much slower mobility that is less prominent. Interestingly, complex 3 was detected when probe A was competed with excess amounts of fragment B. No specific gel shift corresponds to the core promoter region as fragment C, when used as a probe, does not result in mobility shift (data not shown).


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 times 10^6 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.



Methylation Interference Analysis

To more precisely determine the location of nucleotides in the -507 to -467 region that interact with the protein(s), we used methylation interference analysis. Our results (Fig. 6) indicated that methylation of at least three guanine nucleotides on the noncoding strand at positions -484, -478, and -472 (denoted by stars in Fig. 6) interfered with binding. No contact sites were detected on the coding strand by this method (data not shown). Because the majority of guanine nucleotides were more diffuse and less intense in the bound lane versus the free lane, we used site-directed mutagenesis to confirm the three guanine nucleotides that were completely absent in the free lane did represent contact nucleotides for the DNA-protein interaction observed in the EMSA shown in Fig. 4and Fig. 5. Therefore, EMSA was conducted using both wild type and mutant oligonucleotide sequences in which the residues at positions -484, -478, and -472 on the coding strand were changed from cytosine to adenine nucleotides. U1240MG nuclear extract was used in the binding reaction, and the results are shown in Fig. 7. Probes D and E are wild type sequences with boundaries as shown. Probe D is identical to that used in Fig. 5; mutE is identical to probe E with the exception of the three point mutations at positions -484, -478, and -472. As expected, probes D and E are able to form a complex with U1240MG proteins. In contrast, the mutE probe is unable to bind any proteins in the U1240MG extract or compete as effectively for binding as probes D and E. Additionally, the oligonucleotide from -507 to -490 neither shifts itself nor competes with the complexes formed by probe D or E (data not shown). The results confirm that these nucleotides are critical for the protein interaction(s) in RR-2.


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.



Analysis of Binding Activity of FGF-1.B Promoter Mutants

To further address the importance of sequences 3` to -489 in RR-2 activity, we cloned the three point mutations discussed above into the context of the -540 to +31 proximal promoter luciferase construct and conducted EMSA with genomic fragments isolated from the wild type plasmid (probeA in Fig. 8) and from the mutant plasmid (mutA in Fig. 8). The results show that the wild type fragment is much more efficient in binding protein and being retarded through the polyacrylamide gel. As shown previously, the competition analysis indicates that only sequences 5` to -467 are involved since probe C does not compete for binding with probe A. Significantly, probe E (-489 to -467) does compete for binding with probe A, with 95% efficiency. These data support the idea that sequences between -489 and -467 are important in the formation or stability of the protein-DNA interaction(s) observed. Likewise, mutA is much less efficient (<2%) in binding protein (Fig. 8).


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.



Functional Analyses of FGF-1.B Proximal Promoter Mutants

To determine if the sequence-specific protein interaction is responsible for the enhancer activity in RR-2, reporter activity was determined in three different 1.B proximal promoter mutants. The reporter construct containing sequences -540 to +31 with the three point mutations at positions -484, -478, and -472 was designated -540 mut. To assess the importance of the other sequences in RR-2 5` and 3` to position -489, two deletion constructs were cloned. Del-1 is the parent -540 wt, which contains a deletion of nucleotides -512 to -485; Del-2 is the parent -540 wt, which contains a deletion of nucleotides -490 to -467. These three mutant clones diminished the luciferase activity in U1240MG cells by 39, 55, and 31%, respectively, as compared with the -540 wt clone. The luciferase activity of RR-1 (construct -196 to +31) averages 50% of that with -540 wt, which contains RR-2 in addition to RR-1. Therefore, the results using the mutant constructs are consistent with the interpretation of an enhancer element(s) in the -507 to -467-bp region, since the activity is diminished to the level of that seen with only the RR-1 and core promoter regions. Interestingly, the results show that the mutants had no diminishing effect of the minimal luciferase activity as compared with the -540 wt in the FGF-1.B negative cell line, U1242MG (data not shown).

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.



Southwestern Analysis Identified a Specific Nuclear Factor in Brain and U1240MG

Using EMSA, sequences between -507 and -467 result in a unique complex following binding with U1240MG nuclear extract as compared with U1242MG nuclear extract (Fig. 5). However, the mobility difference between U1240MG and U1242MG complexes is very small, and further proof is desired. To further examine the sequence-specific DNA binding activity between -489 and -467, we conducted a Southwestern analysis on partially purified nuclear extract from U1240MG, U1242MG, and human brain tissue. The protein gel was blotted with radiolabeled probe E and mutE (-489 to -467). Fig. 11A showed three minor proteins (118, 62, and 40 kDa) and a major 37-kDa protein from U1240MG nuclear extract, which bound to oligonucleotide probe E. In the brain extract, a 43-kDa protein instead of a 40-kDa protein was shown to bind weakly to probe E. Of particular interest is the 37-kDa protein, which is absent in the U1242MG extract while present in both the U1240MG and brain extracts. Furthermore, this 37-kDa protein did not bind to mutE while the other three minor proteins and an additional 45-kDa protein were found to bind to the mutE probe in all three nuclear extracts (Fig. 11B). These results are consistent with the hypothesis of a tissue-specific protein, which is responsible for the tissue-specific transcription of FGF-1.B. The functional differences seen in the luciferase construct -540 mut between U1240MG and U1242MG may be attributed in part to the 37-kDa protein.

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.


DISCUSSION

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 beta-galactosidase expression vector. However, we found that these glioblastoma cell lines had a high background of beta-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. (^2)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 beta 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. (^3)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 (^4)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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant R01 CA45611. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by National Institutes of Health National Research Service Award T32 CA09338.

Supported by National Institutes of Health Research Career Development Award K04 CA01369. To whom correspondence should be addressed: Dept. of Internal Medicine, Ohio State University, Davis Medical Research Ctr., 480 West Ninth Ave., Columbus, OH 43210. Tel.: 614-293-4803; Fax: 614-293-5631; chiu.1{at}osu.edu.

(^1)
The abbreviations used are: FGF, fibroblast growth factor; bp, base pair(s); EMSA, electrophoretic mobility shift analyses; wt, wild type; mut, mutant; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol.

(^2)
K. Alam and I.-M. Chiu, unpublished results.

(^3)
V. Leybman and I.-M. Chiu, unpublished results.

(^4)
K. Alam and I.-M. Chiu, unpublished results.


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