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(Received for publication, October
27, 1994; and in revised form, December 21, 1994) From the
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.
TNF ( 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- 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- 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.
To test specific TNF promoter 5` sequences for
their ability to activate a heterologous promoter, oligonucleotides TII
and TII 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).
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.
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
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-139
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.
Figure 4:
Binding of Jun to the palindromic element
(TIIa) of the human TNF promoter. A,
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.
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-139
Figure 6:
Ets-related factors bind to the human TNF
promoter.
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-139
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). 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 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, ( 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,
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6577-6583
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)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 
B 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.
, or transforming growth factor 
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) .B 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.
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
, TIIb, TIIb, 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-139
CAT 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) . 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.).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.
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
, 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 
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 10
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) [
C]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 -galactosidase
expression plasmid pSV--galactosidase (Promega Corp.). Expression
of -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).
-galactosidase expression plasmid
pSV--galactosidase in three distinct experiments. Expression of
-galactosidase activity is correlated to the amount of cytoplasmic
protein incubated in each assay.
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-139CAT, 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) resulted in
reduced basal and PMA-inducible CAT activity, suggesting that this site
plays a major role for transcriptional activity.
CAT. TII, TIIa, and TIIb
represent oligonucleotide probes used in gel retardation
assays.
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 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.
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 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 (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 (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).
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-139CAT, 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.
CAT. 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 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.
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 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-139CAT). 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.
CAT. 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).
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) ).)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. (
)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. 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.
)))
We are grateful to E. Serfling for generously
providing the GSTc-jun expression vector and to P. Angel and
A. Meichle for helpful discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
