INTRODUCTION

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Transforming growth factor type- (TGF-)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- (TGF-1, TGF-2, and TGF-3) during mouse embryogenesis, it has been argued that the TGF-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- (13-17), in particular TGF-2, and in embryos that cannot produce functional TGF- receptors due to inactivation of the TR-II gene by gene targeting (18). Given the importance of TGF- during development, it is not surprising that defects in TGF- 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- are more likely to exhibit uncontrolled growth and to undergo neoplastic transformation (20-27).

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

Efforts to define the roles of TGF- 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- until after they are induced to differentiate (38). Equally important, the up-regulation in the expression of TGF- receptors by the EC-derived differentiated cells coincides with the ability of exogenous TGF- 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 TR-II gene both in F9 EC cells and their differentiated counterparts. Our findings demonstrate that few, if any, TR-II transcripts are expressed by F9 EC cells, whereas there is a dramatic increase in the expression of TR-II mRNA when F9 EC cells are induced to differentiate. This observation is supported by the differences in expression of various TR-II promoter/reporter gene constructs in EC cells and their differentiated counterparts, arguing strongly that the large increase in the steady-state levels of TR-II mRNA that accompany differentiation is due, at least in part, to an increase in the transcription of the TR-II gene promoter. In addition, our results identify both positive and negative regulatory regions in the TR-II promoter that appear to contribute significantly to the transcriptional activity of the TR-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 TR-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 TR-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 TR-II promoter in both EC cells and their differentiated counterparts. The TR-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 TR-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 TR-II gene.

	MATERIALS AND METHODS

Top 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.

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Expression Plasmids-- TR-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 TR-II gene as a template and cloned into pGEM4-SVOCAT (42). The constructs were named pTRII-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- (CLONTECH) contains the -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 TR-II promoter/CAT plasmid was co-transfected with 2 µg of the internal standard, pCMV-. 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 -galactosidase activity by the method of Rosenthal (51) to adjust for differences in transfection efficiency (52). In some experiments, 10 µg of pTRII-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).

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	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Expression of TR-II mRNA in F9 EC and F9-differentiated Cells-- TR-II expression is normally associated with ligand binding and growth responsiveness of cells to TGF- (21-23, 58). To identify possible mechanisms for the significant increase in TGF- ligand binding and growth responsiveness that is observed when EC cells are induced to differentiate (38), we initially examined mRNA expression of the TR-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 TR-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 TR-II mRNA in F9 EC cells and F9-differentiated cells and the ability of each cell type to bind and respond to TGF-.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Regulation of TR-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 TR-II cDNA clone, H2-3FF, was used as a hybridization probe. Following autoradiography, the TR-II probe was removed and the same blot was rehybridized with a human GAPDH cDNA probe for normalization.

Transcriptional Regulation of the TR-II Gene-- Due to the large increase in the steady-state levels of TR-II mRNA when F9 EC cells are induced to differentiate, we examined the transcriptional activity of the TR-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 TR-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 TR-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 TR-II promoter-CAT constructs, pTRII-1883/+50, pTRII-274/+50, and pTRII-137/+50 were transiently transfected into F9 EC cells. (These constructs contain a common 3' end located at +50 in relationship to the primary TR-II transcription start site (42, 43), and increasing amounts of the TR-II 5'-flanking sequence ranging from nucleotide 1883 to 137.) Consistent with the virtual absence of TR-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-, 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 pTRII-274/+50 when compared with pTRII-137/+50 suggests the presence of a strong positive regulatory element(s) located in the region between 137 and 274 of the TR-II gene promoter. Furthermore, the 2-fold decrease in transcription of the pTRII-1883/+50 construct relative to pTRII-274/+50 points to a weak negative regulatory element(s) located in the region between 274 and 1883 (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Activity of the TR-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 TR-II promoter/CAT plasmids pTRII-137/+50, pTRII-274/+50, and pTRII-1883/+50, together with the -galactosidase normalizing plasmid, pCMV-. 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.

Identification of a Cis-regulatory Element That Elevates TR-II Promoter Activity in F9-differentiated Cells-- The large difference between the level of CAT activity expressed in the differentiated cells by constructs pTRII-274/+50 and pTRII-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 TR-II gene in HepG2 cells. This observation along with our previous findings demonstrating that a CRE/ATF element located in the TGF-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 TR-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 pTRII-274/+50 and pTRII-137/+50 constructs, there was about a 9-fold difference in the expression of pTRII-219/+2 when compared with pTRII-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).

View larger version (14K): [in this window] [in a new window]   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 TR-II promoter/CAT plasmids pTRII-137/+2, pTRII-219/+2, and pTRII-219 M/+2, together with the -galactosidase normalizing plasmid, pCMV-. The plasmid pTRII-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.

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Analysis of the Binding of Nuclear Proteins from F9 EC Cells and Their Differentiated Cells to the CRE/ATF Site in the TR-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 TR-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 TR-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-2 gene promoter (53, 60) also competed effectively for the formation of the DNA-protein complex binding to the TR-II CRE/ATF (Fig. 4). Similarly, the unlabeled wild-type TR-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-2 gene (data not shown).

View larger version (67K): [in this window] [in a new window]   Fig. 4.   Binding of nuclear proteins from F9 EC and F9-differentiated cells to the CRE/ATF site in the TR-II promoter. Gel mobility shift assay of the 32P-labeled wild-type TR-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) TR-II CRE/ATF site (indicated by the line above the sequence), a mutation 5' of the TR-II CRE/ATF site (M1), or a mutation within the TR-II CRE/ATF site (M2). In addition, competition with a 25-fold molar excess of unlabeled dsODN containing the TGF-2 CRE/ATF site (underlined; 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 TR-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 TR-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 TR-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.

View larger version (98K): [in this window] [in a new window]   Fig. 5.   Identification of nuclear proteins from F9 EC cells that bind to the TR-II CRE/ATF site. Gel mobility supershift assay of the 32P-labeled wild-type TR-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.

View larger version (68K): [in this window] [in a new window]   Fig. 6.   Identification of nuclear proteins from F9-differentiated cells that bind to the TR-II CRE/ATF site. Gel mobility supershift assay of the 32P-labeled wild-type TR-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 TR-II Promoter in Vivo-- Despite the finding that ATF-1 binds to the TR-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 TR-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 pTRII-219/+2 promoter/reporter construct, TR-II promoter activity was up-regulated by over 40-fold compared with the expression of the pTRII-219/+2 construct co-transfected with pSV (an SV40 promoter vector without ATF-1 or ATF-2) (Fig. 7). In contrast, co-transfection of the pECEATF-2 plasmid with pTRII-219/+2 resulted in only a modest (4-fold) induction of TR-II promoter activity (Fig. 7). The increase in pTRII-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 TR-II promoter constructs (pTRII-137/+2 and pTRII-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 TR-II gene in EC-differentiated cells.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effects of ATF-1 and ATF-2 transcription factors on TR-II promoter activity. F9-differentiated cells were co-transfected in monolayer with 10 µg of the TR-II promoter/CAT construct pTRII-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. SV plasmid (7.5 µg), which lacks ATF-1 and ATF-2 expression genes, was used as a control. All transfections included the -galactosidase normalizing plasmid, pCMV-. The bars represent CAT activities relative to the expression of pTRII-219/+2 when transfected with the SV control (1,036 cpm). The experiment was repeated three times with similar results.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Effect of ATF-1 on TR-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 TR-II promoter/CAT constructs pTRII-47/+2, pTRII-137/+2, and pTRII-219/+2 along with 7.5 µg of the pECEATF-1 or pSV plasmids. All transfections included the -galactosidase normalizing plasmid, pCMV-. The pCMV-CAT (5 µg) was also co-transfected with 7.5 µg of either the pECEATF-1 or pSV 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 pTRII-47/+2, pTRII-137/+2, pTRII-219/+2, and pCMV-CAT when transfected with the pSV 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 TR-II Promoter-- Previous studies by Bae et al. (42) identified a regulatory region located between 137 and 47 in the TR-II promoter that had a strong negative effect on the basal transcriptional activity of the TR-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 TR-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 TR-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 TR-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.

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View larger version (99K): [in this window] [in a new window]   Fig. 9.   Binding of nuclear proteins from F9 EC (A) and F9-differentiated (B) cells to the putative CCAAT box motif in the TR-II promoter. Gel mobility shift assay of the 32P-labeled wild-type TR-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 TR-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.

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The Transcription Factor Complex NF-Y Is Present in the DNA-Protein Complexes Formed between the TR-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 TR-II CCAAT box motif raised the possibility that NF-Y may also bind to the TR-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 TR-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 TR-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 TR-II Expression in Vivo-- The results of our transient expression studies, combined with our in vitro binding analyses to the TR-II CCAAT box motif, implies a role for NF-Y in TR-II transcription. However, additional studies are required to address the question of whether NF-Y affects TR-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).

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View larger version (16K): [in this window] [in a new window]   Fig. 10.   Effect of the dominant-negative NFYA29 mutant protein on TR-II promoter activity in F9-differentiated cells. F9-differentiated cells were co-transfected in monolayer with 10 µg of the TR-II promoter/CAT constructs pTRII-219/+2, pTRII-100/+2, and pTRII-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 -galactosidase normalizing plasmid, pCMV-. The bars represent CAT activities relative to the expression of the pTRII-47/+2 construct when transfected with the control vector (38,110 cpm). The experiment was repeated four times with similar results.

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View larger version (18K): [in this window] [in a new window]   Fig. 11.   Effect of the dominant-negative NFYA29 mutant protein on TR-II promoter activity in F9 EC cells. F9 EC cells were co-transfected in monolayer with 10 µg of the TR-II promoter/CAT constructs pTRII-219/+2, pTRII-100/+2, and pTRII-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 -galactosidase normalizing plasmid, pCMV-. The bars represent CAT activities relative to the expression of the pTRII-47/+2 construct when transfected with the control vector (6,750 cpm). The experiment was repeated three times with similar results.