From the
Department of Medicine, Mount Sinai Hospital and the
Department of Pathology, University Health Network, Freeman Centre for Endocrine Oncology and Ontario Cancer Institute, University of Toronto, Toronto, Ontario M5G 2M9, Canada and the ¶Department of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, December 6, 2002 , and in revised form, January 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Full-length FGFR4 has been reported to be expressed mainly in adult lung, liver, kidney, pancreas, muscle, and spleen (5, 6). It initially was considered not to have a significant role in tumorigenesis. However, FGFR4 has been shown to mediate membrane ruffling in breast carcinoma cells (7) and to modulate erythroid cell proliferation (8). Moreover, mutational analyses of the kinase domains of FGFR1, FGFR3, and FGFR4 revealed that all three receptor domains have comparable transforming properties (9). We have shown that targeted expression of a human pituitary tumor-derived FGFR4 isoform (ptd-FGFR4) recapitulates pituitary tumorigenesis (4). This truncated form of FGFR4 is a result of transcription initiation in intron 5 and a translation start site in exon 6 (4). In this report, we investigate an alternative FGFR4 promoter that may be implicated in the genesis of ptd-FGFR4. Sequence analysis of the human FGFR4 gene predicts a possible promoter within intron 4 including a classic TATA box with a possible transcription start site in intron 5 (4, 10). We thus focused on intron 4 and the surrounding region and determined the relevant cis-DNA-binding elements in pituitary cells that may contribute to utilization of this site.
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MATERIALS AND METHODS |
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PlasmidsPromoter analysis of the human FGFR4 gene was performed with the assistance of a gene finder. The convention for sequence coordinates with +1 as the first base of the coding sequence in exon 1 was adopted. Analysis of FGFR4 intronic sequences predicted a possible promoter within intron 4 (nucleotides 50565166) and a classic TATA box (nucleotides 54125417) with a possible transcription start site in intron 5 (nucleotide 5453). To generate the putative intron 4 (In4)-inclusive promoter, a KpnI/XhoI fragment of 313 bp consisting of intron 4 and exon 5 was derived by PCR from PAC# 32C5 (11). The upstream primer was 5'-GGACCTCTCGAATAGGCACA-3' (nucleotides 50215040), and the downstream primer was 5'-GGCTTACGGAGGTTACGCCA-3' (nucleotides 53335314). After cloning into TA vector (Invitrogen), products were subcloned into their corresponding sites of the promoterless firefly luciferase expression vector pGL3 (Promega, Madison, WI) to yield the P313-Luc plasmid. The orientation and sequence of all constructs were verified by restriction analysis and nucleotide sequencing, respectively. The expression vector pcDNA-AP-2 encoding the complete sequence for AP-2
cloned into pcDNA3.1 (Invitrogen) KpnI/XhoI sites was used as described previously (12).
Site-directed MutagenesisSite-directed mutagenesis was performed using a transformer site-directed mutagenesis kit (Clontech Laboratories, Inc., Palo Alto, CA) following the manufacturer's instructions. Mutations of the AP-2 transcription core binding site (-CCCTCCAGC-) substituted by -CTTTCCAGC- and the NFB binding site (-GGGGCCTTCC-) substituted by -GGCGCCTTGC- were introduced separately into the P313-Luc construct and In4 B2 fragment using mutagenic primers for the sites. Another primer containing the mutation of a unique restriction site was used as a selection marker. Mutations were confirmed by restriction digestion and nucleotide sequencing.
Transfection and Luciferase AssaysAll plasmid reporters and expression vectors were prepared by column chromatography (QiaGen, Missisauga, Ontario) for sequencing and transfections. Cells were transfected by the LipofectAMINE method (Invitrogen) according to the manufacturer's protocol. Cells were plated into six-well cluster dishes (7 x 104 cells/well) and were transfected the following day with 3 or 5 µl/well LipofectAMINE and 1 or 2 µg of DNA/well as indicated. The total amount of transfected DNA was kept constant by adding empty vector. Transfection efficiency was monitored by simultaneous cotransfection with a -galactosidase reporter construct (20 ng/well). Histone deacetylase inhibition was performed using trichostatin-A (Sigma) at 100 ng/ml for 24 h. 48 h following transfection, cells were lysed in a buffer containing 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, Triton X-100, and 1 mM dithiothreitol. Luciferase activity was measured for 20 s in a luminometer.
-Galactosidase activity was measured to normalize for variations in transfection efficiency. Promoter activity of each construct was expressed as firefly luciferase/
-galactosidase activity. Each experiment was performed independently on three separate occasions with at least triplicate wells in each experiment.
Preparation of Nuclear ExtractsNuclear extracts were prepared by washing cells in 1x phosphate-buffered saline and lysing in 100 µl of buffer (10 mM HEPES, pH 7.9, 1 mM dithiothreitol, 1 mM EDTA, 60 mM KCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) for 5 min on ice. The pellet was resuspended in 100 µl of the nuclear resuspension buffer (0.25 mM Tris-HCl, pH 7.8, 60 mM KCl, 1 mM dithiothreitol, 1.5 mM phenylmethylsulfonyl fluoride) and lysed with three cycles of freezing and thawing to 37 °C. After centrifugation at 13,000 rpm for 10 min at 4 °C, the clear supernatant was collected and kept at -80 °C for further analysis. Protein concentrations were determined by the Bio-Rad protein assay.
Electrophoretic Mobility Shift Assays (EMSAs)Oligonucleotides were labeled with [-32P]ATP using the T4 DNA polynucleotide kinase. Five µg of nuclear protein extracts and 2 ng of labeled oligonucleotides were allowed to bind for 30 min at room temperature in a final volume of 20 µl of binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM MgCl2, 2% glycerol, and 1 µg of poly(dI-dC) (Amersham Biosciences, Peapack, NJ). Protein-DNA complexes were resolved in 4% polyacrylamide gels containing 0.5x Tris borate/EDTA. Overlapping double-stranded oligonucleotide fragments of intron 4 of FGFR4 (nucleotides 50565166) were used as probes and for competition in EMSAs as shown in Fig. 1 and detailed as follows: fragment A1 (nucleotides 50565085), 5'-GTCAGTAGGTCTCCAAGGACTTGTGTCCCG-3'; fragment A2 (nucleotides 50775115), 5'-TGTGTCCCGCTGCTGCTCATCTGATCACTGAGAAGAGGA-3'; fragment B1 (nucleotides 51065136), 5'-GAGAAGAGGAGGCCTGTGTGGGAACACACGG-3'; and fragment B2 (nucleotides 51275166), 5'-GAACACACGGTCATTCTAGGGGCCTTCCCCTGCCCTCCAG-3'. In some experiments, the B2 fragment was further extended (B2ex) to include the first six nucleotides of exon 5, thereby including the entire AP-2 consensus binding site, 5'-GAACACACGGTCATTCTAGGGGCCTTCCCCTGCCCTCCAGCACCCT-3' (Fig. 1).
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Competitor double-stranded oligonucleotides containing transcription binding sites were as follows: AP-2, sense 5'-CCACAAACGACCGCCCGCGGGCGGT-3' (commercially obtained from Geneka Biotech Inc., Carlsbad, CA) and its complementary strand; the mutant oligonucleotide for mAP-2, sense 5'-CCACAAACGACCGATTGCGGGCGGT-3' and its complementary strand; AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3' and its complementary strand; AP-2, 5'-GATCGAACTGACCGCCCGCGGCCCGT-3' and its complementary strand; AP-4, sense 5'-TTACTCCCAGCTCCAGCCGG-3' and its complementary strand; and NF
B, sense 5'-AGTTGAGGGGACTTTCCCAGGC-3' and its complementary strand (synthesized by Sigma). Complementary strands were annealed in a buffer of 10 mM Tris-Cl, pH 8.0, 50 mM NaCl, and 1 mM EDTA before being used as probes or competitors.
Gel shift probes were purified with a Sephadex G-50 spin column after labeling. For competition assay, 10, 20, 50, or 100 M excess of unlabeled fragment was added as competitor DNA. Samples were electrophoresed on 4% polyacrylamide non-denatured gels containing 0.5% Tris borate buffer and 2% glycerol. Gels were dried under a vacuum and autoradiographed.
Western Blot AnalysisProtein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein (50 µg) from whole cell lysates or nuclear fractions were solubilized in 2x SDS-sample buffer, separated on SDS-8% polyacrylamide gels, and transferred to nitrocellulose. Blots were incubated with polyclonal antisera raised against the carboxyl terminus of AP-2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) or FGFR4 (1:1000; Santa Cruz Biotechnology), or a monoclonal antibody against actin (1:500). The specificity of the detected bands was confirmed by preabsorption of antisera with purified peptide.
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RESULTS |
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Intron 4 of FGFR4 Contains Binding Sites for AP-2To determine which factors potentially may interact with this putative intronic region, we screened potential candidates using EMSA. EMSAs were performed using nuclear extracts from exponentially growing rat pituitary tumor GH4 cells or from primary human pituitary tumors as described previously (13). The overlapping fragments consisted of 3040 bp of intron 4 that were designated as A1, A2, B1, and B2. These fragments were labeled and used as probes for the assessment of specific binding. Fig. 2A depicts an example of an EMSA using GH4 rat pituitary tumor nuclear extract incubated with the In4 A1 probe. We found no evidence of specific complexes that could be competed by putative transcription factors using this In4 A1 fragment. Similar nonspecific findings were obtained with the In4 A2 and In4 B1 fragments. In contrast, incubation of the same nuclear extracts with the In4 B2 fragment yielded distinct complexes that were competed specifically by wild type but not mutant AP-2 oligonucleotides (Fig. 2B). This complex was not competed by oligonucleotides for predicted binding sites for AP-4, OCT-1, Ik, or STAT but was competed partially with an oligonucleotide for NF
B. The putative AP-2 binding site is situated on the intron-exon junction (Fig. 1). Extension of the probe by 6 nucleotides into exon 5 (In4 B2ex) to complete the AP-2 consensus binding site resulted in a more distinct complex that was supershifted by AP-2
antibody (Fig. 2C).
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To determine further the specificity of the putative intron 4 interaction with AP-2, we examined the ability of an AP-2 consensus probe to form complexes with GH4 nuclear extracts. This complex was competed specifically by the In4 B2 fragment but not by other fragments from the same intron (Fig. 2D). Moreover, the In4 B2 probe formed specific complexes with the recombinant GST-AP-2 fusion protein (Fig. 2D). Mutation of the putative AP-2 binding site in the B2 fragment resulted in a complex that was not competed by wild type AP-2 (Fig. 2E), consistent with the presence of a single AP-2 binding site in this fragment of intron 4. This complex, however, was competed partially by NF
B (Fig. 2E). Consistent with these findings, an NF
B probe also formed complexes with GH4 nuclear extracts that were competed specifically by In4 B2 but not by the adjacent In4 A2 sequence (Fig. 2F). These results indicate that the region of the cryptic promoter contained within In4 B2 contains adjacent consensus binding sites for AP-2 and NF
B. Furthermore, GH4 cells express nuclear factors that bind to these sites, and this multiprotein-DNA complex is supershifted with antibody to AP-2
, thus confirming that AP-2
is one factor that targets the region encompassing In4 B2.
Primary Human Pituitary Tumors Express AP-2Given our previous findings of a human pituitary tumor-derived FGFR4 isoform, we sought evidence for a potential role of the AP-2 binding site in intron 4 in primary human pituitary tumors. Nuclear proteins from primary human pituitary tumors formed the same slow migrating complexes with the In4 B2 probe (Fig. 3A). These complexes were competed specifically by an oligonucleotide for wild type AP-2 but not by mutant AP-2.
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To confirm that primary human pituitary tumors express the AP-2 protein, we examined by Western blotting extracts from primary human pituitary adenomas characterized as expressing the truncated ptd-FGFR4 (4). We identified a protein of
50 kDa using an AP-2
-specific antibody in nontumorous pituitary tissue and in pituitary adenomas (Fig. 3B). This product comigrated with protein derived from HEK 293 cells transfected with an expression vector for AP-2
. When adjusted for actin levels, AP-2
protein was more abundant in the pituitary tumors than in nontumorous pituitary tissue.
Functional Contribution of AP-2 in Regulation of FGFR4 To determine the potential functional contribution of the AP-2 binding site in intron 4 (P313-Luc) on promoter activity, we examined the effect of mutation of this site on promoter activity. Disruption of the AP-2 site in the B2 fragment resulted in marked reduction in luciferase activity (Fig. 4). In addition, mutation of the NFB site also resulted in a loss of reporter activity but to a lesser extent than that noted with the loss of the core AP-2 binding site. Conversely, inclusion of exon 5 and intron 5 did not enhance promoter activity (data not shown).
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Transfection of AP-2 in GH4 cells treated with trichostatin-A resulted in enhanced expression of ptd-FGFR4 (Fig. 5). Taken together, these results suggest that AP-2
in pituitary tumors, coupled with chromatin changes such as HDAC inhibition, result in enhanced utilization of a cryptic promoter within intron 4, contributing to the genesis of the truncated receptor ptd-FGFR4.
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DISCUSSION |
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Two earlier reports identified the human and murine FGFR4 promoter regions and their transcription start sites (10, 11). Neither study, however, addressed characterization of putative transcription factor binding or transcriptional activity. Our analysis of the 5' FGFR4 promoter revealed a region 115 bp upstream of the start site that we found to be essential for reporter activity in pituitary cells (13). Overlapping 4050-bp fragments of this minimal functional promoter were examined by EMSAs. Specific DNA-protein complexes were noted with fragments that contained multiple binding sites for Sp1. This was consistent with the role of Sp1 in the regulation of other FGFR promoters lacking TATA and CAAT sequence motifs including FGFR1 (14), FGFR2, and FGFR3 (15). For each of these FGFRs, 100 bp of sequence 5' to the transcription initiation site appear to confer transcriptional activity to the promoters (16). We further characterized fragment C (65 to 25) of FGFR4, as it demonstrated consistently strong binding with nuclear extracts from rat pituitary cells. This fragment contains predicted sites for binding the zinc finger-containing protein Ik, flanked by two sites for Sp1 and Ets-type factors (13). These studies led to the localization of Ik in the pituitary and to identifying it as an important contributor to cell-specific FGFR4 transcriptional regulation.
The current studies point to AP-2 as an important factor that recognizes a downstream cryptic promoter in intron 4 of FGFR4. The AP family of transcription factors consists of four different members known as AP-2
, AP-2
, AP-2
, and AP-2
(12, 17). The carboxyl-terminal half of these proteins contains a basic domain and a highly conserved helix-span-helix motif that mediates DNA binding and dimerization. In particular, it has been suggested that AP-2
is important in regulating cell proliferation through control of a number of genes responsible for terminal differentiation as well as apoptosis (17, 18, 19).
Our current findings also point to an accompanying role for NFB in binding and regulating intron 4. In the pituitary, NF
B has been implicated in mediating a number of hormonal signals, including responsiveness to inhibition by dopamine in prolactin-producing pituitary cells (20). Interestingly, regulation of the cholecystokinin (21) and serum amyloid A1 (22) gene promoters also involves partially overlapping consensus sites for AP-2 and NF
B. These studies demonstrated a complex functional interaction between these two transcription factors. Our promoter analysis similarly indicates that both NF
B and AP-2
are involved coordinately in the regulation of this cryptic promoter. The close proximity of these two sites further suggests a potential interaction between these two distinct factors to regulate coordinately FGFR4 gene expression.
Several pituitary hormone genes, including proopiomelanocortin (23), prolactin (24), the corticotrophin-releasing hormone receptor (25), and somatostatin receptors (26), have been described as containing AP-2 binding sites. The presumed functional role of AP-2 in transcriptional regulation of many of these putative target genes, however, remains to be determined.
The presence of AP-2 binding sites in intronic sequences has been identified only rarely. DNase I footprinting experiments using intron 1 of the peripherin gene revealed at least two protected regions (In 1 A and In 1 B). In 1 A encompasses an AP-2-like binding site that interacts with both neuroblast and fibroblast nuclear proteins as well as with recombinant AP-2 protein. Gel shift experiments, however, suggested that the interacting nuclear factors are distinct from AP-2
but nonetheless probably belong to the AP-2 family (27). Analysis of the laminin receptor precursor promoter similarly revealed that the first intron contains enhancer activity. This region was shown to include a functional AP-2 cis-acting element that is repressed by p53 (28). Our current findings provide evidence for AP-2
as a distinct factor that is expressed in pituitary tumors, is able to recognize the distal region of an intronic sequence, and is a participant in the regulation of a cryptic FGFR4 promoter.
The mechanisms of FGFR action in endocrine cell development and tumorigenesis remain to be elucidated (1). Our previous identification of a novel human pituitary tumor-derived truncated FGFR4 isoform with distinct properties from wild type FGFR4 (4) has provided a unique opportunity to examine the effects of alternative transcription initiation on FGFR4 function. Our current findings on the recognition of an intronic sequence of FGFR4 by AP-2 and the strong expression of this factor by pituitary tumors begin to address the mechanisms that may favor alternative promoter utilization in transformed cells.
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FOOTNOTES |
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|| To whom correspondence should be addressed: University of Toronto-Mount Sinai Hospital, 600 University Ave., #437, Toronto, Ontario M5G 1X5, Canada. Tel.: 416-586-8505; Fax: 416-586-8834; E-mail: sezzat{at}mtsinai.on.ca.
1 The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; ptd-FGFR4, human pituitary tumor-derived FGFR4; In4, intron 4; EMSA, electrophoretic mobility shift assay; B2ex, extended B2 fragment; Ik, Ikaros; GST, glutathione S-transferase; Luc, luciferase; STAT, signal transducers and activators of transcription.
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REFERENCES |
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