Transcriptional Activation of the Type II Transforming Growth Factor-beta Receptor Gene upon Differentiation of Embryonal Carcinoma Cells*

David KellyDagger §, Seong-Jin Kimparallel , and Angie RizzinoDagger §**

From the Dagger  Eppley Institute for Research in Cancer and Allied Diseases and § Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198 and the parallel  NCI, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previously, it has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type-beta receptors. Here, we show that the type II receptor (Tbeta R-II) gene is activated at the transcriptional level when EC cells differentiate. We show that the differentiated cells, but not the parental EC cells, express transcripts for Tbeta R-II. In addition, the expression of Tbeta R-II promoter/reporter gene constructs are elevated dramatically when EC cells differentiate and we identify at least two positive and two negative regulatory regions in the 5' flanking region of the Tbeta R-II gene. Moreover, we identify a cAMP response element/activating transcription factor site that acts as a positive cis-regulatory element in the Tbeta R-II promoter, and we demonstrate that the transcription factor ATF-1 binds to this site and strongly stimulates the expression of the Tbeta R-II promoter/reporter gene constructs when ATF-1 is overexpressed in EC-derived differentiated cells. Equally important, we identify a negative regulatory element in a 53-base pair region that had previously been shown to inhibit strongly the expression of Tbeta R-II promoter/reporter gene constructs. Specifically, we demonstrate that this region, which contains an inverted CCAAT box motif, binds the transcription factor complex NF-Y (also referred to as CBF) in vitro. Furthermore, expression of a dominant-negative NF-YA mutant protein, which prevents DNA binding by NF-Y, enhances Tbeta R-II promoter expression. Together, these studies suggest that the transcription factors ATF-1 and NF-Y play important roles in the regulation of the Tbeta R-II gene.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transforming growth factor type-beta (TGF-beta )1 refers to a complex family of genetically distinct polypeptides that are secreted by virtually all cells and which exert potent effects on cell proliferation, differentiation, extracellular matrix production, and immunoregulation (1-4). Based on these activities and the defined spatial and temporal pattern of expression of the three mammalian isoforms of TGF-beta (TGF-beta 1, TGF-beta 2, and TGF-beta 3) during mouse embryogenesis, it has been argued that the TGF-beta s play important roles in the generation and modification of extracellular signals that direct critical processes during mammalian development (5-12). This is borne out by the induction of fetal defects in embryos that are unable to produce the different isoforms of TGF-beta (13-17), in particular TGF-beta 2, and in embryos that cannot produce functional TGF-beta receptors due to inactivation of the Tbeta R-II gene by gene targeting (18). Given the importance of TGF-beta during development, it is not surprising that defects in TGF-beta signal transduction have also been implicated in the pathological processes of many diseases, including arthritis, ulcerative diseases, atherosclerosis, and glomerulonephritis (2, 19). Equally important, cells that lose the ability to respond to TGF-beta are more likely to exhibit uncontrolled growth and to undergo neoplastic transformation (20-27).

TGF-beta s primarily exert their biological effects through interactions with three distinct high affinity TGF-beta cell surface receptors (designated types I, II, and III). All three receptors have been cloned and characterized (28-33). Both the type I (Tbeta R-I) and type II (Tbeta R-II) receptors are transmembrane serine/threonine kinases that act in concert to mediate intracellular signaling. The type III (Tbeta R-III) receptor (also referred to as betaglycan) is a transmembrane proteoglycan devoid of intrinsic signaling ability, but which may act to present ligand to other signaling receptors (34). The most commonly held model for the activation of the TGF-beta signal transduction cascade proposes the selective binding of TGF-beta to the type II receptor, a constitutively active kinase (35). Ligand binding to Tbeta R-II induces recruitment of Tbeta R-I into a stable complex. Once this complex is formed, Tbeta R-II transphosphorylates Tbeta R-I at serine and threonine residues, resulting in signal transduction to downstream substrates. Thus, loss of responsiveness to TGF-beta could result from changes in the expression of functional TGF-beta receptors rather than defects in the expression or activation of TGF-beta ligand.

Efforts to define the roles of TGF-beta and their receptors have involved the study of embryonal carcinoma (EC) cells, as they are a model system used frequently for studying early mammalian development (36). EC cells resemble cells of the early mouse embryo morphologically and biochemically. Moreover, they can be induced to differentiate into many of the cell types formed during mammalian embryogenesis (37), making them particularly well suited for the investigation of the signal transduction events involved in cellular differentiation. Furthermore, they provide a useful tool for the identification of mechanisms involved in carcinogenesis, as EC cells are tumorigenic whereas their differentiated cells are largely non-tumorigenic.

Using this model system, we demonstrated that EC cells do not express detectable cell surface receptors for TGF-beta until after they are induced to differentiate (38). Equally important, the up-regulation in the expression of TGF-beta receptors by the EC-derived differentiated cells coincides with the ability of exogenous TGF-beta to inhibit their proliferation as well as to their loss of tumorigenic potential (38, 39). In the present study, we examined the expression of the Tbeta R-II gene both in F9 EC cells and their differentiated counterparts. Our findings demonstrate that few, if any, Tbeta R-II transcripts are expressed by F9 EC cells, whereas there is a dramatic increase in the expression of Tbeta R-II mRNA when F9 EC cells are induced to differentiate. This observation is supported by the differences in expression of various Tbeta R-II promoter/reporter gene constructs in EC cells and their differentiated counterparts, arguing strongly that the large increase in the steady-state levels of Tbeta R-II mRNA that accompany differentiation is due, at least in part, to an increase in the transcription of the Tbeta R-II gene promoter. In addition, our results identify both positive and negative regulatory regions in the Tbeta R-II promoter that appear to contribute significantly to the transcriptional activity of the Tbeta R-II gene in both EC and EC-derived differentiated cells. In this regard, mutation of a CRE/ATF motif (-196 to -185) within one of the positive regulatory regions reduces substantially Tbeta R-II promoter activity in the EC-derived differentiated cells. Gel mobility shift analyses demonstrates that the transcription factor ATF-1 is able to bind to this CRE/ATF motif in vitro. We demonstrate further that expression of ATF-1 in vivo up-regulates Tbeta R-II promoter activity in the differentiated cells, most likely through the CRE/ATF motif. Conversely, we have identified an inverted CCAAT box motif (-83 to -74) that appears to negatively influence the transcriptional activity of the Tbeta R-II promoter in both EC cells and their differentiated counterparts. The Tbeta R-II CCAAT box motif is bound in vitro by the transcription factor complex, NF-Y (also referred to as CBF) in both cell types. Importantly, we demonstrate that expression of a dominant-negative NF-YA mutant protein, which prevents DNA binding of the NF-Y transcription factor complex, specifically enhances the expression of Tbeta R-II promoter/reporter gene constructs in both F9 EC cells and their differentiated cells. Together, these studies suggest that ATF-1 and NF-Y play important roles in the regulation of the Tbeta R-II gene.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells and Culture Conditions-- Stock cultures of mouse F9 EC, mouse PYS-2 and mouse PSA-5E cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Intergen Company, Purchase, NY) as reported previously (40, 41). Differentiation of F9 EC cells was induced by treatment with 5 µM all-trans-retinoic acid (RA, Eastman Kodak Co.). Stock cultures and all experimental cultures were maintained at 37 °C in a moist atmosphere of 95% air and 5% CO2.

RNA Isolation and Northern Blot Analysis-- Poly(A)+ RNA was isolated from nearly confluent cultures of F9 EC cells and F9-differentiated cells by the Invitrogen FastTrack 2.0 Kit (Invitrogen, San Diego, CA). F9-differentiated cells were derived from F9 EC cells treated with 5 µM RA for 5 days. For Northern blotting, 3 µg mRNA samples from each cell type were denatured in 1× MOPS running buffer (40 mM MOPS, pH 7, 10 mM sodium acetate, and 1 mM EDTA), 2.2 M formaldehyde, and 50% formamide at 65 °C for 15 min and electrophoresed in a 1% agarose gel containing 1× MOPS running buffer in 0.22 M formaldehyde. After electrophoresis, the fractionated mRNA was transferred to an MSI nylon membrane (Fisher Scientific, Pittsburgh, PA) in 10× SSPE (1.5 M NaCl, 0.1 M NaH2PO4, and 10 mM EDTA, pH 7.4). Following transfer, the membrane was baked at 80 °C for 2 h.

Filters were prehybridized for 3 h at 42 °C in a solution consisting of 5× SSPE, 5× Denhardt's solution, 50% deionized formamide, 1% SDS, and 100 µg/ml denatured salmon testis DNA. Hybridization was performed in the same buffer with 2 × 106 cpm/ml of 32P-labeled probe for 16-18 h at 42 °C. The 32P-labeled probe was obtained by SacI digestion of the human Tbeta R-II cDNA clone, H2-3FF (30). The approximately 1.5-kilobase pair fragment was radioactively labeled with [gamma -32P]dCTP using a random primed DNA labeling kit (Boehringer Mannheim GmbH, Mannheim, Germany) to a specific activity of 1-2 × 109 cpm/µg. Following hybridization, the membranes were washed twice in 2× SSPE, 0.1% SDS for 10 min at room temperature followed by a single wash in 0.5× SSPE, 0.1% SDS for 15 min at 50 °C. Filters were autoradiographed at -80 °C with Kodak X-Omat AR film. All prehybridization, hybridization and wash conditions were the same for hybridizations with the hGAPDH normalization probe. The 780-base pair probe was obtained by PstI and XbaI digestion of the plasmid clone, HcGAP (ATCC, Rockville, MD) and radioactively labeled as described above.

Expression Plasmids-- Tbeta R-II promoter/chloramphenicol acetyltransferase (CAT) reporter gene expression plasmids were generated and amplified by polymerase chain reaction using genomic DNA containing the 5'-untranslated region of the human Tbeta R-II gene as a template and cloned into pGEM4-SVOCAT (42). The constructs were named pTbeta RII-n, where n is the distance in nucleotides from the transcription initiation site identified by Humphries et al. (43) and Bae et al. (42). The expression plasmids pECEATF-1 and pECEATF-2 were provided by Dr. Michael O'Reilly (University of Rochester, Rochester, NY). These plasmids contained the human ATF-1 and ATF-2 cDNAs (44) inserted into the expression plasmid pECE (45). The eukaryotic expression plasmid pNFYA29 was obtained with permission of Dr. R. Mantovani from Dr. Peter Edwards (UCLA School of Medicine). This plasmid uses an SV40 promoter to drive the expression of the NFYA29 mutant protein (46). The normalization plasmid, pCMV-beta (CLONTECH) contains the beta -galactosidase reporter gene under the control of the CMV immediate early promoter (47). The plasmid pDOL-CMV-CAT contains the CAT reporter gene under the control of the CMV immediate early promoter (48) and was used as a positive control to monitor the general transcriptional activity of F9 EC and F9-differentiated cells. All plasmids were verified by sequencing, and purified by Qiagen tip-500 columns.

Transient Transfection Assay-- F9 EC cells, F9-differentiated cells (day 3), PYS-2 cells, and PSA-5E cells were transfected by the calcium phosphate precipitation method as described previously (49). Typically, 20 µg of each Tbeta R-II promoter/CAT plasmid was co-transfected with 2 µg of the internal standard, pCMV-beta . After an overnight incubation with the DNA-CaPO4 precipitate, the cells were washed twice with serum-free medium and refed with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. CAT activities were determined 48 h after transfection by the method of Seed and Sheen (50) and normalized to beta -galactosidase activity by the method of Rosenthal (51) to adjust for differences in transfection efficiency (52). In some experiments, 10 µg of pTbeta RII-n constructs were co-transfected with optimal concentrations of the expression plasmids containing human ATF-1 or ATF-2 cDNAs or the dominant negative mutant, NFYA29.

Preparation of Nuclear Extracts and Gel Mobility Shift Assay-- Nuclear extracts from F9 EC and F9-differentiated (day 5) cells were prepared as described previously (53, 54) with slight modifications of the original method of Dignam et al. (55). Nuclear extracts were prepared in the presence of the following protease inhibitors: pepstatin A, antipain, chymostatin, and leupeptin (all at 1 µg/ml), PMSF (1 mM), soybean trypsin inhibitor (20 µg/ml), benzamidine (2.5 mM), and aprotinin (2.5 kallikrein-inactivating units/ml). Nuclear extracts also contained protein phosphatase inhibitors: (NH4)2MoO4 (1 mM) and NaF (5 mM). Protein concentrations were determined using the Pierce Micro BCA protein assay reagent (Pierce).

Gel mobility shift assays were based on the method of Fried and Crothers (56) as described previously (53). Reaction mixtures were incubated for 20 min at room temperature with 12 µg of F9 EC cell and F9-differentiated cell nuclear extracts. Each reaction mixture contained 1 µg of poly(dI)-poly(dC) (Amersham Pharmacia Biotech) as nonspecific competitor DNA. The double-stranded oligodeoxynucleotide (dsODN) probes containing the wild type or mutant sequences of the human Tbeta R-II promoter region located between -210 and -185 (42) were as follows (the putative CRE/ATF site is indicated by a single underline, while the mutated sequences are double-underlined): wild-type probe: 5'-tgaaCTGTGTGCACTTAGTCAT-3', and its complement, 5'-aagaATGACTAAGTGCACACAG-3'; M1 mutant probe: 5'-tgaaCTGGTGTCACTTAGTCAT-3', and its complement, 5'-aagaATGACTAAGTGACACCAG-3'; M2 mutant probe: 5'-tgaaCTGTGTGCACTGCTGCAT-3', and its complement, 5'-aagaATGCAGCAGTGCACACAG-3'. When annealed, the probes had 5' overhangs (shown in lowercase), which permitted radioactive labeling by Klenow fill-in reaction. The dsODN probe containing the wild-type sequence corresponding to the region located between -104 and -67 of the human Tbeta R-II promoter (42) consisted of 5'-tcGAGGGGCTGGTCTAGGAAACATGATTGGCAGCTACGAG-3', and its complement, 5'-tcgaCTCGTAGCTGCCAATCATGTTTCCTAGACCAGCCC-3'. The putative CCAAT box motif is indicated by a single underline. Competitor dsODNs were as follows: Tbeta R-II promoter sequence between -104 and -81: 5'-tcGAGGGGCTGGTCTAGGAAACATGA-3', and its complement, 5'-tcgaTCATGTTTCCTAGACCAGCCC-3'; Tbeta R-II promoter sequence between -91 and -67: 5'-AGGAAACATGATTGGCAGCTACGAG-3', and its complement, 5'-tcgaCTCGTAGCTGCCAATCATGTT-3'; murine FGF-4 promoter sequence between -125 and -97 (57): 5'-agcttCTCCTCCCCCGGCGGTGATTGGCAGGCGG-3', and its complement, 5'-tcgaCCGCCTGCCATCACCGCCGGGGGAGGAG-3'. These oligonucleotides were also designed so that, when annealed, the probes had 5' overhangs (shown in lowercase), which permitted radioactive labeling by Klenow fill-in reaction.

For gel mobility supershift analyses with the wild-type Tbeta R-II -210 to -185 probe, the binding reactions were incubated for 4 h at 4 °C with 4 µg of the antibodies indicated, prior to the addition of the radiolabeled probe. The ATF-1 specific monoclonal antibody was generated against recombinant ATF-1 and reacts only with complexes containing ATF-1. This antibody was kindly provided by Dr. Steven Hinrichs (University of Nebraska Medical Center, Omaha NE). Polyclonal antibodies specific to ATF-2, c-Jun, CREB, and CREM were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For gel mobility supershift analyses with the wild-type Tbeta R-II -104 to -67 probe, the binding reactions were incubated for 1 h at 4 °C with 1 µg of the antibodies indicated, prior to the addition of the radiolabeled probe. The NF-YA antibody and nonspecific IgG antibodies were purchased from Rockland Inc. (Gilbertsville, PA). The nondenaturing 4% polyacrylamide gels (30:1, acrylamide:bisacrylamide) were electrophoresed at 120 V for 3-5 h at 4 °C in high ionic strength buffer containing 50 mM Tris, 100 mM glycine, and 2 mM EDTA, then dried, and subjected to autoradiography.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of Tbeta R-II mRNA in F9 EC and F9-differentiated Cells-- Tbeta R-II expression is normally associated with ligand binding and growth responsiveness of cells to TGF-beta (21-23, 58). To identify possible mechanisms for the significant increase in TGF-beta ligand binding and growth responsiveness that is observed when EC cells are induced to differentiate (38), we initially examined mRNA expression of the Tbeta R-II gene in F9 EC cells and their differentiated counterparts. Northern blot analysis of poly(A)+ RNA from undifferentiated F9 EC cells detected little or no transcript of approximately 5.2 kilobases that corresponds to the size predicted for the Tbeta R-II gene (Fig. 1)(30). However, the intensity of this transcript increased substantially (>15-fold) after F9 EC cells were induced to differentiate with RA (Fig. 1). Thus, it appears that there is a strong correlation between the expression of Tbeta R-II mRNA in F9 EC cells and F9-differentiated cells and the ability of each cell type to bind and respond to TGF-beta .


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Fig. 1.   Regulation of Tbeta R-II gene expression during the differentiation of F9 EC cells. Northern blot analysis of poly (A)+ RNA from F9 EC cells and F9 EC cells treated for 5 days with 5 µM RA (F9-diff d5) was performed as described under "Materials and Methods." A 1.5-kilobase pair SacI fragment of the human Tbeta R-II cDNA clone, H2-3FF, was used as a hybridization probe. Following autoradiography, the Tbeta R-II probe was removed and the same blot was rehybridized with a human GAPDH cDNA probe for normalization.

Transcriptional Regulation of the Tbeta R-II Gene-- Due to the large increase in the steady-state levels of Tbeta R-II mRNA when F9 EC cells are induced to differentiate, we examined the transcriptional activity of the Tbeta R-II gene promoter in F9 EC cells and their differentiated counterparts. These studies employed chimeric gene constructs in which various amounts of the 5'-flanking region of the human Tbeta R-II gene were inserted upstream of the CAT reporter gene in the plasmid pGEM-SVOCAT (42). Transient transfection of HepG2 cells with these constructs had previously identified several distinct regulatory regions in the Tbeta R-II promoter including: two positive regulatory regions (-219 to -172 and +1 to +35), two negative regulatory regions (-1240 to -504 and -100 to -67), and the core promoter region (-47 to -1)(42). In the current study, the Tbeta R-II promoter-CAT constructs, pTbeta RII-1883/+50, pTbeta RII-274/+50, and pTbeta RII-137/+50 were transiently transfected into F9 EC cells. (These constructs contain a common 3' end located at +50 in relationship to the primary Tbeta R-II transcription start site (42, 43), and increasing amounts of the Tbeta R-II 5'-flanking sequence ranging from nucleotide -1883 to -137.) Consistent with the virtual absence of Tbeta R-II transcripts in EC cells (Fig. 1), virtually no CAT activity was detected over background (Fig. 2). This result is unlikely to be explained by low transfection efficiency, as both the normalizing plasmid, pCMV-beta , and positive control plasmid, pDOL-CMV-CAT, are expressed strongly (data not shown). In stark contrast to our observations in F9 EC cells, all of the constructs expressed substantially greater levels of CAT activity in the F9-differentiated cells (Fig. 2). Equally important, the 9-fold increase in transcriptional activity of pTbeta RII-274/+50 when compared with pTbeta RII-137/+50 suggests the presence of a strong positive regulatory element(s) located in the region between -137 and -274 of the Tbeta R-II gene promoter. Furthermore, the 2-fold decrease in transcription of the pTbeta RII-1883/+50 construct relative to pTbeta RII-274/+50 points to a weak negative regulatory element(s) located in the region between -274 and -1883 (Fig. 2).


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Fig. 2.   Activity of the Tbeta R-II promoter in EC cells and their differentiated counterparts. F9 EC cells, F9-differentiated cells, PYS-2 cells, and PSA-5E cells were transfected in monolayer with the Tbeta R-II promoter/CAT plasmids pTbeta RII-137/+50, pTbeta RII-274/+50, and pTbeta RII-1883/+50, together with the beta -galactosidase normalizing plasmid, pCMV-beta . The bars represent the average normalized CAT activity (cpm) of duplicate plates for each plasmid. All experiments in this figure were repeated at least twice with similar results.

To ensure our observations were not unique to F9-differentiated cells, we also examined the expression of these constructs in two stable EC-derived differentiated cell lines, PYS-2 (parietal endoderm-like) and PSA-5E (visceral endoderm-like). For the most part, these cell lines demonstrated a pattern of expression for each of the constructs that is similar to that observed for the F9-differentiated cells (Fig. 2). One notable difference is that the negative regulatory region between -274 and -1883 appears to have a stronger influence on the expression of the reporter gene in both the PYS-2 and PSA-5E cells than in the F9-differentiated cells.

Identification of a Cis-regulatory Element That Elevates Tbeta R-II Promoter Activity in F9-differentiated Cells-- The large difference between the level of CAT activity expressed in the differentiated cells by constructs pTbeta RII-274/+50 and pTbeta RII-137/+50 suggests the presence of a positive regulatory element(s) located in the region between -274 and -137. Studies by Bae et al. (42) identified a putative CRE/ATF site located between -196 and -190 that contributed significantly to the basal transcriptional activity of the Tbeta R-II gene in HepG2 cells. This observation along with our previous findings demonstrating that a CRE/ATF element located in the TGF-beta 2 gene promoter was essential for its expression in EC-differentiated cells (59) as well as other cell types (60, 61), led us to examine the effect of the putative CRE/ATF site on Tbeta R-II promoter expression in F9-differentiated cells. For these studies, we utilized a set of shorter constructs that eliminated a second positive regulatory region (+1 to +35) identified by Bae et al. (42) in HepG2 cells. Similar to our observations with the pTbeta RII-274/+50 and pTbeta RII-137/+50 constructs, there was about a 9-fold difference in the expression of pTbeta RII-219/+2 when compared with pTbeta RII-137/+2 (compare Figs. 2 and 3), suggesting that in F9-differentiated cells the function of the region between -219 and -137 is not dependent on the putative downstream positive regulatory element(s).


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Fig. 3.   Functional characterization of the CRE/ATF site in F9-differentiated cells. F9-differentiated cells were transfected in monolayer with the wild-type and mutant Tbeta R-II promoter/CAT plasmids pTbeta RII-137/+2, pTbeta RII-219/+2, and pTbeta RII-219 M/+2, together with the beta -galactosidase normalizing plasmid, pCMV-beta . The plasmid pTbeta RII-219 M/+2 contains modifications within the CRE/ATF site of the promoter insert. Specifically, the wild-type sequence located at -196 to -190 was modified from TTAGTCA to TGCTGCA. The bars represent the average normalized CAT activity (cpm) of duplicate plates for each plasmid. All experiments in this figure were repeated at least three times with similar results.

To determine whether the putative CRE/ATF site influenced basal transcriptional activity, the sequence at -196 to -190 in pTbeta RII-219/+2 was modified from TTAGTCA to TGCTGCA. CAT activity was reduced approximately 50% when F9-differentiated cells were transfected with the mutant pTbeta RII-219 M/+2 construct instead of pTbeta RII-219/+2 (Fig. 3). This finding argues that the CRE/ATF site located at -196 to -190 exerts a significant influence on the basal transcriptional activity of the Tbeta R-II promoter in F9-differentiated cells. However, as CAT activity was reduced by only 50%, which is similar to the results observed in HepG2 cells (42), other cis-regulatory elements within the region -219 to -137 are likely to contribute to the activity of the Tbeta R-II promoter.

Analysis of the Binding of Nuclear Proteins from F9 EC Cells and Their Differentiated Cells to the CRE/ATF Site in the Tbeta R-II Promoter-- Previous studies in HepG2 cells (42), as well as studies in F9-differentiated cells (Fig. 3), demonstrate the importance of the CRE/ATF site for expression of the Tbeta R-II gene. However, the factor(s) that binds to this site has not been identified. This led us to initially examine the in vitro binding of nuclear proteins to the CRE/ATF site. Gel mobility shift analysis of radiolabeled dsODNs containing the Tbeta R-II CRE/ATF motif with nuclear extracts prepared from F9 EC and F9-differentiated cells resulted in the formation of a single prominent DNA-protein complex that migrated with similar mobility in each extract (Fig. 4). The protein(s) in each complex appears to bind specifically to the CRE/ATF site of the probe, as a 25-fold excess of both unlabeled wild-type probe and unlabeled probe (M1) mutated slightly upstream (-203 to -200) of the CRE/ATF site competed effectively for the formation of DNA-protein complex, whereas a 25-fold excess of unlabeled probe (M2) mutated within (-195 to -192) the CRE/ATF site competed only very weakly (Fig. 4). Moreover, a 25-fold excess of unlabeled probe containing the essential CRE/ATF element (-74 and -67) from the human TGF-beta 2 gene promoter (53, 60) also competed effectively for the formation of the DNA-protein complex binding to the Tbeta R-II CRE/ATF (Fig. 4). Similarly, the unlabeled wild-type Tbeta R-II probe and not the CRE/ATF mutant counterpart (M2) was able to compete effectively for the factors that bind to the CRE/ATF motif of the TGF-beta 2 gene (data not shown).


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Fig. 4.   Binding of nuclear proteins from F9 EC and F9-differentiated cells to the CRE/ATF site in the Tbeta R-II promoter. Gel mobility shift assay of the 32P-labeled wild-type Tbeta R-II CRE/ATF dsODN was performed with 12 µg of crude nuclear extract prepared from either F9 EC or F9-differentiated cells as described under "Materials and Methods." Competition analysis of the DNA binding activity was performed by the addition of 25-fold molar excess of unlabeled dsODNs containing either the wild-type (WT) Tbeta R-II CRE/ATF site (indicated by the line above the sequence), a mutation 5' of the Tbeta R-II CRE/ATF site (M1), or a mutation within the Tbeta R-II CRE/ATF site (M2). In addition, competition with a 25-fold molar excess of unlabeled dsODN containing the TGF-beta 2 CRE/ATF site (underlined; beta 2) was also performed. This, and all gel shift studies described in this report, were repeated at least once with similar results. In addition, the same results were observed with different preparations of nuclear extracts.

ATF-1 Is Present in the DNA-Protein Complexes Formed between the Tbeta R-II CRE/ATF Site and Nuclear Extracts from F9 EC and F9-differentiated Cells-- To identify the transcription factor(s) present in the DNA-protein complexes formed between the Tbeta R-II CRE/ATF site and nuclear extracts prepared from F9 EC and F9-differentiated cells, we used a battery of antibodies that individually recognize transcription factors that bind to CRE/ATF motifs, including antibodies that recognize ATF-1, ATF-2, c-Jun, CREB, and CREM. Each of these antibodies were incubated with nuclear extracts prepared from F9 EC and F9-differentiated cells and analyzed by gel mobility shift assay with the Tbeta R-II CRE/ATF specific probe. Only ATF-1, or a closely related transcription factor, appears to be present in the DNA-protein complexes formed with the F9 EC (Fig. 5) and F9-differentiated (Fig. 6) cell nuclear extracts, as determined by both the change in migration and intensity of the prominent DNA-protein complex. However, neither of the heterodimeric partners of ATF-1 identified to date, CREB and CREM, were detected in the DNA-protein complex, suggesting that ATF-1 is binding either as a homodimer or as a heterodimer with an as yet unidentified transcription factor.


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Fig. 5.   Identification of nuclear proteins from F9 EC cells that bind to the Tbeta R-II CRE/ATF site. Gel mobility supershift assay of the 32P-labeled wild-type Tbeta R-II CRE/ATF dsODN was performed with 12 µg of crude nuclear extract prepared from F9 EC cells as described under "Materials and Methods." Reaction mixtures containing nuclear extract were left untreated (lane 2) or were preincubated with ATF-1-specific monoclonal antibody (an IgA-type antibody) (lane 3) or polyclonal antibodies (IgG-type antibodies) specific for ATF-2, c-Jun, CREB, and CREM in lanes 4, 5, 6, and 7, respectively. Non-specific mouse IgA and IgG were used as negative controls in lanes 8 and 9.


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Fig. 6.   Identification of nuclear proteins from F9-differentiated cells that bind to the Tbeta R-II CRE/ATF site. Gel mobility supershift assay of the 32P-labeled wild-type Tbeta R-II CRE/ATF dsODN was performed with 12 µg of crude nuclear extract prepared from F9-differentiated cells as described under "Materials and Methods." Reaction mixtures containing nuclear extract were left untreated (lane 2) or were preincubated with ATF-1-specific monoclonal antibody (an IgA-type antibody) (lane 3) or polyclonal antibodies (IgG-type antibodies) specific for ATF-2, c-Jun, CREB, and CREM in lanes 4, 5, 6, and 7, respectively. Non-specific mouse IgA and IgG were used as negative controls in lanes 8 and 9.

The Transcription Factor ATF-1 Up-regulates the Tbeta R-II Promoter in Vivo-- Despite the finding that ATF-1 binds to the Tbeta R-II CRE/ATF site in vitro, it was possible that other members of the CREB/ATF family of transcription factors are responsible for the basal transcriptional activity mediated through the CRE/ATF site in vivo. Therefore, to examine the ability of ATF-1 to influence the expression of the Tbeta R-II gene, we employed eukaryotic expression vectors in transient transfection assays that express either ATF-1 (pECEATF-1) or ATF-2 (pECEATF-2) proteins under the control of the SV40 promoter. When F9-differentiated cells were co-transfected with pECEATF-1 and the pTbeta RII-219/+2 promoter/reporter construct, Tbeta R-II promoter activity was up-regulated by over 40-fold compared with the expression of the pTbeta RII-219/+2 construct co-transfected with pSVDelta (an SV40 promoter vector without ATF-1 or ATF-2) (Fig. 7). In contrast, co-transfection of the pECEATF-2 plasmid with pTbeta RII-219/+2 resulted in only a modest (4-fold) induction of Tbeta R-II promoter activity (Fig. 7). The increase in pTbeta RII-219/+2 expression by ATF-1 does not appear to be due to general effects on cellular transcription, as overexpression of ATF-1 had only a minor effect (<3-fold induction) on two other Tbeta R-II promoter constructs (pTbeta RII-137/+2 and pTbeta RII-47/+2) that do not contain the CRE/ATF site (Fig. 8). Similarly, overexpression of ATF-1 did not have a significant effect on the CMV promoter (Fig. 8). Together, our findings argue that ATF-1 contributes to the expression of the Tbeta R-II gene in EC-differentiated cells.


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Fig. 7.   Effects of ATF-1 and ATF-2 transcription factors on Tbeta R-II promoter activity. F9-differentiated cells were co-transfected in monolayer with 10 µg of the Tbeta R-II promoter/CAT construct pTbeta RII-219/+2 and with optimal concentrations (7.5 µg) of either the ATF-1 or ATF-2 expression plasmids, pECEATF-1 and pECEATF-2, respectively. SVDelta plasmid (7.5 µg), which lacks ATF-1 and ATF-2 expression genes, was used as a control. All transfections included the beta -galactosidase normalizing plasmid, pCMV-beta . The bars represent CAT activities relative to the expression of pTbeta RII-219/+2 when transfected with the SVDelta control (1,036 cpm). The experiment was repeated three times with similar results.


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Fig. 8.   Effect of ATF-1 on Tbeta R-II promoter activity appears to be specific to the CRE/ATF site. F9-differentiated cells were co-transfected in monolayer with 10 µg of the Tbeta R-II promoter/CAT constructs pTbeta RII-47/+2, pTbeta RII-137/+2, and pTbeta RII-219/+2 along with 7.5 µg of the pECEATF-1 or pSVDelta plasmids. All transfections included the beta -galactosidase normalizing plasmid, pCMV-beta . The pCMV-CAT (5 µg) was also co-transfected with 7.5 µg of either the pECEATF-1 or pSVDelta to monitor effects on general transcription. The bars represent relative CAT activities of each of the reporter plasmids in the presence or absence of ATF-1. CAT activities of pTbeta RII-47/+2, pTbeta RII-137/+2, pTbeta RII-219/+2, and pCMV-CAT when transfected with the pSVDelta control were 4,688, 1,003, 6,189, and 58,933 cpm, respectively. The experiment was repeated twice with similar results.

Identification of a Cis-regulatory Element Located between -83 and -74 of the Tbeta R-II Promoter-- Previous studies by Bae et al. (42) identified a regulatory region located between -137 and -47 in the Tbeta R-II promoter that had a strong negative effect on the basal transcriptional activity of the Tbeta R-II gene in HepG2 cells. However, the location of the negative regulatory element and, more important, the transcription factor that binds to this site were not identified. Utilizing a set of Tbeta R-II promoter/CAT deletion constructs that ranged from nucleotide -219 to -47, and each ending at +2, we determined that the region located between -100 and -47 also suppresses Tbeta R-II promoter activity in F9-differentiated cells (data not shown, also see Fig. 10). Sequence analysis of this region identified a 10-base pair putative cis-regulatory element (TGATTGGCAG) located between -83 and -74 that contains an inverted CCAAT box motif. Moreover, expression of Tbeta R-II promoter/reporter constructs with this site mutagenized is elevated when transfected into HepG2 cells.2 Interestingly, this sequence is identical to the core sequence (-789 to -780) of a negative regulatory element identified in the human CYP1A1 gene using HepG2 cells (62, 63). Moreover, this same sequence has been demonstrated to be an essential cis-regulatory element of the fibroblast growth factor-4 (FGF-4) gene in F9 EC cells (64-66). In both the CYP1A1 gene and the FGF-4 gene, it was determined that the transcription factor complex NF-Y was able to bind in vitro to the core CCAAT box motif.

These observations led us to examine the binding of nuclear protein(s) from F9 EC cells and their differentiated counterparts to the -104 to -67 region of the Tbeta R-II promoter. Gel mobility shift analysis of a radiolabeled dsODN containing the Tbeta R-II -104 to -67 region with nuclear extracts prepared from F9 EC and F9-differentiated cells, resulted in the formation of at least two distinct DNA-protein complexes (Fig. 9, compare A and B). It is important to note that a third less distinct and slower migrating DNA-protein complex was consistently formed in multiple nuclear extract preparations of F9 EC cells, but not in their differentiated counterparts. It is also important to note that the two distinct DNA-protein complexes formed with each nuclear extract migrate with very similar mobilities when electrophoresed on the same gel under identical conditions (data not shown). In this regard, the DNA-protein complexes shown in Fig. 9 (A and B) were run on different gels for different lengths of time.


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Fig. 9.   Binding of nuclear proteins from F9 EC (A) and F9-differentiated (B) cells to the putative CCAAT box motif in the Tbeta R-II promoter. Gel mobility shift assay of the 32P-labeled wild-type Tbeta R-II CCAAT box dsODN was performed with 12 µg of crude nuclear extract prepared from either F9 EC cells or F9-differentiated cells as described under "Materials and Methods." Competition analysis of the DNA binding activity was performed by the addition of 50-fold molar excess of unlabeled dsODNs containing either the full length (-104/-67) wild-type probe, or Tbeta R-II sequences between -104/-87 and -91/-67. Competition was also performed with a 50-fold molar excess of unlabeled dsODN containing the FGF-4 promoter sequence between -125 and -97 (FGF-4 CAAT). NF-Y and nonspecific mouse IgG antibodies were used in supershift analysis as described under "Materials and Methods." Similar results were observed with different preparations of nuclear extracts.

Nuclear proteins in each complex from both the F9 EC and F9-differentiated cells appear to bind specifically to the -83 to -74 CCAAT box-containing motif in the Tbeta R-II promoter. This is supported by the finding that specific protein binding is abolished completely by a 50-fold molar excess of dsODNs containing either the -104 to -67 or the -91 to -67 region of the Tbeta R-II promoter, whereas dsODNs containing either the region between -104 to -87 or a mutated CCAAT box consensus sequence were unable to compete for the binding of any of the DNA-protein complexes (Fig. 9, A and B, data not shown). Equally important, dsODNs containing the -125 to -97 region of the murine FGF-4 promoter (which contains the Tbeta R-II TGATTGGCAG motif) competed as effectively for the binding of all of the DNA-protein complexes as that observed by both the Tbeta R-II -104/-67 and -91/-67 competitor dsODNs (Fig. 9A). Similarly, a dsODN containing the human CYP1A1 NRE also was able to compete for the binding of all of the DNA-protein complexes (data not shown).

The Transcription Factor Complex NF-Y Is Present in the DNA-Protein Complexes Formed between the Tbeta R-II Putative CCAAT Box Motif and Nuclear Extracts from F9 EC and F9-differentiated Cells-- Previous studies using gel mobility supershift analysis determined that the transcription factor complex, NF-Y is one of the transcription factors that binds to the TGATTGGCAG sequence in both the FGF-4 gene (64-66) and human CYP1A1 gene (63). The ability of oligonucleotides containing the FGF-4 CCAAT box and the CYP1A1 NRE to compete effectively for the binding of proteins to the Tbeta R-II CCAAT box motif raised the possibility that NF-Y may also bind to the Tbeta R-II CCAAT box motif. As NF-Y is reported to be a trimeric complex containing NF-YA, NF-YB, and NF-YC (also known as CBF-B, CBF-A, and CBF-C, respectively), we used an antibody that specifically recognizes the NF-YA subunit to characterize the DNA-protein complexes that form between the Tbeta R-II CCAAT box motif and nuclear proteins from both F9 EC and F9-differentiated cells. In both cell types, only the slower of the two closely migrating distinct DNA-protein complexes was supershifted by the addition of the NF-YA antibody, whereas the nonspecific IgG antibody had no effect on the mobility of any of the DNA-protein complexes (Fig. 9, A and B). In addition, the NF-YA antibody also appears to recognize the less distinct slowest migrating DNA-protein complex formed with nuclear extract from F9 EC cells (Fig. 9A), suggesting that NF-Y may be interacting with another factor(s). Thus, it appears that the NF-Y transcription factor complex binds the Tbeta R-II CCAAT box motif in vitro and differentiation does not overtly affect the ability of NF-Y to bind to this site. In contrast to the slower migrating DNA-protein complex, the faster migrating complex observed in both F9 EC cells and their differentiated counterparts was not supershifted by the NF-YA antibody (Fig. 9, A and B). Hence, the factor(s) in this DNA-protein complex appears to be distinct from NF-Y and thus far it has not been identified.

NF-Y Influences Tbeta R-II Expression in Vivo-- The results of our transient expression studies, combined with our in vitro binding analyses to the Tbeta R-II CCAAT box motif, implies a role for NF-Y in Tbeta R-II transcription. However, additional studies are required to address the question of whether NF-Y affects Tbeta R-II expression in vivo. To this end, Mantovani et al. (46) described a mutant NF-YA protein, NFYA29, which contains mutations in three amino acids of the DNA binding domain of NF-YA. The NFYA29 protein continues to bind to the YB subunit of NF-Y, but not to DNA, thereby functioning as a dominant negative repressor of NF-Y-mediated effects on transcription (46). Results from studies co-expressing this dominant-negative NF-YA demonstrated an in vivo role of NF-Y in the sterol-dependent expression of the farnesyl diphosphate (FPP) synthase gene, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase gene (67), and the FGF-4 gene (66).

To examine whether NF-Y influences the expression of the Tbeta R-II gene, we co-transfected both F9-differentiated cells (where the endogenous Tbeta R-II gene is expressed) with various Tbeta R-II promoter-CAT constructs and the expression plasmid for the NFYA29 mutant protein. In these cells, the Tbeta R-II promoter-CAT constructs, pTbeta RII-219/+2, pTbeta RII-100/+2 and pTbeta RII-47/+2 are expressed as described previously. Specifically, the deletion of the region between -219 and -100 containing the positive CRE/ATF site resulted in a dramatic reduction (approx. 11-fold) in the transcription of the Tbeta R-II promoter (Fig. 10, also see Fig. 3). However, when the region between -100 and -47 is deleted, pTbeta RII-47/+2 activity returned to levels higher than the pTbeta RII-219/+2 construct, indicating the presence of a strong negative regulatory element in the -100/-47 region (Fig. 10). (The promoter fragment -47/+2 contains a Sp1 site that when mutated, significantly diminishes the activity of the pTbeta RII-47/+2 construct (42), implicating a potential role for Sp1 in the transcriptional activity of this region.) Co-transfection of pNFYA29 with the pTbeta R-II promoter-CAT constructs resulted in a dramatic increase (approximately 17-fold) in the expression of the pTbeta RII-100/+2 construct, which contains the Tbeta R-II CCAAT box motif, but had little or no effect on the expression of the pTbeta RII-47/+2 construct, which does not contain the CCAAT box motif (Fig. 10). In addition, NFYA29 also increased the expression of the pTbeta RII-219/+2 construct to a level similar to the expression of the pTbeta RII-100/+2 construct (Fig. 10). Importantly, expression of pNFYA29 had little or no effect on the expression of a CMV-CAT control vector or a plasmid containing the beta -galactosidase reporter gene under the control of a SV40 promoter suggesting that the NFYA29 mutant protein does not exert general effects on transcription or transfection efficiency (data not shown). The increase in Tbeta R-II promoter activity caused by the expression of mutant NFYA29 in these experiments argues strongly that binding by NF-Y to the Tbeta R-II CCAAT box motif can act to inhibit transcription of this gene in vivo.


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Fig. 10.   Effect of the dominant-negative NFYA29 mutant protein on Tbeta R-II promoter activity in F9-differentiated cells. F9-differentiated cells were co-transfected in monolayer with 10 µg of the Tbeta R-II promoter/CAT constructs pTbeta RII-219/+2, pTbeta RII-100/+2, and pTbeta RII-47/+2 with 5 µg of the pNFYA29 expression plasmid (solid bars) as described under "Materials and Methods." As a control, the parent vector lacking the NFYA29 insert was used to equalize the amount of DNA transfected into the cells (open bars). All transfections included the beta -galactosidase normalizing plasmid, pCMV-beta . The bars represent CAT activities relative to the expression of the pTbeta RII-47/+2 construct when transfected with the control vector (38,110 cpm). The experiment was repeated four times with similar results.

As a result of the above observations, we also transfected F9 EC cells, where the Tbeta R-II gene is not expressed, with the same set of deletion constructs. As might be expected, the pTbeta RII-219/+2 and pTbeta RII-100/+2 constructs were expressed weakly in F9 EC cells; however, removal of the region between -100 and -47 resulted in a significant increase in the basal transcription of the pTbeta RII-47/+2 construct (Fig. 11). As in the case of EC-differentiated cells, co-transfection of EC cells with pNFYA29 and pTbeta RII-100/+2 or pTbeta RII-219/+2 constructs resulted in a substantial increase (6.3- and 3-fold, respectively) in the overall expression of these constructs while having little or no effect on the expression of the pTbeta RII-47/+2 construct (Fig. 11). Thus, the CCAAT box motif may also play a role in limiting the transcription of the Tbeta R-II gene in F9 EC cells.


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Fig. 11.   Effect of the dominant-negative NFYA29 mutant protein on Tbeta R-II promoter activity in F9 EC cells. F9 EC cells were co-transfected in monolayer with 10 µg of the Tbeta R-II promoter/CAT constructs pTbeta RII-219/+2, pTbeta RII-100/+2, and pTbeta RII-47/+2 with 5 µg of the pNFYA29 expression plasmid (solid bars) as described under "Materials and Methods." As a control, the parent vector lacking the NFYA29 insert was used to equalize the amount of DNA transfected into the cells (open bars). All transfections included the beta -galactosidase normalizing plasmid, pCMV-beta . The bars represent CAT activities relative to the expression of the pTbeta RII-47/+2 construct when transfected with the control vector (6,750 cpm). The experiment was repeated three times with similar results.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

TGF-beta ligand binding and cellular responsiveness to TGF-beta increase dramatically when EC cells are induced to differentiate, which suggests strongly that TGF-beta receptors are differentially expressed by EC cells and their EC-derived differentiated counterparts (38). In this report, we examined the expression of the Tbeta R-II gene in F9 EC and F9-differentiated cells, as Tbeta R-II expression is normally associated with ligand binding and growth responsiveness of cells to TGF-beta (21-23, 58). Our studies demonstrate that the expression of Tbeta R-II is closely associated with the induction of differentiation in F9 EC cells. Few, if any, transcripts for the Tbeta R-II gene are detected in F9 EC cells, whereas there is a dramatic increase in the mRNA steady-state levels of this gene in F9-differentiated cells. Equally important, the high levels of expression of Tbeta R-II promoter/CAT reporter chimeric gene constructs in F9-differentiated cells, but not in their undifferentiated parental cells, argues strongly that the increase in the steady state levels of Tbeta R-II mRNA results, at least in part, from increased transcription of the Tbeta R-II gene. Furthermore, this study identifies two different transcription factors, ATF-1 and NF-Y, that bind in vitro and in vivo to a positive regulatory element and negative regulatory element, respectively.

The up-regulation of the Tbeta R-II gene during differentiation coincides with the ability of TGF-beta to both bind and inhibit the growth of the EC-derived differentiated cells as well as to their loss of tumorigenic potential. Therefore, EC cells provide a powerful model system for studying the mechanisms that control Tbeta R-II expression during normal development as well as during the processes of malignant transformation. In regard to the latter, several lines of evidence suggest that Tbeta R-II may act as a tumor suppressor gene. A number of tumors, including small cell lung carcinoma (26), thyroid tumors (27), and prostate adenocarcinoma (25) show a loss of or reduced expression of functional TGF-beta type II receptors. Additionally, transfection of wild-type Tbeta R-II constructs into hepatoma cells and MCF-7 breast carcinoma cells lacking functional type II receptors restores sensitivity to TGF-beta and suppresses their tumorigenic phenotype (21, 22). Moreover, gross structural mutations of both Tbeta R-II alleles has been observed in 71-90% of colorectal tumors and cell lines and in 71% of gastric cancer cell lines with microsatellite instability (23, 24, 68). However, in a number of instances in which cells fail to express the Tbeta R-II gene at the RNA or protein level, no apparent structural mutations within the coding region of the gene were observed. This raises the possibility that defects in the promoter region of the Tbeta R-II gene and/or in the mechanisms regulating the transcription of the Tbeta R-II gene may play important roles in the aberrant expression of Tbeta R-II in certain neoplasms. In this regard, the low levels of Tbeta R-II mRNA expressed by A431 tumor cells is thought to be a result of a point mutation located in the 5' flanking region of the Tbeta R-II promoter (69).

To understand further the mechanism(s) that control the regulation and expression of the Tbeta R-II promoter during differentiation, our studies also examined the expression of Tbeta R-II promoter/reporter gene constructs in the differentiated cells derived from various EC cell lines to identify putative DNA regulatory elements that are required for normal Tbeta R-II promoter expression. The data presented demonstrates the existence of at least four distinct regulatory regions in the Tbeta R-II promoter including, two positive regulatory regions located between -274/-100 and -47/+2, and two negative regulatory regions located between -1883/-274 and -100/-47. In addition, we determined that a putative CRE/ATF site located at -196 to -190 exerts a significant positive influence on Tbeta R-II promoter activity in the differentiated cells, whereas a putative inverted CCAAT box motif located at -83 to -74 exerts a significant negative influence on Tbeta R-II promoter activity in both F9 EC cells and their differentiated counterparts.

In regard to the CRE/ATF motif, mutation of this site reduces transcription of wild-type Tbeta R-II promoter/reporter constructs in the differentiated cells by approximately 50%, which is similar to what was observed in HepG2 cells (42). Gel mobility shift analysis further demonstrated that protein complexes containing the transcription factor ATF-1 specifically recognize and bind the Tbeta R-II CRE/ATF site. Overexpression of ATF-1 in F9-differentiated cells resulted in a dramatic increase in Tbeta R-II promoter activity most likely through the CRE/ATF site, suggesting that ATF-1 not only interacts with this site in vitro but also in vivo. Interestingly, the expression of the TGF-beta 2 gene in EC cells and their differentiated counterparts is also regulated by mechanisms involving the binding of ATF-1 to an essential CRE/ATF element (7, 53, 59, 60).

Another important aspect of the work reported in this study is the demonstration that a 53-base pair negative regulatory region located between -100 and -47 contains an inverted CCAAT box motif that negatively regulates Tbeta R-II promoter activity. We demonstrate further that the transcription factor complex, NF-Y, binds to the Tbeta R-II CCAAT box motif in vitro when nuclear extracts from both F9 EC cells and their differentiated counterparts are used in gel mobility supershift analyses. Importantly, we also demonstrate that expression of the dominant-negative NFYA29 mutant protein in both F9 EC and F9-differentiated cells specifically increases the expression of Tbeta R-II promoter/reporter constructs that contain the CCAAT box motif. In further support of these findings, we have shown that NF-Y in nuclear extracts prepared from HepG2 cells binds to the CCAAT box motif (data not shown) and expression of Tbeta R-II promoter/reporter constructs with this site mutagenized is elevated when transfected into HepG2 cells.2 Thus, our findings argue strongly that in vivo binding of NF-Y to the CCAAT box motif represses the activity of the Tbeta R-II promoter.

CCAAT box motifs are found in the promoter regions of many genes, and a survey of over 500 unrelated promoter sequences determined that most proximally located CCAAT boxes reflect the target sequence for NF-Y rather than C/EBP or NF1 (70). NF-Y was originally identified as a ubiquitously expressed protein that binds to the Y box motif, originally defined as an inverted CCAAT box motif (CTGATTGGYY) in all MHC class II genes that is critical for tissue specific gene expression (71). Although the exact mechanism(s) by which NF-Y regulates transcription is unclear, it has been demonstrated to act as both a positive (66, 72, 73) and a negative (74) transcription factor. Several studies suggest that NF-Y acts by stabilizing or recruiting the binding of additional factors to adjacent promoter elements (75, 76). Whether there are coordinate interactions between ATF-1 and NF-Y that act to regulate the Tbeta R-II promoter in F9 EC and F9-differentiated cells is unknown, however these two factors appear to act in concert to regulate a number of other gene promoters, including the human cyclin A gene and the rat hexokinase II gene (72, 77).

In conclusion, our studies demonstrate clearly that transcription of the Tbeta R-II gene is influenced significantly by both positive and negative cis-regulatory elements and trans-acting factors. However, the exact mechanism(s) by which differentiation turns on the expression of the Tbeta R-II gene remains to be determined. Given the important role of the TGF-beta type II receptor during normal development and in its aberrant expression in certain neoplasms, further study of this gene in this model system is clearly warranted. In addition, it will be interesting to determine whether differentiation also leads to the transcriptional activation of the genes for the type I and type III TGF-beta receptors.

    ACKNOWLEDGEMENTS

We thank Michael O'Reilly for the gift of pECEATF-1 and pECEATF-2 expression plasmids and Steven Hinrichs for the gift of the ATF-1 monoclonal antibody. We thank Robert Mantovani for permission to use the pNFYA29 plasmid, which was generously provided by Peter Edwards.

    FOOTNOTES

* This work was supported in part by Grant CA 74771 from the NCI, National Institutes of Health and by core grants to the Eppley Institute from the American Cancer Society (SIG) and the NCI, National Institutes of Health (Laboratory Cancer Research Center Support Grant CA 36727).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by a postdoctoral fellowship provided by NCI-supported Training Grant T32 CA09476 in Cancer Research.

** To whom all correspondence should be addressed at: Eppley Inst. for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6805. Tel.: 402-559-6338; Fax: 402-559-4651; E-mail: arizzino{at}unmc.edu.

The abbreviations used are: TGF-beta , transforming growth factor-beta ; Tbeta R-II, type II transforming growth factor-beta receptor; EC, embryonal carcinoma; FGF, fibroblast growth factor; RA, retinoic acid; dsODN, double-stranded oligodeoxynucleotide; ATF, activating transcription factor; CRE, cAMP response element; CAT, chloramphenicol acetyltransferase; MOPS, 4-morpholinepropanesulfonic acid.

2 D. Kim and S.-J. Kim, unpublished results.

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Abstract
Introduction
Materials & Methods
Results
Discussion
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