©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Human TNF Promoter by the Transcription Factor Ets (*)

(Received for publication, October 27, 1994; and in revised form, December 21, 1994)

Bernd Krämer Katja Wiegmann Martin Krönke (§)

From the Institut für Medizinische Mikrobiologie und Hygiene, Technische Universität München, Trogerstrasse 32, 81675 München, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor necrosis factor (TNF) affects the growth, differentiation, and function of a multitude of cell types and is viewed as a potent mediator of inflammation and cellular immune responses. In order to delineate functional domains that control TNF gene transcription, we have analyzed a 5` flanking region of the human TNF promoter spanning base pairs -115 to -98. This region contains a PEA3/Ets-1 binding motif 5` GAGGA 3` in direct juxtaposition to an AP-1/ATF-like palindromic sequence motif 5` TGAGCTCA 3`. Specific binding of Ets and Jun to their respective elements is demonstrated by competition analysis as well as by supershift assays. As shown by promoter deletion analysis, these two binding sites were essential for both basal promoter activity and responsiveness to the phorbol ester phorbol 12-myristate 13-acetate. Co-transfection of c-ets or c-jun expression plasmids along with TNF promoter-CAT reporter constructs revealed the participation of both transcription factors in the regulation of TNF gene transcription. Correspondingly, site-specific mutation of either Ets or Jun sites led to a complete loss of responsiveness to the respective transcription factor. These data suggest an essential role of Ets for the activation of TNF gene transcription.


INTRODUCTION

TNF (^1)plays a key role in the regulation of host defense responses against microbial infections. However, an adequate functioning of host defense mechanisms requires a stringent and balanced control of the regulation of TNF production (1, 2, 3) . Deregulated (over)production of TNF contributes to the pathophysiology of a number of disease states such as autoimmune diabetes, septic shock, graft versus host disease, or cachexia accompanying chronical parasitic infections(1, 2, 3, 4, 5, 6) . In addition, several lines of evidence suggest that TNF can stimulate HIV replication by activating kappaB enhancer elements within the viral LTR and thus might function as a disease progression factor in AIDS(7) . This ambivalent biological significance of TNF actions has raised considerable interest in the mechanisms controlling TNF gene transcription.

TNF synthesis and secretion are regulated at several levels (for review, see (8) ). TNF production was shown to be inducible in a variety of different cell types including not only macrophages, B- and T-lymphocytes, but also NK cells, mast cells, and a number of tumor cell lines(9, 10, 11, 12) . TNF production is regulated in part at post-transcriptional levels(13) . For example, AU sequences within the 3`-untranslated region of the TNF mRNA predispose for mRNA degradation by RNases and regulate translational efficiency(14, 15) . Furthermore, a post-translational control mechanism regulates the proteolytic cleavage of the membrane-bound 26-kDa TNF precursor molecule that is required for the release of soluble TNF from the cell surface(16) . Major regulatory mechanisms also operate at the level of TNF gene transcription. Several stimuli such as lipopolysaccharide, PMA, TNF, interferon-, or transforming growth factor beta have been shown to enhance TNF gene expression(3, 17, 18, 19) . In contrast, interleukin-4 or increased intracellular cAMP levels can trigger negative regulatory signals inhibiting TNF gene expression at the level of mRNA transcription(20, 21) .

To date, the nature of these transcriptional control mechanisms is not fully understood. Even though the human TNF promoter contains motifs with similarity to NF-kappaB binding sites, these sequences seem neither required nor sufficient for virus or lipopolysaccharide induction(22) . We, as well as others, recently have localized a PMA-responsive DNA region between bp -286 and -101(23, 24) . A GC-rich sequence was identified between position -170 and -155 with overlapping binding sites for the transcription factors Sp1 and Egr-1(25) . Rhoades et al.(26) describe an AP-1-, as well as an AP-2-binding element between bp -66 and -26. Leitman et al. (27) identified a palindromic, Jun-binding element between bp -109 and -100. A NF-AT binding sequence is located directly adjacent to this motif(28) . Here we address a previously unrecognized binding site for the Ets family of transcription factors, which adjoins upstream to the palindromic, AP-1/ATF-related element.

The Ets multigene family shares a common DNA-binding domain that specifically interacts with sequences containing the common core trinucleotide sequence GGA. About 30 Ets-related proteins have now been found in many species ranging from flies to humans. Most of the Ets-related proteins have been shown to be transcription activators, although some of them may have other functions such as in DNA replication (for review, see (29) ).

Intriguingly, Ets is known to cooperate with AP-1 in the transcriptional regulation of genes like IL-2(30) and collagenase(31) . Here we analyze the functional significance of a previously unrecognized Ets-1 binding element and a recently identified Jun binding element in the human TNF promoter. Both transcription factors are shown to strongly enhance TNF gene transcription; moreover, the Ets and Jun binding elements appear to cooperate in trans-activation of the human TNF promoter.


MATERIAL AND METHODS

Cell Lines and Culture Conditions

Jurkat and HuT78 cell lines were maintained in culture medium consisting of a mixture of Click's/RPMI (50/50 volume percent) supplemented with 10% fetal calf serum and 50 µg/ml each of streptomycin and penicillin.

Oligonucleotides and Plasmids

Sequences of the oligonucleotides used in gel retardation assays (TIIa, TIIa(m), TIIb, TIIb(m), PEA3, ATF, AP-1) are shown in the respective figure legend. For each oligonucleotide, upper and lower strands were synthesized on a 380A DNA synthesizer (AB/I Weiterstadt, Federal Republic of Germany) and purified on OPC columns (Applied Biosystems, Weiterstadt, FRG). 5` deletions of the TNF gene promoter were generated from a pUC13pML plasmid containing the human genomic TNF sequences(32, 25) . pTNF-139CAT and pTNF-101CAT contain the first 139 or 101 bp, respectively, of the human TNF promoter upstream of the transcriptional initiation site (+1). pTNF-139DeltaCAT was obtained by digestion of pTNF-139CAT with SstI, following S1 nuclease treatment and religation. This resulted in a deletion of bp -117 to -95, verified by sequencing according to the method described by Maxam and Gilbert(33) .

To test specific TNF promoter 5` sequences for their ability to activate a heterologous promoter, oligonucleotides TII and TII(m) were cloned into plasmid pJ21CAT ((34) ; kindly provided by Dr. J. Pierce, Boston) containing a minimal mouse c-fos promoter upstream of the CAT gene. To determine both the number and orientation of inserts, plasmids were sequenced by the dideoxy chain termination method using Sequenase(TM) (U. S. Biochemical Corp.).

The c-jun expression vector pRSVcJun contains c-jun cDNA sequences from position +181 (SalI) to +1804 (ScaI) between the Rous sarcoma virus long terminal repeat and SV40 sequences necessary for RNA splicing and polyadenylation(35) . pUCRSV lacks c-jun sequences and was used as a control vector. The c-ets-1 expression vector pCRNCMcEts was constructed by inserting the coding region into the expression plasmid pCRNCM adjacent to the cytomegalovirus immediate-early promoter ((36) .; generously provided by Dr. T. Graf).

Site-directed Mutagenesis

Site-directed mutagenesis of the TNF promoter CAT construct pTNF-139CAT was performed using the Altered Sites(TM) in vitro mutagenesis system (Promega Corp. Madison, WI). Briefly, a mutagenic oligonucleotide containing substituted nucleotides was annealed to the single-stranded DNA template, followed by the synthesis of the mutated strand. Positive mutants are selected by restored antibiotic resistance and verified by sequencing. pTNF-139 m1CAT contains a mutated Ets site, pTNF-139 m2CAT, a mutated Jun site, and in pTNF-139 m3CAT, both Ets and Jun binding sites were mutated. The oligonucleotides used for site-directed mutagenesis are shown in Fig. 8.


Figure 8: Representation of the TNF promoter mutants used for the functional analysis of the neighboring Ets and Jun binding sites. Asterisks mark mutated base pairs.



Bacterial Expression of GSTJun Protein

The plasmid pGEX-3x/c-Jun was used to express the glutathione S-transferase Jun fusion protein as described by Smith and Johnson(37) . For purification of the fusion protein, glutathione-Sepharose was used according to the instructions of the manufacturer (Pharmacia, Uppsala, Sweden).

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts were prepared as described(38) . The protein concentration of nuclear extracts was measured using a BCA assay (Pierce, Hamburg, FRG) with bovine serum albumin as the standard. For each oligonucleotide, the full-length complementary oligonucleotide strands were end-labeled with [-P]ATP (Amersham Corp., Braunschweig, FRG) using polynucleotide kinase (Boehringer, Mannheim, FRG). 5-10 µg of nuclear proteins (amount of protein was kept constant for each assay) were preincubated for 10 min at 24 °C with 500 ng of poly(dI-dC) (Pharmacia, Freiburg, FRG) in a binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl(2), 50 mM KCl, 5 mM dithiothreitol, 10% glycerol, 20 µl final volume). When indicated, unlabeled competitor oligonucleotides were preincubated with nuclear proteins 10 min prior to the addition of labeled oligonucleotide. For supershift assays, anti-Ets ((36) ; kindly provided by Dr. T. Graf) and anti-Jun (sc-44 X, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were added to the reaction mixture and incubated for 1 h at room temperature. Samples were loaded onto a 0.25 times TBE, 6% polyacrylamide gel and electrophoresed at 20 V/cm. Gels were dried and exposed to Kodak XAR films at -70 °C using intensifying screens.

Cell Transfection and CAT Assays

Cells were transfected using the DEAE-dextran method as described(24) . Briefly, 5 times 10^6 cells were incubated with 10 µg of plasmid DNA for 30 min in serum-free Click's/RPMI medium containing 0.25 mg/ml DEAE-dextran (Pharmacia, Uppsala, Sweden). 50 µg/ml chloroquine (Sigma, München, FRG). 48-h post-transfection, cells were harvested and cytosolic proteins were quantified by a BCA assay (Pierce, Hamburg, FRG). 150-200 µg of protein were incubated for 4 h at 37 °C in a buffer containing 0.25 M Tris, pH 7.5, 1 mM acetyl-CoA (Boehringer) and 0.45 nmol (25 nCi) [^14C]chloramphenicol (Amersham). Amounts of protein were kept identical for each probe of an assay in order to ensure corresponding transfection efficiencies. This was verified by transfection of HuT78 cells with a beta-galactosidase expression plasmid pSV-beta-galactosidase (Promega Corp.). Expression of beta-galactosidase activity (measured by a spectrophotometric assay, Promega Corp.) correlated with the amount of cytoplasmic protein incubated (Fig. 2C). Acetylated forms of chloramphenicol were separated by thin layer chromatography. For quantification, the appropriate spots were cut out and the radioactivity was determined by liquid scintillation counting. Alternatively, autoradiographs were analyzed by two-dimensional laser scanning (Personal Densitometer with ImageQuant 3.22, Molecular Dynamics, Krefeld, FRG).


Figure 2: Functional analysis of a 5` human TNF promoter region. A, schematic representation of the human TNF promoter-CAT hybrids. B, TNF promoter-CAT constructs were transiently transfected into Jurkat and HuT78 cells. CAT activity was measured in transfected cells left untreated (open bars) or stimulated with PMA (filled bars). Relative CAT activities are representative for three to four independent experiments. C, analysis of plasmid expression. HuT78 cells were transfected with 5 µg of a beta-galactosidase expression plasmid pSV-beta-galactosidase in three distinct experiments. Expression of beta-galactosidase activity is correlated to the amount of cytoplasmic protein incubated in each assay.




RESULTS

Identification of a Functional Regulatory Element within the Human TNF Gene Promoter

Previous studies identified in the 5` region of the human TNF promoter one element between bp -139 and -101 that had a strong impact on transcription activity(25) . This region contains potential binding sites for the transcription factors Ets and AP-1 (Fig. 1). Ets is known to bind to the sequence (A/C)GGAA(39, 29) , present at positions -118 to -144 of the human TNF promoter. The palindromic motif 5` ATGAGCTCAT 3` between bp -109 and -100 shows similarities to binding sites for AP-1 or ATF/CREB-like transcription factors(40) . This element has been shown to bind the transcription factor Jun; however, the functional significance remained unclear(27) . To determine the regulatory impact of these two sequences, human acute leukemic Jurkat T cells and cutaneous T cell lymphoma HuT78 cells were transfected with TNF promoter deletion mutants fused to the bacterial CAT gene (Fig. 2A). These cell lines were chosen based on our previous observation that the endogenous TNF gene is inducible in these cell types(41) . PMA was used as a stimulus because this phorbol ester proved to be a potent inducer of TNF gene expression in many cell types(11) . As shown in Fig. 2B, 5` deletion from position -139 to -101 (pTNF-101CAT) resulted in drastically reduced TNF promoter activity. Furthermore, this construct was only scarcely inducible by PMA in HuT78 cells. To address the participation of the two detected potential binding sites for Ets and AP-1/ATF, bps -117 to -95 were deleted (pTNF-139DeltaCAT, Fig. 1and Fig. 2). Basal promoter activity and inducibility of this deletion mutant was markedly reduced. Three copies of a 34-bp TNF promoter-derived oligonucleotide, TII, containing both Ets and AP-1/ATF sites ( Fig. 1and Fig. 3), conferred to a minimal c-fos promoter CAT construct both elevated basal expression and responsiveness to PMA. Mutation of the AP-1/ATF binding site (TII(m)) resulted in reduced basal and PMA-inducible CAT activity, suggesting that this site plays a major role for transcriptional activity.


Figure 1: Nucleotide sequence of the human TNF promoter from bp -130 to -90 and schematic representation of the putative binding sites for the transcription factors Ets and AP-1 (gray boxes). The bold sequence represents the internal deletion in plasmid pTNF-139DeltaCAT. TII, TIIa, and TIIb represent oligonucleotide probes used in gel retardation assays.




Figure 3: Activation of a heterologous c-fos promoter. A, schematic representation of the heterologous c-fos/TNF promoter-CAT hybrids. B, CAT constructs were transiently transfected into HuT78 cells. CAT activity was measured in transfected cells left untreated (open bars) or stimulated with PMA (filled bars). Relative CAT activities are representative for three independent experiments.



Characterization of the Factor(s) Binding to the Palindromic Element

The palindromic sequence motif 5` ATGAGCTCAT 3` shows similarities to known binding sites for AP-1 or ATF/CREB-like transcription factors, although the GC core is inverted. In order to characterize the factor(s) binding to this element, EMSA were performed using a 19-bp oligonucleotide, TIIa, corresponding to bp -113 to -95 of the TNF gene. When radiolabeled TIIa was incubated with nuclear extracts prepared from HuT78 cells, retarded protein-DNA complexes were detected (Fig. 4A, lanes 1-4). Competition analysis was performed using excess of unlabeled TIIa or TIIa(m) where the binding site was mutated. As shown in Fig. 4A, only the wild-type oligonucleotide competed for proteins binding to the labeled TIIa probe, indicating specific and high-affinity binding of nuclear factor(s) to the AP-1/ATF related binding site. An ATF binding sequence from the human somatostatin enhancer (Fig. 4A, lanes 5 and 6) competed as efficiently as TIIa for complex formation. An AP-1 binding sequence from the human collagenase enhancer also interfered with the formation of the TIIa-protein complex, although with less efficiency (Fig. 4A, lanes 7 and 8). These findings suggest, that the nuclear factor(s) recognizing the TNF promoter sequence 5` ATGAGCTCAT 3` can bind to ATF/CRE and AP-1 consensus sequences.


Figure 4: Binding of Jun to the palindromic element (TIIa) of the human TNF promoter. A, P-labeled oligonucleotide probe TIIa 5` GCAGATGAGCTCATGGGTG 3` was incubated with nuclear extracts from HuT78 cells. Competition was performed using unlabeled TIIa (lanes 2 and 3), TIIa(m) 5` GCAGATGtctaCATGGGTG 3` (lane 4), the ATF binding site of the somatostatin promoter 5` GTGGCTGACGTCAGAGAGG 3` (lanes 5 and 6) and the AP-1 binding site of the collagenase promoter 5` GAAGCATGAGTCAGACACG 3` (lanes 7 and 8). B, P-labeled oligonucleotide probes TIIa (lanes 1-4), AP-1 (lanes 5-9) and ATF (lanes 10-14) were incubated with nuclear extracts from HuT78 cells left untreated (lanes 1, 5, and 10) or stimulated for 2 h with 20 ng/ml PMA (lanes 2-4, 6-9, and 11-14). For competition analysis, extracts were incubated in the presence of 40-fold excess of unlabeled oligonucleotides TIIa (lanes 3, 9, and 14), TIIa(m) (lanes 4, 8, and 13), AP-1 (lane 7), and ATF (lane 12). C, P-labeled probe TIIa was incubated with bacterially expressed GST protein (lane 1) and GSTJun fusion protein (lanes 2-4). For competition, 40-fold excess of unlabeled TIIa (lane 3) or TIIa(m) (lane 4) was used. D, P-labeled oligonucleotide TIIa was incubated with nuclear extracts of HuT78 cells stimulated for 2 h with 20 ng/ml PMA (lanes 1 and 2). 100 ng of anti-Jun antiserum was added and incubated for 1 h at room temperature prior to EMSA (lane 2).



The formation of protein complexes with TIIa or the AP-1 binding motif could be enhanced by PMA (Fig. 4B, lanes 2 and 6). In contrast, the factors binding to the ATF sequence were constitutively present (Fig. 4B, lanes 10 and 11). The participation of Jun could be demonstrated using a bacterially expressed GSTcJun fusion protein that bound with high specificity to the palindromic sequence in the TNF promoter (Fig. 4C, lanes 2-4). Moreover, the binding of Jun to TIIa was supported by a supershifted complex that was formed by an anti-Jun antibody (Fig. 4D, lane 2). These results confirm a previous report by Leitman et al.(27) , who identified Jun as one member of this PMA-inducible complex.

Jun Induces TNF Gene Transcription via the Palindromic Element 5` ATGAGCTCAT 3`

To investigate whether Jun is able the trans-activate the human TNF promoter via the Jun binding element, we performed co-transfection experiments, using a c-jun expression plasmid pRSVcJun. Both the homologous TNF promoter CAT construct pTNF-139CAT (Fig. 5A), as well as the heterologous reporter plasmid p3xTIIJ21CAT (Fig. 5B) were markedly stimulated by co-transfection of the c-jun expression plasmid, whereas internal deletion (pTNF-139DeltaCAT, Fig. 5A), or mutation (p3xTIImJ21CAT, Fig. 5B) of the Jun binding element completely abolished responsiveness to pRSVcJun. These findings indicate that Jun trans-activates the TNF promoter via the palindromic element.


Figure 5: Trans-activation of the human TNF promoter via the palindromic element. A, HuT78 cells were co-transfected with increasing amounts of c-jun expression plasmid pRSVcJun and 5 µg of either pTNF-139CAT or pTNF-139DeltaCAT. B, the c-jun expression plasmid was co-transfected in HuT78 cells along with the heterologous reporter constructs p3xTIIJ21CAT or p3xTIImJ21CAT. The total amount of DNA transfected was kept constant at 6 µg using the empty expression vector pUCRSV. Relative CAT activities are representative for three independent experiments.



Identification of Binding Sites for the Transcription Factor Ets

Computer analysis identified an Ets binding motif between bp -118 to -114 adjacent to the palindromic Jun binding element. In order to reveal binding of Ets or a related factor to this sequence, we performed electrophoretic mobility shift assays using a TNF promoter-derived oligonucleotide, TIIb (Fig. 6, lanes 1-4), and an oligonucleotide containing a well characterized Ets binding site of the PEA3 promoter (36) (Fig. 6, lanes 5-8). Incubation of nuclear extracts from HuT78 cells with either TIIb or PEA3 resulted in virtually identical retardation patterns (Fig. 6, lanes 1 and 5). The protein-DNA complex could be efficiently cross-competed for by excess of either TIIb or PEA3 (Fig. 6, lanes 2, 3, 6, and 7), whereas the oligonucleotide TIIb(m) containing a mutated Ets binding sequence did not compete (Fig. 6, lanes 4 and 8). Binding of Ets was confirmed by an Ets-specific antiserum. Addition of anti-Ets resulted in further retardation of the protein-DNA complex (Fig. 6, lane 10). These results identified an Ets-related factor binding to the ``GGA'' element.


Figure 6: Ets-related factors bind to the human TNF promoter. P-Labeled oligonucleotide probes TIIb 5` ACCGCTTCCTCCAGATGA 3` (lanes 1-4, 9, and 10) and PEA3 5` CGAGCAGGAAGTTCGACG 3` (36) (lanes 5-8) were incubated with nuclear extracts of HuT78 cells. For competition, 50-fold excess of unlabeled TIIb (lanes 2 and 6), PEA3 (lanes 3 and 7), and TIIb(m) 5` ACCGCTgttgtCAGATGA 3` (lanes 4 and 8) was used. For supershift assays, anti-Ets antiserum (36) was added and incubated for 1 h at room temperature prior to EMSA (lane 10). The arrow indicates the supershifted complex.



Overexpression of Ets Trans-activates the Human TNF Promoter

In order to examine whether the TNF gene can be activated by exclusive Ets stimulation, pTNF-139CAT was co-transfected into HuT78 cells along with a c-ets expression plasmid, pCRNCMcEts. As shown in Fig. 7A, pCRNCMcEts markedly stimulated pTNF-139CAT, whereas internal deletion of the Ets binding element resulted in loss of responsiveness to c-ets (pTNF-139DeltaCAT). pCRNCMcEts also stimulated the heterologous reporter plasmid p3xTIIJ21CAT containing the Ets binding site. In contrast, the minimal fos promoter pJ21CAT was not activated (Fig. 7B). These findings indicate that c-ets overexpression is sufficient to activate TNF gene transcription.


Figure 7: Trans-activation of the human TNF promoter by Ets. A, HuT78 cells were co-transfected with c-ets expression plasmid pCRNCMcEts and 5 µg of either pTNF-139CAT or pTNF-139DeltaCAT. B, the heterologous reporter constructs p3xTIIJ21CAT and pJ21CAT were co-transfected in HuT78 cells along with the c-ets expression plasmid pCRNCMcEts. Total amounts of DNA transfected were kept constant at 6 µg using the empty expression vector pCRNCM. Relative CAT activities are representative for three independent experiments.



Functional Analysis of the Adjacent Ets and Jun Binding Elements

To investigate the individual regulatory function of either the Ets or AP-1/ATF binding site, three different mutants of the TNF promoter region between bp -117 and -95 were generated (Fig. 8). Mutations were introduced into the Ets binding site (pTNF-139 m1CAT) or the Jun binding site (pTNF-139 m2CAT) and finally into both Ets and Jun binding sites (pTNF-139 m3CAT). Co-transfection experiments were performed using either c-jun (pRSVcJun) or c-ets (pCRNCMcEts) expression plasmids. As shown in Fig. 9A, pRSVcJun stimulated the wild-type TNF promoter sequences (pTNF-139CAT) in a dose-dependent manner. As expected, the reporter plasmid containing the mutated Jun binding element (pTNF-139 m2CAT) could not be trans-activated. Correspondingly, overexpression of c-ets strongly stimulated the wild-type reporter plasmid pTNF-139CAT, whereas pTNF-139 m1CAT containing a mutated Ets sites proved unresponsive (Fig. 9B).


Figure 9: Ets and Jun trans-activate the human TNF promoter. A, HuT78 cells were co-transfected with the c-jun expression plasmid pRSVcJun and 5 µg of either pTNF-139CAT or pTNF-139 m2CAT. The amount of DNA added was kept constant at 7 µg using the empty expression plasmid pUCRSV. B, the TNF promoter derived TNF promoter CAT constructs pTNF-139CAT or pTNF-139 m1CAT were co-transfected with increasing amounts of the c-ets expression vector pCRNCMcEts. The amount of DNA added was kept constant at 15 µg using the empty expression vector pCRNCM. Values of CAT expression were calculated as -fold induction compared with co-transfection of empty expression vector alone. The results shown are representative for four to five independent experiments. C, HuT78 cells were transfected with 5 µg of the TNF promoter-derived mutants and either left untreated or 24-h post-transfection-stimulated with 20 ng/ml PMA for 24 h. Values of CAT expression were calculated as -fold induction compared with unstimulted cells.



To address the possibility of a functional cooperation of both regulatory elements, HuT78 cells were transfected with the wild-type TNF promoter CAT construct (pTNF-139CAT) or the mutant reporter plasmids pTNF-139 m1CAT, pTNF-139 m2CAT, or pTNF-139 m3CAT (Fig. 9C). As expected, mutation of both binding sites (pTNF-139 m3CAT) produced an almost complete loss of responsiveness to PMA, indicating that both elements are essential for induced TNF promoter activity. Mutation of the Ets site (pTNF-139 m1CAT) or the Jun binding element (pTNF-139 m2CAT), on the other hand, reduced stimulation by only half as compared with the wild-type reporter plasmid (pTNF-139CAT). Similarly, mutation of either one or both regulatory elements reduced basal promoter activity (not shown).


DISCUSSION

In this study we have identified a previously unrecognized Ets binding element in the 5`-flanking region of the human TNF promoter that functions as a major important positive regulatory element.

Computer analysis identified the core Ets binding motif GGA between bp -118 and -114 of the human TNF promoter. Binding of Ets protein to this sequence was confirmed by cross-competition studies and serologic identification by an Ets-specific antiserum. The family of Ets proteins consists of DNA binding factors with homology over a region of 85 amino acids(29) . This so-called ETS domain confers the ability to bind to the DNA motif (A/C)GGAA in the middle of 10 bp(39) . The flanking sequences are variable and there is growing evidence that they may determine which member of the Ets protein family will bind. The overall Ets binding sequence in the human TNF promoter is not identical to other known binding sites for Ets family proteins, but high similarities to Ets-1, Ets-2, Elf-1, and PU-1 responsive elements can be detected. It is therefore not yet clear which Ets protein binds to the TNF promoter. On the other hand, different ets motifs appear to vary in their selectivity for binding proteins(42) , and conversely, different Ets-like proteins vary in their selectivity for a given motif(43) . Co-transfection experiments demonstrated trans-activation of the human TNF promoter by a c-ets expression plasmid. Furthermore, responsiveness to Ets could also be conferred to the heterologous reporter construct p3xTIIJ21CAT. Ets proteins are well known transcriptional activators. They have been implicated in the regulation of gene expression during a variety of biological processes, including growth control, transformation, T cell activation, and developmental programs in many organisms. In addition, they often co-operate with other transcription factors in regulation of gene transcription (for review, see (29) ).

Directly 3` to the Ets responsive element the human TNF promoter contains the sequence 5` ATGAGCTCAT 3`. This palindromic motif resembles both the ATF/CRE consensus sequence TGACGTCA (44) and the AP-1/TRE consensus sequence TGA(G/C)TCA(45) . Competition studies clearly indicated that the TNF promoter binding factor(s) can bind to both ATF/CRE and AP-1 consensus sequences ( Fig. 4and (27) ). The PMA responsiveness of this TNF promoter elements may be distinctive. While ATF/CRE sequence motifs have been shown to mediate cAMP responsiveness of a number of cellular genes(44, 46) , they seem incapable of mediating transcriptional activation by phorbol esters via protein kinase C-dependent pathways. Unlike ATF/CREB, the factor binding to the TNF promoter element TIIa was not responsive to agents that raise intracellular cAMP levels, (^2)yet could be activated by PMA. Thus, induction characteristics indicate a resemblance to AP-1/TRE-responsive elements. Two further findings support this conclusion. First, recombinant Jun protein binds with high specificity and avidity to this element, and second, Jun could be identified as part of the binding complex by anti-Jun antiserum. Co-transfection experiments, using a c-jun expression plasmid, demonstrated trans-activation of the human TNF promoter via the palindromic sequence motif. Notably, the participation of Fos in activating the human TNF promoter has been excluded previously by Leitman et al.(27) . This implicates dimerization of Jun with some other protein which may belong to the ATF/CRE superfamily of transcription factors. Interestingly, dimerization of Jun with ATF/CREB proteins increases affinity for CRE(47) . We would like to emphasize, however, that binding of recombinant ATF-2 protein (kindly provided by Drs. S. Wagner and M. Green) to the palindromic sequence 5` ATGAGCTCAT 3` could neither be detected in the absence nor in the presence of recombinant Jun. (^3)Clearly, the protein that forms a heterodimer with Jun and binds to the human TNF promoter has yet to be identified. A NF-AT binding motif was recently identified in direct juxtaposition downstream of the palindromic motif(28) . NF-AT is known to form complexes with Jun and Fos proteins in activated T cells (48) . It will be interesting to investigate possible cooperation of ATF/AP-1 and NF-AT elements in controlling TNF gene transcription.

Deletion of the identified Ets and Jun binding sequences resulted in markedly reduced TNF promoter activity (Fig. 2B and 3B). Accordingly, mutation of the core binding sequences completely abolished trans-activation by the respective transcription factor. Although synergistic activation could not be demonstrated directly by co-transfection of c-jun and c-ets expression plasmids,^2 mutation of the Ets site or the Jun site markedly reduced PMA-induced TNF promoter activity. On the other hand, mutation of both elements produced an almost complete loss of responsiveness to PMA, indicating that both elements are essential for induced TNF promoter activity. Down-modulation of transcriptional activity by deletion of only one of the two adjacent regulatory elements could be explained by a combined regulatory impact on TNF gene transcription. Cooperation of Ets with AP-1 has been shown in promoters of the genes for collagenase(31) , urokinase-type plasminogen activator (uPA)(49) , and in the polyoma virus enhancer(50) . Ets appears to play an essential role with regard to the regulation of TNF promoter activity. In the absence of the Ets binding element, the TNF promoter proved less responsive not only to trans-activation by Jun. We have recently shown that a Sp1 and Krox-24/Egr-1 binding element exerts its function only in the presence of the Ets element(25) . Further work is required to completely understand the co-operative role of Ets in controlling TNF gene transcription.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft and the Deutsche Krebshilfe. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut für Medizinische Mikrobiologie und Hygiene, Technische Universität München, Trogerstr. 32, 81675 München, FRG. Tel.: 49-89-4140-4141; Fax: 49-89-4140-4942.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; AP-1, activator protein 1; CREB, cAMP-responsive element-binding protein; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate; ATF, activating transcription factor.

(^2)
B. Krämer, K. Wiegmann, and M. Krönke, unpublished observation.

(^3)
B. Krämer, K. Wiegmann, and M. Krönke, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to E. Serfling for generously providing the GSTc-jun expression vector and to P. Angel and A. Meichle for helpful discussion.


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