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• W2015145164 abstract "The fibroblast growth factor-binding protein (FGF-BP) stimulates FGF-2-mediated angiogenesis and is thought to play an important role in the progression of squamous cell, colon, and breast carcinomas. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induction of the FGF-BP gene occurs through transcriptional mechanisms involving Sp1, AP-1, and CCAATT/enhancer-binding protein sites in the proximal FGF-BP gene promoter. The level of TPA induction, however, is limited due to the presence of a repressor element that shows similarity to a non-canonical E-box (AACGTG). Mutation or deletion of the repressor element led to enhanced induction by TPA or epidermal growth factor in cervical squamous cell and breast carcinoma cell lines. Repression was dependent on the adjacent AP-1 site, without discernible alteration in the binding affinity or composition of AP-1. We investigated the following two possible mechanisms for E-box-mediated repression: 1) CpG methylation of the core of the E-box element, and 2) binding of a distinct protein complex to this site. Point mutation of the CpG methylation site in the E-box showed loss of repressor activity. Conversely, in vitro methylation of this site significantly reduced TPA induction. In vitro gel shift analysis revealed distinct and TPA-dependent binding of USF1 and USF2 to the repressor element that required nucleotides within the E-box. Furthermore, chromatin immunoprecipitation assay showed that USF, c-Myc, and Max proteins were associated with the FGF-BP promoter in vivo. Overall, these findings suggested that the balance between trans-activation by AP-1 and repression through the E-box is an important control mechanism for fine-tuning the angiogenic response to growth factor-activated pathways. The fibroblast growth factor-binding protein (FGF-BP) stimulates FGF-2-mediated angiogenesis and is thought to play an important role in the progression of squamous cell, colon, and breast carcinomas. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induction of the FGF-BP gene occurs through transcriptional mechanisms involving Sp1, AP-1, and CCAATT/enhancer-binding protein sites in the proximal FGF-BP gene promoter. The level of TPA induction, however, is limited due to the presence of a repressor element that shows similarity to a non-canonical E-box (AACGTG). Mutation or deletion of the repressor element led to enhanced induction by TPA or epidermal growth factor in cervical squamous cell and breast carcinoma cell lines. Repression was dependent on the adjacent AP-1 site, without discernible alteration in the binding affinity or composition of AP-1. We investigated the following two possible mechanisms for E-box-mediated repression: 1) CpG methylation of the core of the E-box element, and 2) binding of a distinct protein complex to this site. Point mutation of the CpG methylation site in the E-box showed loss of repressor activity. Conversely, in vitro methylation of this site significantly reduced TPA induction. In vitro gel shift analysis revealed distinct and TPA-dependent binding of USF1 and USF2 to the repressor element that required nucleotides within the E-box. Furthermore, chromatin immunoprecipitation assay showed that USF, c-Myc, and Max proteins were associated with the FGF-BP promoter in vivo. Overall, these findings suggested that the balance between trans-activation by AP-1 and repression through the E-box is an important control mechanism for fine-tuning the angiogenic response to growth factor-activated pathways. Mitogen-induced expression of the fibroblast growth factor-binding protein is transcriptionally repressed through a non-canonical E-box element.Journal of Biological ChemistryVol. 275Issue 50PreviewPage 28544: Figure 5 D is incorrect. The corrected Figure 5 is shown below. Full-Text PDF Open Access fibroblast growth factor fibroblast growth factor-binding protein squamous cell carcinoma 12-O-tetradecanoylphorbol-13-acetate CCAATT/enhancer-binding protein epidermal growth factor upstream stimulatory factor aryl hydrocarbon receptor nuclear translocator basic helix-loop-helix leucine zipper chromatin immunoprecipitation polymerase chain reaction improved minimum essential medium mutated AP-1 Angiogenesis, or the growth of new blood vessels, is an early and necessary event in the growth of a tumor. Understanding the mechanisms that underlie the switch to an angiogenic phenotype of a tumor is an integral part of the development of anti-angiogenic therapy. One way in which the angiogenic pathway can be stimulated is through the activation of fibroblast growth factors (FGF-1 and FGF-2)1 that are present at high levels in most tissues where they are bound to heparan sulfate proteoglycans and sequestered in the extracellular matrix (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Vlodavsky I. Bashkin P. Ishai-Michaeli R. Chajek-Shaul T. Bar-Shavit R. Haimovitz-Friedman A. Klagsbrun M. Fuks Z. Ann. N. Y. Acad. Sci. 1991; 638: 207-220Crossref PubMed Scopus (39) Google Scholar). Tumor cells can release FGF-2 activity through the induced expression of an FGF-binding protein (FGF-BP), which is secreted from tumor cells and binds and mobilizes stored FGF-2, leading to the activation of FGF-2-dependent processes such as angiogenesis (3Wu D. Kan M. Sato G.H. Okamoto T. Sato J.D. J. Biol. Chem. 1991; 266: 16778-16785Abstract Full Text PDF PubMed Google Scholar, 4Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google Scholar). FGF-BP is found in only a limited number of epithelial tissues where its expression is tightly regulated. During mouse embryonic development, FGF-BP expression is up-regulated in the epithelial layers of the skin, intestine, and lung where it coincides with development of these tissues (5Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google Scholar). After peak FGF-BP expression at embryonic day 18, levels drop significantly after birth and remain low in most tissues of the adult mouse (5Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google Scholar). In human tissues, FGF-BP expression is low but was found to be significantly up-regulated in certain tumors including squamous cell carcinomas (SCC) derived from skin, cervix, lung, or head and neck region (4Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google Scholar). FGF-BP is also highly expressed in some colon cancers (6Czubayko F. Liaudet-Coopman E.D.E. Aigner A. Tuveson A.T. Berchem G. Wellstein A. Nat. Med. 1997; 3: 1137-1140Crossref PubMed Scopus (218) Google Scholar) and breast adenocarcinomas. 2A. T. Riegel and A. Wellstein, unpublished data. A functional role for FGF-BP in these tumors has been shown through the use of ribozyme targeting, where as little as 20% reduction in FGF-BP steady-state mRNA levels led to a decrease in tumor growth and angiogenesis of xenografted cervical SCC and colon tumors (6Czubayko F. Liaudet-Coopman E.D.E. Aigner A. Tuveson A.T. Berchem G. Wellstein A. Nat. Med. 1997; 3: 1137-1140Crossref PubMed Scopus (218) Google Scholar). Thus it appears that for at least some tumors, FGF-BP expression is rate-limiting for tumor growth and angiogenesis. A relationship between FGF-BP expression and tumor formation has also been established by the observation that levels of FGF-BP increase during 7,12-dimethylbenz[a]anthracene- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin carcinogenesis (5Kurtz A. Wang H.L. Darwiche N. Harris V. Wellstein A. Oncogene. 1997; 14: 2671-2681Crossref PubMed Scopus (56) Google Scholar). We subsequently found that FGF-BP gene transcription is directly induced by TPA or epidermal growth factor (EGF) treatment of SCC cell lines (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Analysis of the FGF-BP promoter showed that TPA and EGF induction is mediated within the first 118 base pairs of the proximal promoter and requires several positive regulatory cis-elements in the FGF-BP promoter including Sp1, AP-1, and C/EBP (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In addition, we identified a region of the promoter between the AP-1 and C/EBP sites that mediated a repressive effect on FGF-BP transcription. Deletion or mutation of the repressor region had no effect on basal activity of the promoter but significantly enhanced the level of TPA induction (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), indicating that this region of the promoter functions to limit the overall response to TPA induction of FGF-BP gene expression. In this study, we investigate more closely the mechanisms by which the FGF-BP repressor region can limit transcriptional induction of this gene in response to either TPA or EGF. The human cell lines ME-180 and HeLa (cervical squamous cell carcinoma), BT-549 (ductal breast carcinoma), and MCF-7 (breast adenocarcinoma) were obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in improved minimum essential medium (IMEM) (Biofluids Inc., Rockville, MD) without phenol red and supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Human FGF-BP promoter fragments were cloned into the pXP1 promoterless luciferase reporter vector and have been described elsewhere (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The FGF-BP promoter constructs from −118 to +62 carrying a mutated AP-1 site (mAP-1 and mAP-1/m-58) or mutated E-box (m-55/−56) were generated by PCR-based site-directed mutagenesis as described previously (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, point mutations were introduced into complementary overlapping PCR primers that convert the AP-1/E-box site from GTGAGTAACGTG (−66 to −55) toTGGAGCAACGTG orTGGAGCAATGTG to generate the mAP-1 Luc and mAP-1/m-58 Luc constructs, respectively. For the m-55/−56 Luc construct, PCR primers were generated to introduce point mutations at positions −55 and −56 of the E-box, converting the site to GTGAGTAACGGT (−66 to −55). Primary and secondary PCRs were carried out exactly as described previously (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and cloned into the pXP1 BamHI site. pRL-CMV Renilla luciferase vector (Promega) was used as transfection efficiency control. 24 h before transfection, cells were plated in 6-well plates at a density of either 750,000 cells/well (ME-180) or 250,000 cells/well (all other cell lines). For each transfection, 1.0 μg of FGF-BP promoter-luciferase construct, 0.2 ng of pRL-CMV (transfection efficiency control), and 8 μl of LipofectAMINE (Life Technologies, Inc.) were combined and added to cells for 3 h in serum-free IMEM. The transfected cells were treated for 18 h in serum-free IMEM containing vehicle alone (Me2SO, 0.1%), TPA (100 nm), or EGF (5 ng/ml). Cells were lysed and assayed for luciferase activity as described previously (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Due to a slight background TPA or EGF induction of pRL-CMV, results shown have been normalized for protein content and not for Renillaluciferase activity. Protein content of cell extracts was determined by the Bradford assay (Bio-Rad). Results from transient transfection of all FGF-BP promoter/luciferase constructs were consistent using multiple plasmid preparations. All results were analyzed for statistical significance using t test analysis. ME-180 cells were grown to 80% confluency on 150-mm dishes, serum-starved for 16 h, and treated with vehicle alone (Me2SO, 0.1%) or with 100 nm TPA for 1 h. Nuclear extract preparation, probe labeling, and binding reactions were carried out exactly as described previously (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), using 5 μg of ME-180 nuclear extracts and 200 ng of poly(dI-dC). Reactions were incubated 10 min on ice. 50-Fold molar excess (unless indicated otherwise) of unlabeled competitor oligonucleotides or 2.0-μg supershift antibodies were added and incubated for another 10 min before adding 20 fmol of labeled probe. 0.2 μg of USF-1 or USF-2 blocking peptides were added concurrently with the antibodies. Reactions were carried out 40 min on ice and analyzed by 6% polyacrylamide gel electrophoresis. All promoter fragments for gel shift were generated by annealing synthetic oligonucleotides. Sequence of the consensus AP-1 element was 5′-CTAGTGATGAGTCAGCCGGATC-3′. Supershift antibodies were purchased from Santa Cruz Biotechnology and included antibodies for c-Myc (N-262), Max (C-17), Mad-1 (C-19), Mad-2 (C-17), Mad-3 (E-20), Mad-4 (C-20), USF-1 (C-20), and USF-2 (N-20); the Fos-specific antibodies c-Fos (K-25), c-Fos (4Czubayko F. Smith R.V. Chung H.C. Wellstein A. J. Biol. Chem. 1994; 269: 28243-28248Abstract Full Text PDF PubMed Google Scholar), Fos B (102), Fra-1 (R-20), and Fra-2 (Q-20); and the Jun-specific antibodies c-Jun/AP-1 (D), c-Jun/AP-1 (N), JunB (N-17), and JunD (329). Blocking peptides for USF-1 and USF-2 were also purchased from Santa Cruz Biotechnology. Anti-ARNT polyclonal antibody was kindly provided by Dr. Chris Bradfield (McCardle Laboratory). 20 μg of FGF-BP promoter constructs −118/+62 and m-58 were CpG-methylated in vitro with 10 units of SssI methylase (CpG methylase) (New England Biolabs), 160 μm S-adenosylmethionine (New England Biolabs), and magnesium-free buffer containing 50 mm NaCl, 10 mm Tris-HCl, and 10 mmEDTA. Methylation reaction was carried out for 5 h at 37 °C. Plasmids were purified by phenol/chloroform and ethanol precipitation prior to transfection. Complete CpG methylation was confirmed by digestion with the methylation-sensitive restriction enzymeHpaII (New England Biolabs). Approximately 107 ME-180 cells were serum-starved for 16 h followed by treatment for 1 h with 10−7m TPA. Proteins were cross-linked to DNA by adding formaldehyde directly to culture medium to a final concentration of 1% for 15 min at room temperature. Cells were subsequently washed and scraped into 1 ml of 1× phosphate-buffered saline containing 1× protease inhibitor mixture (Roche Molecular Biochemicals). Cell pellets were lysed in 200 μl of lysis buffer (1% SDS, 10 mm EDTA, 50 mmTris-HCl, pH 8.0, 1× protease inhibitor mixture) for 10 min on ice. Lysates were sonicated on ice to an average DNA length of 100–500 base pairs and centrifuged to remove cell debris. Supernatant was diluted 5-fold in immunoprecipitation buffer (0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mm NaCl, 1× protease inhibitors) and pre-cleared with 50 μl of GammaBind TMPlus SepharoseTM (Amersham Pharmacia Biotech), 20 μg of salmon sperm DNA, and 50 μg of bovine serum albumin for 30 min at 4 °C. Beads were pelleted, and 10 μg of antibody (see previous section) was added to supernatant and incubated overnight at 4 °C. Immune complexes were collected with GammaBindTM Plus SepharoseTM and washed (9Orlando V. Strutt H. Paro R. Methods. 1997; 11: 205-214Crossref PubMed Scopus (513) Google Scholar). DNA was eluted with 1% SDS, 0.1 m NaHCO3 for 15 min at room temperature. Cross-links were reversed by incubating eluates at 65 °C for 4 h in 0.2 m NaCl, followed by digestion with 40 ng/μl proteinase K in 10 mm EDTA, 40 mmTris-HCl, pH 6.5, for 2 h at 45 °C. DNA was recovered by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. PCRs contained 4% of input DNA or 20% of immunoprecipitated DNA along with 10 pmol of primers, 1.5 mm MgCl2, 0.2 mm dNTPs, 1× PCR buffer (Life Technologies, Inc.), and 5 units of Taq DNA polymerase (Life Technologies, Inc.). Primers used were from −369 to −350 and from −47 to −73 of the FGF-BP promoter (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Plasmid containing −1829 to +62 of the FGF-BP promoter was used as a control template. After 22 cycles of PCR, samples were run on a 1% agarose gel, transferred to nylon membrane, and probed with an FGF-BP-specific primer from −118 to −99 end-labeled with T4 kinase. Band intensities were quantitated by PhosphorImager, and the amount immunoprecipitated was expressed as percent of total input. We previously discovered that deletion of the region between −57 and −47, situated between the AP-1 and C/EBP sites, resulted in enhanced TPA induction of transcription (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), indicating the involvement of a negative regulatory element in the TPA regulation of FGF-BP. This region has no homology to any known transcription factor binding sites except for the presence of an imperfect E-box between −60 and −55. To characterize further the FGF-BP repressor element, we tested whether mutations in this region would disrupt repressor activity and lead to enhanced TPA or EGF induction of the promoter. The region of the FGF-BP promoter between −118 and +62 harbors all of the necessary elements for full induction by both TPA (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and EGF (8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Internal deletion from −57 to −47 generated in the context of the −118 to +62 promoter showed significantly increased induction by both TPA and EGF when transfected into ME-180 cervical squamous carcinoma cells (Fig. 1 A). In addition, introduction of a C to T point mutation at position −58 within the E-box showed a dramatically increased response to TPA, going from a 7- to 18-fold induction (Fig. 1 A). EGF induction of the −58 mutant was also highly increased, going from a 5.5- to 10-fold induction (Fig. 1 A). Loss of repression was not reflected at the level of basal promoter activity since the repressor mutant constructs −57/−47 and m-58 had the same uninduced promoter activity as the wild-type −118/+62 (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Therefore, the region between −58 and −47 of the FGF-BP promoter appears to function in limiting the overall transcriptional response to both TPA and EGF. In order to determine whether repressor activity on the FGF-BP promoter existed in other cell types, we tested the TPA and EGF response of the wild-type −118/+62 or the E-box point mutant (m-58) promoter constructs in other cell lines. The −58 mutant showed significantly enhanced TPA induction in HeLa (cervical squamous carcinoma) and MCF-7 (breast cancer) cell lines (Fig. 1 B). In cell lines where the FGF-BP promoter was EGF-responsive, such as HeLa and BT-549 (breast), the fold EGF induction of the −58 mutant was also consistently higher (Fig. 1 C). These data indicate that repression of the FGF-BP promoter is a general mechanism of FGF-BP transcriptional regulation in response to TPA or EGF stimulation. The proximity of the AP-1 site to the repressor mutations raised the possibility that the observed increase in the TPA response was due to its impact on the juxtaposed AP-1 site. To test the possible influence that the −58 mutation may have on the AP-1 site, we generated double mutant constructs carrying the −58 mutation in conjunction with mutations in either the AP-1 site or the C/EBP site. We have shown previously that EGF or TPA induction of FGF-BP partly depends on the AP-1 and C/EBP sites in the promoter, since mutation of the AP-1 site (mAP-1), or deletion of C/EBP (delta C/EBP) results in a significant decrease in the amount of induction by EGF and TPA with no effect on basal promoter activity (Fig. 2 and Refs. 7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar and 8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Mutations in the AP-1 site have also been shown to disrupt AP-1 binding (7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) (Fig. 5 B). As shown in Fig. 2, the −58 mutation alone showed an enhanced TPA response, whereas mutation of the −58 and AP-1 sites together showed no loss of repression. Instead, the m-58/mAP-1 double mutant displayed a similar level of TPA or EGF induction as that of the mAP-1 single mutant construct. Conversely, the −58 mutation in combination with a deleted C/EBP site resulted in loss of repression and increased TPA and EGF induction (Fig. 2). Therefore, the effect of the −58 mutation is not dependent on the C/EBP site but is dependent on an intact AP-1 site since the mAP-1/m-58 construct does not result in a loss of repression.Figure 5Characterization of transcription factor binding to the FGF - BP promoter repressor element. A, diagram illustrating protein binding to FGF-BP promoter fragments used as probes in gel shift analysis. The indicated binding of AP-1 (complex 1) and binding to the repressor element (complex 2) represents results from gel shifts (B–D). Sequences of all promoter fragments used are shown in Fig. 6 A. B, gel shift analysis of protein binding to the FGF-BP promoter fragment between −70 and −51. Labeled probe was incubated in the presence of nuclear extracts from untreated (lane 1) or TPA-treated (lanes 2–4) ME-180 cells. Binding reactions were incubated in the presence of 50-fold molar excess of unlabeled mutated promoter fragments as indicated.Complexes 1–4 are indicated to the side of the panel.C, gel shift analysis of protein binding to the FGF-BP promoter fragment which is cytosine-methylated at position −58 (top strand) and −57 (bottom strand). Labeled unmethylated (lanes 1 and 2) and methylated (lanes 3 and 4) probes were incubated in the presence of nuclear extracts from TPA-treated ME-180 cells. Binding reactions were incubated in the absence (lanes 1 and3) or presence (lanes 2 and 4) of 50-fold molar excess of unlabeled methylated promoter fragment.D, gel shift analysis of protein binding to the FGF-BP repressor element. Labeled −70/−51 (lane 1) or mAP-1 (lanes 2–6) probes were incubated in the presence of nuclear extracts from untreated (lanes 1 and 2) or TPA-treated (lanes 3–6) ME-180 cells. Competition for complex 2 binding was carried out in the presence of 50-fold unlabeled promoter fragment as indicated. Comp, competitor DNA.View Large Image Figure ViewerDownload (PPT) The AP-1 dependence of repressor activity prompted us to confirm that the −58 mutant phenotype was not simply a consequence of altered AP-1 binding affinity to the FGF-BP AP-1 site. We carried out gel shift competition analysis of the AP-1 site in the presence or absence of the −58 mutation. By using a promoter fragment that spans the AP-1/repressor element (−70 to −51) as a probe, we could detect AP-1 binding (upper complex) and binding of additional proteins that are specific to the repressor element (Fig.3 A, upper panel and Refs. 7Harris V.K. Liaudet-Coopman E.D.E. Boyle B.J. Wellstein A. Riegel A.T. J. Biol. Chem. 1998; 273: 19130-19139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholarand 8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). When the −58 point mutation was introduced into the repressor site, the lower complexes disappeared, and only the AP-1 complex was bound (Fig. 3 A, lower panel). Competition with an unlabeled consensus AP-1 element (Fig. 3 A, lanes 2–4) or with the FGF-BP AP-1 element (lanes 5–7) could effectively compete for AP-1 binding. Both competitors reduced AP-1 binding to the probe at concentrations between 10- and 20-fold molar excess, as determined by quantitation of the AP-1 band intensity. Similarly, competition for AP-1 bound in the presence of the −58 mutation also occurred at concentrations between 10- and 20-fold molar excess of unlabeled AP-1 elements (Fig. 3 A, lanes 8–14). Although quantitation of AP-1 binding to the m-58 probe suggests a decreased AP-1 binding affinity, this may be due to the difficulty in obtaining accurate quantitation of band intensity in the presence of multiple bands (Fig.3 A, upper panel). Nevertheless, these results indicate that the enhanced TPA induction caused by the −58 mutation cannot be explained by increased AP-1 binding affinity. Alternatively, the −58 mutation could potentially alter the composition of AP-1 by flanking the AP-1 site with nucleotides that favor binding of different AP-1 family members, leading to increased transcriptional activation. We have published elsewhere that EGF activation of ME-180 cells led to increased binding of c-Fos and JunD proteins to the FGF-BP AP-1 site (8Harris V.K. Coticchia C.M. Kagan B.L. Ahmad S. Wellstein A. Riegel A.T. J. Biol. Chem. 2000; 275: 10802-10811Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We asked which AP-1 family members were bound to the FGF-BP promoter after TPA treatment and whether the composition of AP-1 changed in the presence of the −58 mutation. Gel supershift analysis was carried out using the −70/−51 FGF-BP promoter fragment as a probe in the presence of TPA-treated ME-180 extracts and antibodies specific for individual members of the AP-1 family (Fig. 3 B, lanes 1–10). Supershift of the AP-1 complex occurred in the presence of cross-reactive Fos and Jun antibodies (lanes 2 and 7, respectively) as well as with specific antibodies for c-Fos (lane 3), Fra2 (lane 6), and JunD (lane 10). The binding of c-Fos, Fra2, and JunD was also prevalent in the presence of the −58 mutation (Fig. 3 B, lanes 13, 16 and 20). Overall, these experiments demonstrate that although repressor activity mediated through the −58 site is dependent on the adjacent AP-1 site, the −58 mutation had no obvious impact on either AP-1 binding affinity or the composition of the AP-1 complex. We hypothesized that one possible effect of the −58 mutation could be that the C to T point mutation leads to loss of transcriptional repression through disruption of a CpG methylation site. The −57/−47 deletion is consistent with this hypothesis since this mutation also destroys the −58 CpG methylation site. In vivo cytosine methylation occurs preferably at CpG dinucleotides and is closely associated with transcriptional repression of genes with CpG sites in their promoter region (10Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar). In order to determine whether methylation of the −58 CpG site could mediate repression of FGF-BP promoter induction, we tested the effect of in vitro methylation of the FGF-BP promoter constructs on their transcriptional response to TPA. The wild-type −118/+62 and the m-58 promoter constructs were methylated in vitro with SssI methylase so that each plasmid would differ in its CpG methylation pattern only at the −58 site (Fig. 4). Complete methylation of each plasmid construct was confirmed by digestion withHpaII (data not shown). Transfection of the methylated −118/+62 plasmid into ME-180 cells resulted in a significant 50% decrease in the level of TPA induction compared with the unmethylated −118/+62 plasmid (Fig. 4). On the other hand, methylation of the m-58 promoter construct, which is unmethylated at position −58, demonstrated a similar level of TPA induction compared with the unmethylated m-58 construct (Fig. 4). Both methylated constructs displayed an equivalent decrease in basal promoter activity which was 20% lower than the unmethylated plasmids (data not shown) and w" @default.
• W2015145164 created "2016-06-24" @default.
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• W2015145164 creator A5031466900 @default.
• W2015145164 creator A5039372111 @default.
• W2015145164 creator A5076845153 @default.
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• W2015145164 date "2000-09-01" @default.
• W2015145164 modified "2023-09-27" @default.
• W2015145164 title "Mitogen-induced Expression of the Fibroblast Growth Factor-binding Protein Is Transcriptionally Repressed through a Non-canonical E-box Element" @default.
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