Fibroblast Growth Factor Receptor 4 Is a Target for the Zinc-Finger Transcription Factor Ikaros in the Pituitary

ShunJiang Yu, Sylvia L. Asa and Shereen Ezzat

Department of Medicine (S.Y., S.E.), Mount Sinai Hospital and University of Toronto, and Department of Pathology (S.L.A.), University Health Network and University of Toronto, The Freeman Centre for Endocrine Oncology and The Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9

Address all correspondence and requests for reprints to: Dr. Shereen Ezzat, Mount Sinai Hospital, 600 University Avenue, 437 Toronto, Ontario, Canada M5G 1X5. E-mail: sezzat{at}mtsinai.on.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of endocrine cell hormonal and proliferative properties, and FGFR4 is differentially expressed in normal and neoplastic pituitary. We therefore examined the functionally important cis-DNA elements and multiprotein complexes implicated in the cooperative control of expression of the human FGFR4 gene in pituitary cells. Using deletional mapping, we defined a 214-bp (-115/+99) promoter that was functional in pituitary GH4 and PRL 235 cells. Overlapping 40- to 50-bp fragments of this minimal promoter were examined by EMSA. Interestingly, fragment C (-64/-26) included potential binding sites for the hematopoietic zinc finger-containing transcription factor Ikaros (Ik) flanked by binding sites for Sp and Ets-type factors. DNA binding by Ik, Sp, and Ets-like factors was confirmed by oligonucleotide competition and supershifting with specific antibodies. Transcriptional regulation of FGFR4 by Ik was demonstrated by cotransfection of Ik1 with or without Sp1 or Ets overexpression and by disruption of the Ik binding site. Although both Ets-1 and Sp1 overexpression stimulated promoter activity, mutation of the Ik-binding site completely eliminated the Ik1 effect. Specific Ik expression was identified by Western blotting of pituitary GH4 and PRL235 cells and localized in primary mouse hormone-producing anterior pituitary cells by immunocytochemistry. Our findings point to a new role for Ik outside the hematopoietic system and suggest a novel transcriptional contribution with Ets and Sp1 in regulation of FGFR4 in the pituitary.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PITUITARY IS the site of synthesis and the target of several growth factors that modulate hormone production and cell proliferation (1). We have focused on the fibroblast growth factor (FGF) and its receptors (FGFRs) in the pituitary for a number of reasons. FGF-2 (basic FGF) is overexpressed in primary human pituitary tumors with the highest levels noted in the most aggressive tumors (2, 3). FGF-2 immunoreactivity is detected in the circulation of patients with pituitary tumors but not in normal subjects in whom the levels decline after surgical removal of the tumors (2). We have also identified FGF-like autoantibodies in the circulation of patients with multiple endocrine neoplasia type 1 and pituitary tumors; protein A eluates from the sera of these patients stimulate cell proliferation (4). Another member of the FGF family, FGF-4, has been found in transforming DNA sequences from human PRL-secreting pituitary tumors (5) and facilitates lactotroph tumorigenesis (6). Moreover, FGF-2 overexpression directed to pituitary cells results in hyperplasia in transgenic mice (7).

FGF signaling is mediated through one of four FGFRs. There are currently four known mammalian FGFR genes encoding a complex family of transmembrane receptor tyrosine kinases (8). Each prototypic receptor is composed of three Ig-like extracellular domains, a single transmembrane domain, a split tyrosine kinase, and a COOH-terminal tail with multiple autophosphorylation sites (8). Multiple forms of cell-bound or secreted forms of FGFR1, -2, and -3 have been characterized; they are produced by the same gene using alternative initiation sites, alternative splicing, exon switching, or variable polyadenylation. In studying FGFR expression in the pituitary, we found that the normal pituitary expresses FGFR1, -2, and -3 but not FGFR4 (9). In contrast, in pituitary tumors, we have identified an FGFR4 isoform containing the third Ig-like loop and the transmembrane and kinase domains (10).

FGFR4 has been reported to be expressed mainly outside the brain and nervous system, in adrenal, lung, kidney, pancreas, muscle, and spleen (11, 12). It was initially considered to have no significant role in tumorigenesis. It has, however, been shown to mediate membrane ruffling in breast carcinoma cells (13) and to modulate erythroid cell proliferation (14). Given our identification of a potential role for FGFR4 in pituitary tumor development (10), we sought to obtain insight into the regulatory mechanisms governing FGFR4 gene expression. We mapped the elements required for minimal promoter activity and identified relevant transcription factors and their functional interactions. Our studies point to a novel role for a zinc finger-containing transcription factor that has generally been regarded as exclusive to the hematopoietic system (15).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of the Human FGFR4 Promoter
Interactive elements, such as TATA or CAAT boxes, often regulate the assembly and efficiency of the basic transcriptional machinery. As with other FGFRs, the FGFR4 gene lacks a classic TATA box but includes a 5'-upstream region that is rich in G + C residues. The latter contains many consensus motifs that would predict recruitment of RNA polymerase II and initiate gene transcription. The 5'-upstream region of the human FGFR4 gene was isolated from PAC#32C5 (16). The region -1,933 to +99 from the transcription start site (16, 17) was placed upstream of the luciferase-reporter vector pGL3-basic. A series of deletion analyses were used to define the minimal promoter and fragments essential for functional activity. To determine potential tissue-specific interactions, these promoters were transfected into nonendocrine NIH 3T3 fibroblasts and GH4 and PRL 235 cells as examples of endocrine pituitary cells that express FGFR4 (see below). Figure 1Go depicts promoter activity of 5'-deletional fragments of the FGFR4 5'-flanking region in transiently transfected NIH 3T3, GH4, and PRL 235 cells, respectively. These cell lines exhibited a similar pattern with promoter activity maintained within a minimal region of 214 bp [P (-115/+99)] (Fig. 1Go). Serial 5'-deletions of the FGFR4 promoter region to +13/+99 markedly abrogate promoter activity whereas the 3'-deleted construct P (-1,133/-173)-Luc showed no significant promoter activity.



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Figure 1. Identification of Transcriptional Activity of the Human FGFR4 Gene

The effects of sequences spanning the 5'-region of FGFR4 from -1,133 to +99 on luciferase activity were assessed in NIH 3T3 (a), GH4 (b), and PRL 235 (c) cells, respectively. A series of deleted promoter fragments were generated by PCR as described in Materials and Methods, ligated, and subcloned upstream of the promoterless luciferase reporter gene pGL3-basic vector as indicated. Note that serial 5'-deletions of the FGFR4 promoter region to +13/+99 markedly abrogates luciferase activity, and that the 3'-deleted construct P (-1,133/-173)-Luc shows minimal activity. Data are presented as the mean luciferase activity adjusted for ß-galactosidase activity (±SD) and compared with control wells of three independent transfections. (P < 0.005).

 
The FGFR4 Promoter Contains Binding Sites for Ikaros (Ik)
Many transcription factors can be simultaneously involved in the regulation of target genes. To determine which factors regulate promoter activity, we screened potential candidates using EMSA. EMSAs were performed using nuclear extracts from exponentially growing NIH 3T3, GH4, and PRL 235 cells with overlapping oligonucleotide fragments derived from the 214-bp minimal FGFR4 promoter as labeled probes. Figure 2Go represents a schema of the overlapping fragments. DNA-protein complexes were detected when nuclear extracts were incubated with fragments B, C, and G but not the other indicated fragments. Fragment B contains multiple Sp1 binding sites that were confirmed by Sp1 oligonucleotide competition and supershifting (data not shown). Fragment G contains multiple binding sites for several ubiquitous transcription factors including members of the Sp and AP families.



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Figure 2. Characterization of Transcription Factor-Binding Elements to the Promoter Region of the hFGFR4 Gene

The minimal promoter of the hFGFR4 gene is divided into overlapping fragments (A–G) that were used to further characterize promoter activity. Fragments B and C formed DNA-protein complexes with nuclear extracts of NIH 3T3 cells, GH4 cells, and PRL 235 cells. Note the Ik binding site in fragment C flanked by Sp1 and Ets binding sites.

 
Fragment C of the FGFR4 promoter includes a unique Ik-like binding site (-GGGA-) that is flanked by a Sp1 and an Ets-type binding site (Fig. 2Go). Given this overlap, we examined the possibility of a nonspecific Ik-like interaction by Sp1 and/or Ets. Probes were composed of the entire fragment C (data not shown) or a smaller Ik oligonucleotide (Fig. 3Go). Specific binding of Ik in pituitary GH4 cells was confirmed by oligonucleotide competition and supershifting (Fig. 3Go). Ik-containing complexes were specifically competed by oligonucleotides encoding the wild-type Ik (Fig. 3aGo). Oligonucleotides encoding binding sites for Ets, Sp1, or mutated Ik binding sites did not appreciably interfere with Ik binding (Fig. 3Go, a and b). Moreover, these Ik-containing complexes migrated further than Ets-containing complexes, consistent with the smaller mol wt of Ik (18) (data not shown). Ik-containing complexes were supershifted by a specific monoclonal anti-Ik antibody that recognizes the C terminus of Ik proteins (19) but not by antibodies to Sp1 or to Pit1 that are highly abundant in this pituitary cell line (Fig. 3cGo). Conversely, Sp1, Ets, and Pit-1 probes could not be supershifted by the anti-Ik antibody (data not shown).



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Figure 3. Ik Forms Specific DNA-Protein Complexes in the Pituitary

a, A DNA fragment corresponding to the Ik binding site in the endogenous FGFR4 proximal promoter was labeled as a probe and incubated with nuclear extracts from GH4 cells in the presence of competing fragments representing wild-type fragment C (C) or Ik. This Ik-generated complex could not be competed in the presence of mutated Ik in fragment C (mC), mutated Ik (mIk), Ets or Sp1 (b) fragment in molar excess as indicated. Note effective competition by Ik and fragment C (arrow) but not Ets or Sp1. c, A DNA probe consisting of the same Ik probe was incubated with nuclear protein from GH4 cells in the absence or presence of antisera to Sp1, Pit-1, or Ik as indicated. Note attenuation and supershifting by the Ik antibody.

 
The Pituitary Expresses Ik
To further demonstrate that the pituitary expresses Ik protein, we examined GH4 and PRL 235 cell extracts by Western blotting. Nuclear fractions identified a signal of approximately 52 kDa that was recognized by the Ik antibody, consistent with the size of wild-type Ik-1. This protein comigrated with thymus-derived protein and was not appreciably present in cytoplasmic protein fractions (Fig. 4aGo). Both GH4 and PRL 235 pituitary cell lines also express FGFR4 (Fig. 4bGo). To determine the cell type responsible for Ik expression in the pituitary, normal mouse pituitary was examined by immunocytochemistry (Fig. 4cGo). Specific nuclear staining was identified in hormone-producing cells of the adenohypophysis, most prominently in cells containing PRL (Fig. 4dGo) and cells containing GH (not shown).



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Figure 4. Ik Expression in the Pituitary

a, Western blotting of GH4 and PRL 235 protein and thymic-derived protein. Note the expression of Ik in nuclear fractions (N) but not cytoplasmic fractions (C) of pituitary cells. Thymocytes from normal mice (T) and Rag2 -/- animals (T-/-) contain abundant Ik 1/2. b, Localization of FGFR4 in GH4 and PRL 235 protein. Both cell lines contain FGFR4 immunoreactivity that comigrates with FGFR4 in the positive control (+) but not in negative HEK293 control cells (-). c, Immunocytochemical localization of Ik in the pituitary. A normal mouse pituitary (top) contains positivity in nuclei of scattered cells (arrows) in the adenohypophysis. Staining is more diffuse in thymic tissue (bottom), which served as the positive control, but is specific to lymphoid components and is negative in endothelial cells (arrowheads). d, In the top photomicrograph, double immunostaining for Ik (blue) and PRL (brown) localizes Ik immunoreactivity in the nuclei of cells that are negative for PRL (arrows) as well as in scattered PRL-immunoreactive cells (double arrow), but most cells that contain PRL exhibit no nuclear reactivity for Ik (arrowheads). In the bottom photomicrograph, double immunostaining for Ik (blue) and ß-TSH (brown) localizes Ik immunoreactivity in the nuclei of cells that are negative for ß-TSH (arrows), but the cells that contain ß-TSH exhibit no nuclear reactivity for Ik (arrowheads).

 
Functional Contribution of Ik with Sp and Ets in Regulation Of Pituitary FGFR4
To determine the functional contribution from Ik in regulating pituitary FGFR4, we examined the wild-type P(-115/+99)-Luc FGFR4 or the same promoter with mutated Ik-binding site [mIk P(-115/+99)-Luc FGFR4] and the effects of cotransfection with Ik, Sp1, and Ets1 factors independently and in combination. Figure 5Go demonstrates the effect of Ik1 transfection on stimulation of wild-type FGFR4 promoter activity in GH4 cells. Disruption of the Ik binding site (mIk) diminished basal promoter activity by approximately 30% and completely eliminated the response to Ik1 transfection (Fig. 5Go). Overexpression of Sp1 resulted in activation of the wild-type FGFR4 reporter, an effect that was attenuated with disruption of the Ik site (Fig. 5Go). Cotransfection of Sp1 and Ik resulted in a greater degree of wild-type promoter stimulation compared with Sp1 alone. Moreover, disruption of the Ik site significantly diminished the combined effect of Ik and Sp1 (122% vs. 320%).



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Figure 5. Ik Contributes to FGFR4 Transcriptional Activity

GH4 cells were transiently cotransfected with the minimal FGFR4 P(-115/+99) (open bars) or the same promoter construct with mutated Ik site P(-115/+99) (mIk) (closed bars) and expression vectors encoding Ik1 alone, Sp1 alone or in combination with Ik1 (a), or Ets1 alone or in combination with Ik1 (b). Note activation of wild-type FGFR4 promoter by Ik1, Sp1, and Ets1. Disruption of the Ik-binding site (mIk) completely abrogates the effect of Ik1 and significantly attenuates the combined effect of Ik1 with Sp1 or with Ets1. All transfections included corresponding empty control vectors along with 20 ng of plasmid cytomegalovirus-ß-galactosidase to normalize for transfection efficiency. Data presented represent fold-induction relative to corresponding empty vector controls. Results are mean + SD and represent the mean of three separate experiments, each performed in triplicate.

 
Similarly, overexpression of Ets1 resulted in activation (~3-fold) of the wild-type FGFR4 reporter, an effect that was minimally attenuated with disruption of the Ik site (Fig. 5Go). Cotransfection of Ets1 and Ik1 resulted in a greater degree of wild-type promoter stimulation compared with Ets1 alone. Again, disruption of the Ik site significantly diminished the combined effect of Ik1 and Sp1 (175% vs. 350%). Taken together, these data provide evidence for a transcriptional role of Ik1, in functional association with Sp1 and Ets, in pituitary FGFR4 regulation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recognizing the importance of FGFR4 in pituitary tumorigenesis (10), we set out to examine functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of the FGFR4 gene. Our analysis of the human FGFR4 promoter indicated that a 214-bp fragment of the FGFR4 5'-region is important for promoter activity. Overlapping 40- to 50-bp fragments of this minimal functional promoter were examined by EMSAs. Specific DNA-protein complexes were noted with fragments B, C, and G. Fragment B includes multiple Sp1 binding sites as determined by oligonucleotide competition. We concentrated on fragment C (-64/-25) as it demonstrated consistently strong binding with nuclear extracts from pituitary GH4 and PRL 235 cells. Moreover, this fragment contains multiple predicted binding sites including those for Ap1, Ap2, Ap4, cAMP response element binding protein, and Yy-1. Direct testing of these factors, however, did not reveal evidence for significant binding with nuclear proteins from pituitary cells. Thus, we further characterized only those factors that resulted in shifting on EMSA screening using GH4 and PRL 235 nuclear extracts. The fragment C region also contains predicted sites for binding the zinc finger-containing protein Ik flanked by two sites for Sp1 and Ets-type factors. Here we show for the first time specific DNA binding and transcription regulation of FGFR4 by Ik in general and in the pituitary in particular.

The identification of Sp1 as an important factor in FGFR4 regulation was not surprising. Indeed, this ubiquitous factor has been shown to play a significant role in the regulation of other FGFRs. For example, analysis of the mFGFR1 gene reveals the presence of consensus sequences for binding sites of the transcription factors Sp1, AP1, and AP2, and the absence of TATA and CAAT sequence motifs (20). Transfection of 5'-regulatory region into NIH 3T3 cells defined a minimal promoter within the region defined by -106 and +104 of FGFR1 (20). The functional contribution of the indicated transcription factors to this promoter, however, remains to be fully characterized. Similarly, deletion analysis of the avian FGFR1 promoter reveals a 78-bp region containing multiple Sp1 binding sites that confer a high level of FGFR1 promoter activity in myoblasts (21). The FGFR2 (22) and FGFR3 (23) promoters have also been identified to reside in CpG islands encompassing the 5'-end, lacking classical cis-regulatory motifs. As little as 100 bp of sequence 5' to the initiation site confers significant transcriptional activity to these FGFR promoters (23). Sequence analysis of the FGFR3 promoter also reveals multiple transcription binding sites including five classical Sp1 sites (23). These sites are all situated within the first 200 bp of the transcription start site where Sp1 can facilitate organization of the transcription initiation complex.

The identification of Ets as a transcription factor in pituitary FGFR4 regulation is interesting, because the pituitary is well known to express Ets as an important regulator of hormone gene expression (24). However, expression and action of an Ik-like factor outside of the hematopoietic system was most intriguing.

Ik was initially described as a transcription factor that binds to regulatory sequences of several genes expressed in murine and human lymphoid cells (25, 26). Members of this family of DNA-binding factors have been shown to be critical for the activity of hematopoietic stem cells in the mouse. Mice homozygous for an Ik-null mutation display a marked reduction in long-term repopulation units. These Ik mutant strains exhibit progressive reduction in pluripotent colony-forming unit-spleen (CFU-S) progenitors and the earliest erythroid-restricted burst-forming unit-erythroid (BFU-E) precursors, consistent with the reduction in hematopoietic stem cells (27). These changes are accompanied by a decrease in expression of the tyrosine kinase receptors flk-2 and c-kit (27). Interestingly, FGFR4 has also been implicated as a modulator of erythroid cell proliferation (14). Given our current findings in the pituitary, we now propose that FGFR4 may represent an important target for Ik action in the pituitary as well as hematopoietic systems.

The Ik gene undergoes alternative splicing leading to at least eight different isoforms, all of which contain a bipartite activation domain (19, 28, 29, 30) and two common C-terminal zinc fingers that are involved in homo- and heteromeric interactions (19). Ik isoforms differ in the number of N-terminal DNA-binding zinc fingers that distinguish them into members with or without DNA binding properties (29, 30). Only three of the eight Ik isoforms contain the requisite three or more amino (N)-terminal zinc fingers that confer high-affinity binding to an Ik-specific core DNA sequence motif in the promoters of target genes (19). For example, the Ik-1 and Ik-2 proteins can bind to the same recognition sequences present in a number of lymphocyte-specific regulatory elements. Ik-3 and Ik-4 proteins interact only with a subset of these motifs. Ik-1 and Ik-2 proteins can strongly stimulate in vitro transcription, whereas Ik-3 and Ik-4 are relatively weak activators. Only isoforms with DNA-binding domains when bound in cis to Ik-binding sites can activate gene transcription (19, 29, 31). In contrast, Ik represses transcription when recruited to DNA through a heterologous DNA-binding domain (32). This repression appears to be cell type and promoter specific and is mediated through two repression domains, which interact with the histone deacetylase complexes containing mSin3 (32) and Mi-2 proteins (33). Furthermore, histones have been noted to be underacetylated in the vicinity of Ik recruitment sites whereas the histone deacetylase inhibitor, trichostatin, abrogates transcriptional repression mediated by Ik (32).

In addition to histone deacetylase-dependent mechanisms of repression, Ik-mediated repression has been noted through Ik interaction with the C-terminal binding protein (CtBP). Ik appears to interact with CtBP, thus representing a histone deacetylase activity-independent mechanism of Ik repression. CtBP was first discovered as a protein that interacts with exon 2 of the E1A adenovirus; the fact that CtBP interacts with Ik makes it relevant to note that disruption of Ik-CtBP interaction may occur in the presence of the viral oncoprotein E1A (34). Thus, whether Ik plays an activator or repressive role in the pituitary will require detailed characterization of Ik isoforms and its partners in normal and tumorous pituitary cells.

The mechanisms of FGFR action in endocrine cell development and tumorigenesis remain to be elucidated. Our previous identification of novel tumorigenic isoforms of FGFR4 (10) has provided a unique opportunity to examine the role of the FGFR4 gene in endocrine cell signaling and function. Given the established functions of Ik in T cell development and some forms of leukemia coupled with our current findings of Ik interactions in the pituitary, it is imperative to determine the potentially important functions of Ik outside of the hematopoietic system. Moreover, the regulation of FGFR4 by Ik and Ets should undoubtedly draw new parallels and contrasts between the role of distinct transcription factor partnerships in developmental and neoplastic transitions in the immune and endocrine systems.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
NIH 3T3, the rat pituitary tumor-derived PRL 235, and GH4C1 cell lines were used as models of nonendocrine and endocrine cells, respectively, with well described FGF responses. Cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) with high glucose supplemented with 10% FBS (Sigma, Oakville, Ontario, Canada), 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml of streptomycin. Twenty-four hours before transfection, cells were plated with DMEM containing 10% serum.

Plasmids
Promoter analysis of the human FGFR4 gene was performed with the assistance of gene finder (http://genome.cbs.dtu.dk/htbin/nph-webface). Possible transcription start sites were detected with TRANSFAC-Promoter 2.0 Prediction Program (gene finder). The convention for sequence coordinates with +1 as the first base of the coding sequence in exon 1 was adopted. Only one transcription start site was identified, consistent with ribonuclease protection assay findings previously reported (17). To generate the -1,133/+99 fragment, PAC# 32C5 (16) was used for PCR using an upstream primer containing a KpnI restriction site and a downstream primer containing a BglII or HindIII site, permitting subcloning into the corresponding sites of the promoterless firefly luciferase expression vector pGL3 (Promega Corp., Madison, WI) to produce a P (-1,133/+99)-Luc construct.

Other reporters P(-855/+99)-Luc, P(-173/+99)-Luc, and P(-115/+99)-Luc were generated by further restriction of the P(-1,133/+99)-Luc construct with StuI, XhoI, and SmaI, respectively. P (-535/+37)-Luc was constructed by PCR using a primer containing an FspI restriction site. The P(+13/+99)-Luc construct was synthesized (Sigma) and similarly positioned into the pGL3 basic vector. The construct P (-1,133/-173)-Luc was generated by deleting the 3'-end of the (-1,133/+99) construct using XhoI with subsequent ligation into the corresponding KpnI and XhoI sites in pGL3. The orientation and sequence of all constructs were verified by restriction analysis and nucleotide sequencing.

The expression vector pSG5-Ets-1 encoding the chicken Ets-1 isoform was obtained from Drs. A. Bradford (University of Colorado, Boulder, CO) and M. Ostrowski (Ohio State University, Columbus, OH) (24), the pPac Sp1 encoding Sp1 and their corresponding empty vector controls were provided by Dr. P. Marsden (University of Toronto, Toronto, Ontario, Canada) (35), and the Ik-1 encoding expression vector was provided by Dr. K. Georgopoulos (Harvard University, Cambridge, MA) (15).

Site-Directed Mutagenesis
Site-directed mutagenesis was performed using a transformer site-directed mutagenesis kit (CLONTECH Laboratories, Inc. Palo Alto, CA) following the manufacturer’s instructions. Mutation of the Ik transcription binding site (-cgggac-), substituted by (-cacgac-), was introduced into the minimal promoter fragment P(-115/+99)-Luc construct using mutagenic primers for the site. Another primer containing the mutation of a unique restriction site was used as a selection marker. The mutation was confirmed by nucleotide sequencing.

Transfection and Luciferase Assays
All plasmid reporters were prepared by column chromatography (QIAGEN, Missisauga, Ontario, Canada) for sequencing and transfections. Cells were transfected by the Lipofectamine method (Life Technologies, Inc.) according to the manufacturer’s protocol. Cells were plated into either 12 wells (3–5 x 105 cells) (for NIH 3T3) or six-well cluster dishes (7 x 105 cells per well), transfected the following day with 3 µl or 5 µl/well of lipofectamine and 1 or 2 µg of DNA per 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 ß-galactosidase control expression plasmid cytomegalovirus-ß-galactosidase (20 ng/well). Forty-eight hours after transfection, cells were lysed in buffer containing 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1% Triton X, and 1 mM dithiothreitol (DTT). Luciferase activity was measured for 20 sec in a luminometer. ß-Galactosidase activity was measured to normalize for variations in transfection efficiency. Promoter activity of each construct was expressed as firefly luciferase/ß-galactosidase activity. Each experiment was independently performed on three separate occasions with triplicate wells in each experiment.

Preparation of Nuclear Extracts
Nuclear extracts were prepared by washing cells in 1x PBS and lysis in 100 µl of buffer (containing 10 mM HEPES, pH 7.9, 1 mM DTT, 1 mM EDTA, 60 mM KCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride) 5 min on ice. The pellet was resuspended into 100 µl of the nuclear resuspension buffer (0.25 mM Tris-HCl, pH 7.8, 60 mM KCl, 1 mM DTT, 1.5 mM phenylmethylsulfonylfluoride) 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 for further analysis. Protein concentrations were determined by the Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay.

EMSA
Oligonucleotides were end-labeled with [{alpha}-32P]dCTP using the Klenow fragment of DNA polymerase. Five micrograms 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 DTT, 0.5 mM MgCl2, 2% glycerol, and 1 µg poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ)]. Protein-DNA complexes were resolved in 4% polyacrylamide gels containing 0.5x Tris-borate-EDTA. Overlapping double-stranded oligonucleotide fragments of FGFR4 between -115/+99 were used as probes and for competition in EMSAs as follows: fragment B (-95 to-56), 5'-GAAGGAGGGGCGGGCCCGAGCAGGAGGGGGCGGGCCCGAG-3', fragment C (-65 to-26), sense: 5'-CGGGCCCGAGGGGCGGGGCGGGACAGGAGGTGGGCCGATC-3', fragment D (-35 to +4), 5'-TGGGCCGCTCGCGGCACGCCGCCGTCGCGGGTACATTCCT-3'; fragment E (-5 to +24), 5'-GTACATTCCTCGCTCCCGGCCGAGGAGCGC-3'; fragment F (+15 to +54), 5'-CGAGGAGCGCTCGGGCTGTCTGCGGACCCTGCCGCGTGCA-3'; and fragment G (+49 to +99), 5'-CGTGCAGGGGTCGCGGCCGGCTGGAGCTGGGAGTGAGGCGGCGGAGGAGC-3'. Fragment A containing long stretches of G’s (-115 to -86), sense: 5'-GGGTGGGGGGGGGGGGCGTGGAAGGAGGGG-3' could not be technically synthesized.

Probes and competing oligonucleotides containing transcription binding sites were as follows: Sp1: 5'-ATTCGATCGGGGCGGGGCGAGC-3' and its complementary strand; Ik: 5'-AAGAAGCGGGAGTGACAGG-3' and its complementary strand; Ets-1: 5'-GGGCTG CTTGAGGAAGTATAAGAAT-3' and its complementary strand; Ap-1: 5'-CGC TTGATGAGTCAGCCGGAA-3' and its complementary strand; Ap-2a: 5'-GATCGA ACTGACCGCCCGCGGCCCGT-3' and its complementary strand; Yy-1: 5'-CGCTCCGCGGCCATCTTGGCGGCT-3' and its complementary strand; cAMP response element binding protein: 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and its complementary strand; Ap-4:5'-TTACTCCCAGCTCCAGCCGG-3' and its complementary strand (all synthesized by Sigma). The complementary strands were annealed in the annealing buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl, 1 mM EDTA).

Gel shift probes were radiolabeled using Klenow fragment I (Roche Molecular Biochemicals, Indianapolis, IN), and purified with G50 spin column. For EMSA, 100 cpm of 32P-labeled probe was incubated with 5 µg nuclear extracts at room temperature for 30 min in a binding reaction consisting of 20 mM HEPES (pH 7.9), 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 mM MgCl2, 2% glycerol, and 1 µg poly(dI-dC) (Pharmacia Biotech) in a final volume of 20 µl. For competition assay, 10 or 100 molar excess of unlabeled fragment was added as competitor DNA or mouse monoclonal antibody (4E9; kindly provided by K. Georgopolous, Boston, MA), which recognizes the C-terminal fragments of Ik proteins (19) 30 min before addition of radiolabeled probe. Samples were electrophoresed on 4% polyacrylamide nondenatured gels containing 0.5% Tris-borate buffer and 2% glycerol. Gels were dried under vacuum and autoradiographed.

Western Blot Analysis
Protein concentrations were determined by the Bio-Rad Laboratories, Inc. protein assay. Equal amounts of protein (50 µg) from whole-cell lysates, cytoplasmic, or membrane fractions were solubilized in 2x SDS sample buffer, separated on SDS-8% polyacrylamide gels, and transferred to nitrocellulose. Blots were incubated with a mouse monoclonal antibody (4E9; kindly provided by K. Georgopolous) that recognizes the C-terminal fragments of Ik proteins (19) or a polyclonal affinity-purified rabbit antiserum directed against the C terminus of FGFR4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Immunocytochemical Localization of Ik in the Pituitary
Immunolocalization of Ik was performed with a mouse monoclonal antibody (4E9; kindly provided by K. Georgopoulos) that recognizes the C-terminal tail of Ik (19). Primary mouse pituitary tissues were fixed in formalin and embedded in paraffin; after microwave antigen retrieval, immunolocalization was performed with the primary antibody at a dilution of 1:400 and detected with the streptavidin-biotin-peroxidase complex technique and 3,3'-diaminobenzidine. Colocalization with pituitary hormones was performed with double staining as previously described (36). Negative controls were performed with normal mouse ascites or normal rabbit serum replacing the primary monoclonal or polyclonal antibody, respectively, and after preabsorption of the primary antibody or antiserum with purified antigen.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the advice of Dr. H. Elsholtz and Dr. K. Georgopoulos and the technical assistance of Mr. Kelvin So.


    FOOTNOTES
 
This work was supported by the Canadian Institutes of Health Research (CIHR) (Grant MT-14404 to S.E. and S.L.A.).

Abbreviations: CtBP, C-terminal binding protein; DTT, dithiothreitol; FGFR, fibroblast growth factor receptor; Ik, Ikaros.

Received for publication November 19, 2001. Accepted for publication January 11, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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