(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 ()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.
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
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-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.
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 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).
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 -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.
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-139CAT. 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.
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
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).
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-139CAT. 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.
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
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.
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-139CAT. 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.
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 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, ()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.
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, 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.