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J. Biol. Chem., Vol. 279, Issue 19, 19407-19420, May 7, 2004

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Unique and Selective Effects of Five Ets Family Members, Elf3, Ets1, Ets2, PEA3, and PU.1, on the Promoter of the Type II Transforming Growth Factor- Receptor Gene^*

Janel L. Kopp¶, Phillip J. Wilder¶, Michelle Desler, Jae-Hwan Kim||, Jingwen Hou, Tamara Nowling**, and Angie Rizzino

From the Eppley Institute for Research in Cancer and Allied Diseases and the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805

Received for publication, December 23, 2003 , and in revised form, February 12, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Previous studies have shown that the promoter of the type II TGF- receptor gene (TR-II) is strongly stimulated by Elf3, a member of the Ets transcription factor family. The TR-II gene behaves as a tumor suppressor and it is expressed in nearly all cell types, whereas Elf3 is expressed primarily in epithelial cells. Hence, the TR-II gene is likely to be regulated by other Ets proteins in nonepithelial cells. In this study, we examined the effects of four other Ets family members (Ets1, Ets2, PEA3, and PU.1) on TR-II promoter/reporter constructs that contain the two essential ets sites of this gene. These studies employed F9 embryonal carcinoma cells and their differentiated cells, because transcription of the TR-II gene increases after F9 cells differentiate. Here we demonstrate that Ets2, which is expressed in F9-differentiated cells along with Elf3, does not stimulate or bind to the TR-II promoter in these cells. In contrast, PEA3 stimulates the TR-II promoter in F9-differentiated cells, but it inhibits this promoter in F9 cells. Thus, the effects of PEA3 on the TR-II promoter are cell context-dependent. We also show that the effects of Elf3 are cell context-dependent. Elf3 strongly stimulates the TR-II promoter in F9-differentiated cells, but not in F9 cells. In contrast to Elf3 and PEA3, Ets1 strongly stimulates this promoter in both F9 cells and F9-differentiated cells. Finally, we show that PU.1 exerts little or no effect on the activity of the TR-II promoter. Together, our findings indicate that Elf3 is not the only Ets protein capable of stimulating the TR-II promoter. Importantly, our findings also indicate that each of the five Ets proteins influences the TR-II promoter in a unique manner because of important differences in their biochemical properties or their patterns of cellular expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TGF-¹ consists of three isoforms that exert potent, pleiotropic effects during growth and differentiation (1, 2). TGF- also plays critical roles in many diseases. In the case of cancer, TGF- has been shown to exert complex effects on tumor growth and metastasis (3, 4). TGF- has also been implicated in the suppression of the immune system (5–9), and decreased expression of functional TGF- receptors by cells of the immune system in an animal model has been shown to suppress tumor metastasis and enhance host survival (8, 9). TGF-s exert their biological effects through interactions with three distinct high affinity TGF- cell surface receptors, which are designated types I, II, and III (1, 2). The type III receptor, also known as betaglycan, is a transmembrane proteoglycan devoid of intrinsic kinase activity. The type I (TR-I) and type II (TR-II) receptors are transmembrane serine/threonine kinases that act in concert to mediate intracellular signaling. Ablation of any of the three TGF- receptors by gene targeting causes lethal defects during development (10–12), and defects in these receptors, in particular TR-I and TR-II, are strongly associated with tumorigenicity (1, 2, 13). Although TR-I and TR-II clearly behave as tumor suppressor genes, inactivation of these genes exerts different effects on tumor growth and tumor metastasis (3, 4).

During the past several years, transcriptional regulation of the TR-II gene has been studied extensively. The TR-II promoter lacks a consensus TATA box, but it has been reported to be regulated by a negative regulatory element and by at least six positive cis-regulatory elements located within 400 base pairs of its major transcription start site (14–25). The positive cis-regulatory elements include a CRE/ATF site located 200 base pairs upstream of the major transcription start site. More than one transcription factor has been reported to activate the TR-II promoter through this cis-regulatory element (16, 24). A CCAAT box, which binds the transcription factor complex NF-Y, has been identified 80 base pairs upstream of the major transcription start site (16, 22). Other studies have identified at least two conserved GC boxes that are located close to the CCAAT box. Sp1 appears to be the prime candidate for activating the TR-II promoter via these GC boxes (19–22). In addition, the murine TR-II 5'-flanking region contains a functional Egr-1 binding site (25). The activity of the TR-II promoter is also influenced heavily by a region containing two overlapping ets sites located just downstream of the major transcription start site (17, 23). Several studies have demonstrated that the transcription factor Elf3 (also known as ESX, ESE-1, Jen, and ERT) can strongly activate the TR-II promoter via these ets sites in epithelial cells (17, 23).

Elf3 is a member of the Ets family of transcription factors, which is composed of over 25 members (26). This family of transcription factors is characterized primarily by a highly conserved DNA binding domain, known as an ETS domain (26). Elf3 not only activates TR-II promoter/reporter constructs in epithelial cells, it also has been shown by chromatin immunoprecipitation studies to bind to the TR-II promoter in vivo (23). Like many other Ets family members, Elf3 appears to contain a potent autoinhibitory domain that limits its binding to DNA in vitro (23). Although the position of this domain within Elf3 has not been determined, it appears to be located N-terminal of its DNA binding domain. Other studies using athymic nude mice and the breast cancer cell line Hs578t, which exhibits little or no endogenous Elf3, have shown that forced expression of Elf3 elevates expression of TR-II and diminishes tumor growth (27). Moreover, Elf3 knockout mice, which exhibit severe defects in the developing gut, express lower levels of TR-II in their enterocytes (absorptive cells of the gut) (28).

Elf3 is expressed by all epithelial cell types examined thus far, but not by other cell types (29, 30). Given that TR-II is expressed by nearly all cell types, it is likely that other members of the Ets family of transcription factors stimulate the transcription of the TR-II gene in cell types that do not express significant levels of Elf3. In this regard, Ets proteins have been found in all cells, and several Ets proteins are expressed ubiquitously (26, 31–33). Furthermore, the DNA binding domains of Ets proteins appear to be structurally very similar. X-ray crystallographic structures of six Ets proteins demonstrate that their DNA binding domains share a common structural fold, which contains three -helices and four -strands (34–38). This raises the possibility that the TR-II gene is stimulated indiscriminately by other members of the Ets family. However, this is unlikely to be the case. Work with other promoters argues that Ets proteins do not promiscuously stimulate promoters that contain ets sites. In fact, a high degree of specificity is observed, such that only one, or at most a few, Ets family members activate any given promoter with ets sites. Currently, the mechanisms responsible for this specificity are only partially understood. However, autoinhibitory domains, which are present in at least eight Ets family members and which limit the binding of these Ets proteins to DNA in vitro (23, 39–44), appear to play critical roles in controlling access to ets sites in vivo. In addition, in several well documented studies, cooperative binding with other transcription factors that bind to adjacent DNA regulatory sequences has been shown to overcome the negative effects of the autoinhibition in vitro (45–47).

Several tumor model systems, including embryonal carcinoma (EC) cells and their differentiated counterparts, have been used to investigate the transcriptional regulation of the TR-II gene. EC cells are used widely, because their differentiation in culture mimics important stages of mammalian embryogenesis (48). EC cells also provide an excellent model system for studying the regulation of TGF- receptors, because differentiation of EC cells suppresses their tumorigenicity (49–51) and leads to the appearance of cell surface TGF- receptors (52). Accordingly, EC cells do not respond to TGF-, whereas, TGF- inhibits the growth of their differentiated cells (52). It is also significant that the differential expression of TGF- receptors by EC cells and their differentiated counterparts parallels the onset of TGF- receptor expression by similar cell types in the early embryo (53). Although TR-I mRNA does not appear to change significantly with the differentiation of EC cells (23), the transcription of the TR-II gene increases when EC cells differentiate (16, 23). Moreover, the mRNA for TR-II and the mRNA for Elf3 increase in parallel, and forced overexpression of Elf3 in EC-differentiated cells strongly increases TR-II promoter activity (23). Together, these findings suggest that the up-regulation of Elf3 plays a key role in transcriptional activation of the TR-II gene when EC cells differentiate into nontumorigenic cells.

To understand the transcription of the TR-II gene more fully, this study compared the abilities of five Ets family members to regulate TR-II promoter activity. Specifically, the effects of Elf3, PEA3, Ets2, Ets1, and PU.1 on the TR-II promoter were compared in F9 EC cells and F9-differentiated cells. The effects of Ets2 were examined, because Ets2 (54) and Elf3 (23) are both expressed in F9-differentiated cells. PEA3 was examined, because it is expressed in EC cells, but its expression is down-regulated strongly when F9 EC cells differentiate (54). Thus, PEA3 expression decreases as the transcription of the TR-II gene increases (16, 54). Ets1 and PU.1 were included in this study as candidate Ets proteins that may be involved in regulating the transcription of the TR-II gene in nonepithelial cells. In this regard, Ets1 and PU.1 are expressed in the cells of the immune system, which express TR-II but not Elf3 (31, 32, 55–57). Together, our findings indicate that each of these Ets family members influences the TR-II promoter in a unique manner because of important differences in their biochemical properties or their patterns of cellular expression. This study also provides further characterization of the ets sites of the TR-II promoter and demonstrates that these two overlapping ets sites are both required for the synergistic stimulation of the TR-II promoter by Ets proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen. Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). Unless indicated otherwise, all chemicals were obtained from Sigma.

Cell Culture—F9 EC cells were cultured on gelatin-coated tissue culture plastic in DMEM containing 10% FBS, as described previously (16, 23). F9 cells were induced to differentiate by a 72-h treatment with 5 µM retinoic acid, and 293T cells were cultured in DMEM containing 10% FBS, as described previously (23). All cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air.

Promoter/Reporter Gene Constructs—Promoter/reporter construct mTR-II–108/+56(B/A), containing both ets sites, and promoter/reporter construct mTR-II–108/+56(X/X), with both ets sites disrupted (previously referred to as TR-II–108/+56(EBSmut2)), have been described previously (23). Constructs mTR-II–108/+56(B/X) and mTR-II–108/+56(X/A) were generated from mTR-II–108/+56(B/A) by site-directed mutagenesis using the "Quick Change" method, as described previously (23). The X/A mutant construct was generated with the primer 5'-GTTGGCGAGGAGAGCGCTGTTTCCCTCTCG-3' and its complement (the mutant sequence is underlined). The B/X mutant construct was generated with the primer 5'-GGAGTTTCCTGTTCTAGACTCGGCGCCCG-3' and its complement (the mutant sequence is underlined). Construct mTR-II–108/+56(B/B) was generated from mTR-II–108/+56(B/A) by site-directed mutagenesis using the primer 5'-GGAGTTTCCTGTTTCCTGTTCGGCGCCCG-3' and its complement. The mutant ets site sequences for mTR-II–108/+56(B/X), mTR-II–108/+56(X/A) and mTR-II–108/+56(B/B) were verified by sequence analysis at the Genomic Core Research Facility (University of Nebraska, Lincoln, NE) or at the Molecular Biology Core Facility of the Eppley Institute.

Ets Protein Expression Constructs—Expression constructs for Elf3 (pcDNA3.0-mElf3), Elf3N233 (Elf3(ATH+DBD)), and Elf3N270 (Elf3(DBD)) have been described previously (23). The expression construct GFP-Elf3 was generated by insertion of the coding sequence for Elf3, from the Elf3 expression vector pcDNA3.0-mElf3, into the GFP (green fluorescent protein) vector, pEGFP-C1 (Clontech Laboratories, Palo Alto, CA). Expression vectors Ets2 and Ets2N331 were obtained from Craig Hauser (Birnham Institute, La Jolla, CA) (58). The expression vector Ets2C453 was generated from Ets2 by replacing the codon for amino acid 453 with a stop codon using the Quick Change method. This was accomplished using the primer pairs 5'-TGCTGGGCTTCACTTGAGAGGAACTGC-3' and 5'-GTTCCTCTCAAGTGAAGCCCAGCAAGT-3' (the underlined sequences refer to the mutation generated to insert a stop codon and eliminate 16 amino acids from the C-terminal tail of Ets2). The absence of a BspEI site, which was deleted to generate the mutation, was used to screen for mutant clones. The mutant sequence was verified by sequence analysis at the Genomic Core Research Facility (University of Nebraska, Lincoln, NE). The expression vector for Ets1, pCMV-Tag2a-FlagEts1, was obtained from Barbara Graves (University of Utah, Salt Lake City, UT) (59). YFP-Ets1 was generated by inserting the coding sequence for Ets1, from pCMV-Tag2a-FlagEts1, into the yellow fluorescent protein (YFP) vector, pEYFP-C1 (Clontech Laboratories, Palo Alto, CA). The expression vector for PEA3 was obtained from John Hassell (McMaster University, Hamilton, Ontario, Canada) (44), and the expression vector PU.1 was obtained from R. Kawahara (University of Nebraska Medical Center) (61).

Transfection and Activities of Promoter/Reporter Gene Constructs—F9 EC cells and F9-differentiated cells were transiently transfected by the calcium phosphate precipitation method, as described previously (16, 23). In addition to 15 µg of TR-II–108/+56 promoter/reporter gene constructs, the cells were transfected with 1 µg of the internal control plasmid, pCMV--gal, to normalize for any differences in transfection efficiency or general effects on transcription. In this regard, Ets1, Elf3, and PU.1, but not PEA3, Ets2, Elf3N233, or Elf3N270, appeared to decrease the activity of pCMV--gal more than 3-fold when their expression vectors were used at concentrations greater than 3 µg. This was also true for other normalizing plasmids tested, which were driven by the SV40 promoter. Hence, expression vectors for Ets1, Elf3, and PU.1 were not used at concentrations above 3 µg. At 3 µg or less, the effects of these Ets proteins on pCMV--gal were generally less than 3-fold. Chloramphenicol acetyltransferase (CAT) and -galactosidase activities were determined 48 h after transfection of F9 EC cells and 72 h after transfection of F9-differentiated cells, as described previously (16).

Transfection and Expression of GFP and YFP Fusion Proteins—F9 EC cells or F9-differentiated cells were transiently transfected with expression vectors for Ets-fluorescent fusion proteins using LipofectAMINE in conjunction with the LipofectAMINE PLUS reagent (Invitrogen) as described previously (62). This transfection protocol was used, because previous studies determined that only small differences in transfection efficiency are observed when these reagents are used (62). Expression of fluorescent proteins was determined 24 h after transfection by flow cytometry using a Becton Dickinson FACSCalibur flow cytometer. The data were collected and analyzed as described previously (62). Examination of YFP-Ets1 and GFP-Elf3 expression by fluorescence microscopy demonstrated that these proteins were localized to the nuclear compartment.

Electrophoretic Mobility Shift Assays (EMSA)—Nuclear extracts were prepared from 293T cells that had been transiently transfected with 20 µg of one of the flag-tagged Ets protein expression vectors, as described previously (23). Double-stranded oligonucleotide probes were radiolabeled, as described previously (16, 23). The amounts of flag-tagged Ets proteins in the nuclear extracts were determined by Western blot analysis using the monoclonal antibody (M2) that recognizes the flag epitope, as described previously (23). For each EMSA, the amount of nuclear extract added to the binding buffer (23) was adjusted so that equal amounts of flag-tagged Ets proteins were compared. In cases where supershift assays were performed, the monoclonal antibody M2 was preincubated with the nuclear extract for 40 min at room temperature prior to adding the radiolabeled DNA probe. In all experiments shown, the DNA-protein complexes were shown to be specific by virtue of competition with an unlabeled double-stranded DNA oligonucleotide with the same sequence as the radiolabeled DNA probe and by the lack of competition with an unlabeled double-stranded DNA oligonucleotide with a different sequence. The DNA-protein complexes were resolved on 6% nondenaturing polyacrylamide gels, as described previously (16, 23). The gels were dried and visualized by a PhosphorImager (Amersham Biosciences). The DNA probes used corresponded to nucleotides 3–32 of the mTR-II gene (23): B/A probe, 5'-TGGCGAGGAGTTTCCTGTTTCCCTCGGCGC-3' and its complement; X/A probe, 5'-TGGCGAGGAGAGCGCTGTTTCCCTCGGCGC-3' and its complement; B/X probe, 5'-TGGCGAGGAGTTTCCTGTTCTAGACGGCGC-3' and its complement; B/B probe, 5'-TGGCGAGGAGTTTCCTGTTTCCTGTGGCGC-3' and its complement. In each case, the mutant sequence is underlined. For each probe, the sequence, TAGC, was added to the 5' end of each oligonucleotide for use in radiolabeling.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Elf3 Synergistically Increases the Activity of the TR-II Promoter via Two Overlapping Ets Sites—Although Ets proteins bind to an essential core sequence of 5'-GGA(A/T)-3' (63), their binding to DNA is affected by flanking sequences. Based on the DNA binding preferences of eight Ets proteins (64–68), Ets proteins appear to bind to a 9-base pair consensus ets site that consists of 5'-(A/G)(C/G/T)(C/A)GGA(A/T)(G/A)(C/T)-3'. Analysis of the region 12–27 relative to the major transcription start site of the mouse TR-II gene indicates that it contains two potential ets sites that overlap by 2 base pairs (15, 23). These sites are arranged in the same 5' to 3' direction. The sequence of the upstream ets site (5'-ACAGGAAAC-3'; designated the B-site) matches the sequence of a consensus site, whereas the sequence of the downstream ets site (5'-GAGGGAAAC-3'; designated the A-site) differs from the consensus sequence above by 2 base pairs, which are shown in bold (Fig. 1A). Except for 1 base pair in the putative A-site of the human gene (5'-GGGGGAAAC-3'), which is shown in bold, the two overlapping ets sites in the mouse and human TR-II genes are conserved fully.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Both ets sites of the TR-II promoter are required for maximal response to Elf3. A, the sequences of the wild-type and mutated ets sites in mTR-II–108/+56(B/A), mTR-II–108/+56(X/A), mTR-II–108/+56(B/X), and mTR-II–108/+56(X/X) are shown. These sequences and the relative positions of the two ets sites are underlined. The mutated sequences of ets sites are boxed and shaded. B, F9-differentiated cells were transiently transfected with 15 µg of each construct along with 1 µg of an internal control plasmid, pCMV--gal. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case. C, F9-differentiated cells were transiently transfected with 15 µg of the mTR-II promoter/reporter construct shown, an Elf3 expression vector (0, 1, or 3 µg), and 1 µg of an internal control plasmid, pCMV--gal. The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case.

To further characterize the contributions of the B-site and the A-site, we compared the ability of Elf3 to stimulate the activities of the parent TR-II promoter/reporter construct, mTR-II–108/+56(B/A), and the two mutant constructs, mTR-II–108/+56(B/X) and mTR-II–108/+56(X/A). For this purpose, F9-differentiated cells were transiently transfected with two concentrations of the Elf3 expression vector and one of the TR-II promoter/reporter gene constructs (Fig. 1C). At the highest concentration tested, Elf3 stimulated the activity of the parent construct, mTR-II–108/+56(B/A), 23-fold. In contrast, Elf3 at the same concentration stimulated the activity of the mTR-II–108/+56(B/X) construct and the mTR-II–108/+56(X/A) construct only 3.7- and 4.7-fold, respectively. This demonstrated that both sites are essential to respond fully to Elf3. More importantly, it argues that the two ets sites function cooperatively to enhance synergistically the expression of the parent mTR-II–108/+56(B/A) construct.

Ets1, but Not PU.1, Strongly Stimulates the TR-II Promoter—Given the impact of Elf3 on the activity of the TR-II promoter, we examined whether other Ets proteins could stimulate the activity of this promoter in F9-differentiated cells. Because the TR-II gene is transcribed in cells that do not express Elf3, we initially examined the ability of Ets1 to stimulate TR-II promoter activity. Ets1, the founding member of the Ets family, is not expressed in F9 EC cells or F9-differentiated cells (54). It is expressed primarily in lymphoid tissues, which express TR-II, but not Elf3 (32, 56). Like Elf3, Ets1 strongly stimulated the parent mTR-II–108/+56(B/A) construct in F9-differentiated cells (Fig. 2A). Furthermore, the strong response to Ets1 required both ets sites, because Ets1 barely stimulated either mTR-II–108/+56(B/X) or mTR-II–108/+56(X/A). These findings led us to examine the ability of a third Ets family member, PU.1, to stimulate the activity of the TR-II promoter. PU.1 is expressed in B-cells and myeloid cells, which express TR-II, but not Elf3 (57). Unlike Elf3 and Ets1, PU.1 exerted only a modest effect on the activity of mTR-II–108/+56(B/A) (Fig. 2B). This modest effect of PU.1 does not appear to be the result of its inability to bind to the TR-II promoter, because PU.1 reduced the response of mTR-II–108/+56(B/A) to Elf3 (Fig. 2B). As a control, we determined that the inhibitory effect of PU.1 was not the result of an effect on Elf3 expression. Using a GFP-Elf3 fusion protein, which we have shown to be fully active in the stimulation of the TR-II promoter, we determined that PU.1 does not reduce the expression of GFP-Elf3 in F9-differentiated cells (Fig. 2C). If anything, PU.1 appeared to enhance the level of GFP-Elf3. However, this enhancement appears to be nonspecific, because it also enhanced the level of GFP itself. Together, these findings argue that Elf3 and Ets1 can strongly stimulate the TR-II promoter, whereas PU.1 exerts little if any effect on the TR-II promoter in F9-differentiated cells.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Ets1 can strongly stimulate the TR-II promoter, but PU.1 does not. A, F9-differentiated cells were transiently transfected with 15 µgofthe mTR-II promoter/reporter gene construct shown and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with an Ets1 expression vector at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case. B, F9-differentiated cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with an expression vector for Elf3 or PU.1 at the amounts shown. The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case. C, F9-differentiated cells were transiently transfected with 2 µg of an expression vector for either GFP-Elf3 or GFP as indicated using LipofectAMINE and the PLUS reagent, as described under "Experimental Procedures." Where indicated, the cells were also transfected with 2 µg of an expression vector for PU.1. GFP fluorescence was measured by flow cytometry, as described under "Experimental Procedures." Positive cells were determined as the percentage of cells expressing fluorescence above that of the mock transfection control. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed twice, and similar results were obtained.

View larger version (13K): [in this window] [in a new window]   FIG. 3. PEA3 can strongly stimulate the TR-II promoter. F9-differentiated cells were transiently transfected with 15 µgofmTR-II–108/+56(B/A) or mTR-II–108/+56(X/X) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with an expression plasmid for PEA3 at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case.

View larger version (18K): [in this window] [in a new window]   FIG. 4. PEA3 does not stimulate the mTR-II promoter in F9 EC cells. A, F9 EC cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) and 1 µg of an internal control plasmid pCMV--gal. Where indicated, the cells were also transfected with an expression vector for Ets1 or PEA3 at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case. B, F9 EC cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with an expression vector for Ets1 or PEA3 at the amounts shown. The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case. C, F9 EC cells were transiently transfected with 2.5 µg of an expression vector for either YFP-Ets1 or YFP using LipofectAMINE and the PLUS reagent, as described under "Experimental Procedures." Where indicated, the cells were also transfected with 2.5 µg of an expression vector for PEA3 or Elf3N270. YFP fluorescence was measured by flow cytometry, as described under "Experimental Procedures." Positive cells were determined as the percentage of cells expressing fluorescence above that of the mock transfection control. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed twice, and similar results were obtained.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Elf3 does not strongly stimulate the mTR-II promoter in F9 EC cells. A, F9 EC cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with an expression vector for Elf3 or Ets1 at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed four times, and similar results were obtained in each case. B, F9 EC cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated the cells were also transfected with an expression vector for Ets1, Elf3, or Elf3N233 at the amounts shown. The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case.

View larger version (22K): [in this window] [in a new window]   FIG. 9. Ets2 is unable to stimulate a TR-II promoter/reporter gene construct containing two B-sites. A, F9-differentiated cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) or mTR-II–108/+56(B/B) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with an expression vector for Elf3 or Ets2 at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case. B, F9-differentiated cells were transiently transfected with 15 µg of mTR-II–108/+56(B/B) and 1 µg of an internal control plasmid pCMV--gal. Where indicated, the cells were also transfected with an expression vector for Elf3N270 or Elf3N233 at amounts shown. The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/B), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed twice, and similar results were obtained in each case.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Ets2 does not stimulate TR-II promoter in F9-differentiated cells. A, F9-differentiated cells were transiently transfected with 15 µg of the mTR-II promoter/reporter construct shown and 1 µg of an internal control plasmid, pCMV--gal. Where indicated the cells were also transfected with an expression vector for Elf3 or Ets2 at the amounts shown. Activities were assayed and normalized as described under "Experimental Procedures." Data shown are means and S.D. for duplicate samples. This experiment was performed three times, and similar results were obtained. B, F9-differentiated cells were transiently transfected with 15 µgofmTR-II–108/+56(B/A) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated, the cells were also transfected with 1 µg of Elf3 and Ets2 or Ets2N331 at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from a representative experiment. This experiment was performed twice, and similar results were obtained. C, F9-differentiated cells were transiently transfected with 2 µg of the expression vector for GFP-Elf3 or GFP as shown using LipofectAMINE and the PLUS reagent, as described under "Experimental Procedures." Where indicated, the cells were also transfected with 2 µg of the Ets2N331 expression vector. GFP fluorescence was measured by flow cytometry, as described under "Experimental Procedures." Positive cells were determined as the percentage of cells expressing fluorescence above that of the mock transfection control. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed twice, and similar results were obtained.

View larger version (20K): [in this window] [in a new window]   FIG. 7. A C-terminal tail truncated form of Ets2 is unable to stimulate the TR-II promoter. F9-differentiated cells were transiently transfected with 15 µg of mTR-II–108/+56(B/A) or mTR-II–108/+56(X/X) and 1 µg of an internal control plasmid, pCMV--gal. Where indicated the cells were also transfected with an Elf3 expression and/or an Ets2C453 expression vector at the amounts shown. CAT and -galactosidase activities were assayed and normalized, as described under "Experimental Procedures." The promoter activity of each construct is calculated relative to the CAT activity observed with mTR-II–108/+56(B/A), which was set to 1. Data shown are means and S.D. for duplicate samples from one representative experiment. This experiment was performed three times, and similar results were obtained in each case.

View larger version (47K): [in this window] [in a new window]   FIG. 8. Full-length Ets2 does not bind in vitro to a DNA probe containing the two ets sites of the mTR-II promoter. A, EMSA was performed with nuclear extracts prepared from 293T cells transfected with expression plasmids for Ets2 or Ets2N331, as described under "Experimental Procedures." For the purposes of comparison, equal amounts of Ets2 and Ets2N331 were added to reaction mixtures of the lanes shown. The amounts of these flag-tagged Ets2 proteins were determined by Western blot analysis using the M2 antibody, as described under "Experimental Procedures." Lane 1, nuclear extract from mock transfected 293T cells. Lanes 2–4, nuclear extract from cells transfected with a full-length Ets2 expression vector. Lanes 5–7, nuclear extract from cells transfected with the Ets2N331 expression vector. Lanes 3 and 6, M2 antibody was added to the reaction mixtures. Lanes 4 and 7, a control IgG was added to the reaction mixtures. The positions of the free probe (P), of the binary Ets2N331/DNA complex (B), and supershifted complex (SS) are indicated. B, EMSA was performed with nuclear extracts prepared from 293T cells transfected with an expression plasmid for Elf3N270 or Ets2N331, as described under "Experimental Procedures." Equal amounts of flag-tagged Elf3N270 and flag-tagged Ets2N331 were added to reaction mixtures of the lanes as shown. The probe added to the reaction mixture in each lane is shown (B/A, B/X, or X/A). The positions of the free probe (P), binary complexes (B-1 or B-2), and ternary complex (T) are shown. Nuclear extract from mock-transfected 293T cells was added to the control lane. C, EMSA was performed with nuclear extracts prepared from 293T cells transfected with an expression plasmid for Ets2, Ets2C453, or Ets2N331. Nuclear extract from mock-transfected 293T cells was added to lane 1. Nuclear extract from 293T cells transfected with Ets2, Ets2N331, and Ets2C453 were added to lanes 2, 3, and 4, respectively. Equal amounts of the flag-tagged Ets2 proteins were added to the reaction mixtures. The DNA probe used contained the sequence for two B-sites (B/B). The positions of the free probe (P), binary complex (B), and ternary complex (T) are shown.

Ets2 Does Not Activate a TR-II Promoter/Reporter Gene Construct Containing Two Consensus Ets Sites—To determine whether the inability of Ets2 to activate the TR-II promoter was because of low affinity for the A-site, we performed two additional studies. First, we examined the in vitro binding of Ets2N331 to a DNA probe, referred to as the B/B probe, in which the sequence of the A-site in the B/A DNA probe was changed to a second B-site. Ets2N331 formed both a binary and a ternary complex with this probe, whereas Ets2C453 and Ets2 failed to form any detectable DNA-protein complexes (Fig. 8C). As expected, Elf3N270 also formed both a binary and a ternary complex with the B/B probe in an EMSA (data not shown).

The finding that Ets2N331 formed a ternary complex with the B/B probe formed the basis for the design of the second study, in which we examined the ability of Ets2 to stimulate the activity of the mTR-II–108/+56(B/B) construct in F9-differentiated cells. This promoter/reporter gene construct contains a second B-site in place of the A-site. Not surprisingly, the basal activity of mTR-II–108/+56(B/B), in the absence of exogenously expressed Ets proteins, was elevated 8-fold in comparison to the parent mTR-II–108/+56(B/A) construct (Fig. 9A). This elevated activity is apparently because of activation of the TR-II promoter by endogenous proteins, because overexpression of Elf3N270 or Elf3N233, which we previously demonstrated behave as a dominant-negative (23), reduced the basal activity of the mTR-II–108/+56(B/B) construct (Fig. 9B). Interestingly, overexpression of Elf3 in F9-differentiated cells increased the activity of the mTR-II–108/+56(B/B) construct 5-fold, whereas overexpression of Ets2 exerted little or no effect on mTR-II–108/+56(B/B) in F9-differentiated cells (Fig. 9A). Thus, the inability of Ets2 to stimulate the TR-II promoter in F9-differentiated cells does not appear to be the result of its inability to bind to the A-site in vivo.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The findings described in this study demonstrate that the TR-II promoter is not only strongly stimulated by Elf3, it is also highly responsive to Ets1 and PEA3 in F9-differentiated cells. Each of these Ets proteins mediates its effects via the two ets sites, which are conserved in the mouse and the human TR-II gene. It is evident that both ets sites are functional and that both sites are required for the synergistic stimulation of the TR-II promoter by Elf3, Ets1, and PEA3. Moreover, we demonstrate that five Ets family members, Elf3, Ets1, PU.1, Ets2, and PEA3, influence the activity of the TR-II promoter in distinctly different ways. Differential expression of these Ets proteins in F9 EC cells and their differentiated counterparts is one of the important mechanisms for determining which Ets protein regulates the TR-II promoter. Importantly, we demonstrate that the effects of at least two of the five Ets proteins, Elf3 and PEA3, are cell type-dependent. We also demonstrate that there are important differences in the ability of two Ets proteins, Elf3 and Ets2, which are both expressed in F9-differentiated cells, to bind to the two ets sites of the TR-II promoter, both in vitro and in vivo. Moreover, our studies argue that differential binding of Ets proteins to the two ets sites can contribute significantly to the differential activation of the TR-II promoter (see below). Hence, nature uses a wide variety of mechanisms to control which members of the Ets family of transcription factors regulate the TR-II promoter.

Two previous studies demonstrated that the activity of the TR-II promoter was reduced significantly when both ets sites were disrupted in combination (17, 23). Neither study specifically examined the effect of disrupting each site individually. In the case of the human TR-II promoter/reporter gene constructs, mutations introduced to disrupt the A-site also introduced changes in the B-site (17). As a consequence, the earlier data appear to indicate that a larger reduction in promoter activity occurs when the A-site is disrupted. In the work presented in this report, care was taken to disrupt each site separately. Given that the B-site matches a 9-base pair consensus sequence for an ets site, but that the sequence of the A-site differs by 2 base pairs of the 9, it was not surprising that a larger decrease in promoter activity was observed with the mTR-II–108/+56(X/A) construct than with the mTR-II–108/+56(B/X) construct, 60% versus 40% (Fig. 1B). Furthermore, disruption of both sites in mTR-II–108/+56(X/X) reduced promoter activity by at least 80%, which is similar to what had been observed in mouse TR-II promoter/reporter gene constructs when the region 3–56 was deleted (23). What is particularly interesting is the effect of overexpressing Elf3 on the activity of these constructs. Although the TR-II promoter is stimulated more than 20-fold by Elf3 when both sites are intact, disruption of either site reduces the response of the promoter to less than 5-fold. This argues that the two ets sites work cooperatively to respond synergistically to Elf3, as well as Ets1 and PEA3. The mechanism(s) responsible for this cooperative effect has not been determined, but it may involve cooperative binding of these Ets proteins to the TR-II promoter. In this regard, Ets1 has been shown to bind cooperatively to two adjacent ets sites in the stromelysin promoter (71).

It is evident from the work presented in this study that the two overlapping ets sites differ in at least two important respects. First, as discussed above, the B-site matches the sequence of a consensus ets site, whereas the A-site differs by 2 base pairs from the consensus sequence. Not surprisingly, the 2 base pairs in the A-site that differ from the consensus sequence are outside the essential core 5'-GGA(A/T)-3' sequence (Fig. 1A). Interestingly, conversion of the A-site to a second B-site elevates the expression of the activity of our mTR-II–108/+56 promoter/reporter construct 8-fold (Fig. 9A). Thus, we suspect that the presence of a nonconsensus ets site limits the activity of the endogenous TR-II promoter and helps to achieve the appropriate level of TR-II transcription in vivo. Given the potent and pleiotropic effects of TGF-, elevating TR-II expression would likely be detrimental to normal cellular physiology.

A second potentially important difference between the B-site and A-site is the differential binding of Ets2. Although it is evident that Ets2N331 can bind to the B-site, it exhibits significantly lower affinity for the A-site. In this regard, when Elf3N270 and Ets2N331 were tested at the same concentration, Elf3N270 formed a prominent DNA-protein complex with the X/A probe, whereas the DNA-protein complex formed between Ets2N331 and the X/A probe was barely detectable (Fig. 8B). In addition, Elf3N270 formed a ternary DNA-protein complex in an EMSA with a DNA probe tha