From the Oxford University Department of Paediatrics,
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3
9DS, United Kingdom, the
Engelhardt Institute of Molecular
Biology, Russian Academy of Sciences and Belozersky Institute of
Physico-Chemical Biology Moscow State University, Moscow, Russia, and
the ** Intramural Research Support Program, SAIC Frederick,
Frederick, Maryland 21702
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ABSTRACT |
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We describe a dense cluster of DNA-protein
interactions located 600 nucleotides upstream of the transcriptional
start site of the human tumor necrosis factor (TNF) gene. This area was
identified as being of potential importance for
lipopolysaccharide-inducible TNF expression in the human monocyte cell
line Mono Mac 6, based on reporter gene analysis of point mutations at
a number of nuclear factor B (NF-
B)-like motifs within the human
TNF promoter region. The area contains two NF-
B sites, which are
here shown by DNase I and methylation interference footprinting to
flank a novel binding site. UV cross-linking studies reveal that the
novel site can also bind NF-
B as well as an unknown protein(s) of
approximately 40 kDa. We show that these three adjacent
B-binding
sites differ markedly in their relative affinities for p50/p50,
p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears
capable of binding up to three NF-
B heterodimers simultaneously.
Reporter gene studies indicate that each element of the cluster
contributes to lipopolysaccharide-induced transcriptional activation in
Mono Mac 6 cells. These findings suggest that NF-
B acts in a complex manner to activate TNF transcription in human monocytes.
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INTRODUCTION |
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A fundamental problem in infectious disease is to understand the
mechanisms by which microbial stimuli such as bacterial
lipopolysaccharide (LPS)1
regulate the expression of human inflammatory mediators such as tumor
necrosis factor- (TNF). Production of TNF by macrophages and
monocytes occurs early in infection and orchestrates much of the
subsequent inflammatory response (1). Because excessive production of
TNF is responsible for many of the pathological consequences of severe
infection, its regulation is critical. This occurs at both the
transcriptional and post-transcriptional levels (2), with
transcriptional regulation showing specificity for both stimulus and
cell type (3). Reporter gene studies of the human TNF promoter using a
range of stimuli and cell types have identified a number of regulatory
elements upstream of the transcriptional start site, mostly within the
proximal 200 base pairs (4-13). However, we still have a poor
understanding of the precise regulatory events that are responsible for
the rapid induction of high level TNF expression in human monocytes and
macrophages during acute bacterial infection.
Much debate has centered on the role of nuclear factor-B (NF-
B)
in transcriptional activation of the human TNF gene in monocytes. Although there is strong evidence that murine TNF expression is influenced by a number of NF-
B binding sites (14-16), the situation in humans is less clear. Several lines of circumstantial evidence suggest that NF-
B could play an important role. A number of
NF-
B-like binding motifs are located between the start of
transcription of the TNF gene and the 3' terminus of the
lymphotoxin-
gene: they are here denoted
B1 (
873 to
864 nt),
B2 (
627 to
618 nt),
B2a (
598 to
589 nt), CK-1 (
213 to
204 nt (17)), and
B3 (
98 to
89 nt). It is worth noting that
site
B2 is 100% conserved between mouse, rabbit, and human TNF
promoters (18). Moreover, LPS induces nuclear translocation of NF-
B
in human monocytes, and LPS-inducible TNF expression is suppressed by
specific inhibitors of NF-
B mobilization (19, 20). However, studies of the role of NF-
B in the transcriptional activation of the human
TNF gene have yielded a conflicting picture. Initial gene reporter
studies suggested that NF-
B binding in the human TNF promoter region
is of little functional importance when tested in the murine macrophage
cell line P388D1 (5) or when analyzed in other contexts such as phorbol
12-myristate 13-acetate stimulation of U937 (myeloblastoid) cells (4)
and K562 (erythroblastoid) cells (21). However, recent studies indicate
that site
B3 contributes to transcriptional activation of the TNF
gene by superantigen (6) and cooperates with an adjacent
cAMP-responsive element site to provide an enhancement of TNF
transcription in LPS-stimulated human monocyte cell line THP-1
(13).
The present investigation focuses on the role of the other NF-B
binding sites, in particular the conserved site
B2 and its adjacent
site
B2a. Several studies have shown that NF-
B-specific complexes
are formed when site
B2a is tested against nuclear extracts from
stimulated monocytes and macrophages (13, 20, 22), but there has been
doubt about their functional importance. For example, reporter gene
studies in human THP-1 cells concluded that the region containing
B2
and
B2a does not contribute to LPS inducibility, even though
NF-
B-specific complexes were formed at the site
B2a with greater
affinity than at the apparently functional site
B3 (13). However, a
different picture emerged when reporter constructs containing the human
TNF promoter region were transiently expressed in the murine macrophage
line ANA-1. In this cell line, which resembles mature bone marrow
macrophages, LPS inducibility was found to fall 5-10-fold as a result
of deleting the region from
656 to
442 nt (22, 23).
These discrepant observations raise the intriguing possibility that the
functional activity of the region around B2 and
B2a has complex
determinants that are specific not only for stimulus and cell type but
for the stage of cell differentiation as discussed in Ref. 6. Because
the primary concern of the present study is the mechanism of
LPS-induced TNF expression in human monocytes, here we have used Mono
Mac 6, the human cell line whose surface markers and functional
properties most closely resemble those of a mature monocyte (24). In
this cell line, we find that mutations in sites
B2 and
B2a sites
have a more profound effect on LPS-inducible reporter gene expression
than mutations at other NF-
B sites in the TNF promoter region. We
further show that sites
B2 and
B2a flank a novel binding site,
such that this 39-nt segment of DNA is capable of binding up to three
NF-
B heterodimers plus an unknown constitutive factor(s). Each
element of this cluster appears to contribute to transcriptional
activation of the TNF gene.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
Human TNF promoter sequence (1173 to +130 nt)
derived from
1173-CAT construct (19) was used to generate the wild
type TNF promoter construct wt (
1173)-pXP1 in eukaryotic expression vector pXP-1 (ATCC), and corresponding fragments with point mutations at sites
B2,
B2a and
B32 were used to generate
the mutant TNF promoter constructs
B2-mt-pXP1,
B2a-mt-pXP1, and
B3-mt-pXP1. A set of site-directed mutations at the
site, a
mutation at site
B1, and +6 and +10 insertions between the sites
B2 and
were generated by PCR using
1173-CAT construct as a
template and oligonucleotides bearing nucleotide substitutions:
-mt1
(F:agctCCGGGGaTccTTTCACTCCCCG; R:agctCGGGGAGTGAAAggAtCCCCGG);
-mt2
(F:agctCCGGGGGTGATTTggaTCCCCG; R:agctCGGGGAtccAAACACCCCCGG);
-mt3
(F:agctCCGGGGGGTGgaTcCACTCCCCG; R:agctCGGGGAGTGgAtcCACCCCCGG); 1-mt
(F:agctGAGgATccGGACCCCCCCTTAA; R:agctTTAAGGGGGGGTCCggATcCTC); +6
(F:atggatccTCCCCGGGGCTGTCC; R:atggatccGTGAAATCACCCCCGGG); +10
(F:atggatccatTCCCCGGGGCTGTCC; R:atggatccatGTGAAATCACCCCCGGG) along with vector-specific primers HindIII (CTCGCCAAGCTTGGAAGAG) for F
primer or KpnI (CACACCGGGTACCGTGCAC) for R
primer. PCR products were cloned at HindIII/KpnI
of pXP-1 vector. All constructs were verified by DNA sequencing.
Nuclear Extracts and Electrophoretic Mobility Shift
Assay--
Oligonucleotide probes were radiolabeled with
[-32P]dCTP (Amersham Pharmacia Biotech): 1 (F:
agctGAGTATGGGGACCCCCCCTTAA; R: agctTTAAGGGGGGGTCCCCATACTC); 2 (F:
agctGGGTCTGTGAATTCCCGGGGGT; R: agctACCCCCGGGAATTCACAGACCC); 2a (F:
agctTCCCCGGGGCTGTCCCAGGCTT; R: agctAAGCCTGGGACAGCCCCGGGGA); 3 (F:
agctGCTCATGGGTTTCTCCACCAAG; R: agctCTTGGTGGAGAAACCCATGAGC);
(F:
agctCCGGGGGTGATTTCACTCCCCG; R: agctCGGGGAGTGAAATCACCCCCGG); CK-1 (F:
agctGTGTGAGGGGTATCCTTGATGC; R: agctGCATCAAGGATACCCCTCACAC); 2 mt (F:
agctGGGTCTtTGAATTCCCGGGGGT; R:
agctACCCCCGGGAATTCAaAGACCC); 2a mt (F:
agctTCCCCGtaGCTGTCCCAGGCTT; R:
agctAAGCCTGGGACAGCtaCGGGGA); 3 mt (F:
agctGCTCATtaGTTTCTCCACCAAG; R:
agctCTTGGTGGAGAAACtaATGAGC); and
-mt1,
-mt2,
-mt3, and 1-mt (as described above). Mono Mac 6 cells (10 × 106 to 20 × 106) were stimulated with 100 ng/ml LPS for 1 h, and nuclear extracts were prepared as described
previously (25). The binding reaction contained 12 mM
HEPES, pH 7.8, 80-100 mM KCl, 1 mM EDTA, 1 mM EGTA, 12% glycerol, and 0.5 µg of poly(dI-dC)
(Amersham Pharmacia Biotech). Nuclear extracts (1-4 µg) or
recombinant proteins (10-50 ng) were mixed in an 8-µl reaction with
0.2-0.5 ng of labeled probe (1-5 × 104 cpm) and
incubated at room temperature for 10 min. The reaction was analyzed by
electrophoresis in a nondenaturing 5% polyacrylamide gel at 4 °C in
0.5× TBE buffer. For supershift analysis, the reaction mixture was
preincubated with appropriate antiserum (Santa Cruz) at room
temperature for 5-10 min prior to addition of the labeled probe.
Indicated gels were quantified using the PhosphorImager (Molecular
Dynamics).
UV Cross-linking and Immunoprecipitation--
The binding
reaction was performed with radiolabeled oligoduplex corresponding to
the site in which three central dT nucleotides were substituted
with bromodeoxyuridine. The EMSA gel was UV illuminated at 302 nm for
30 min at 4 °C and exposed to autoradiography for 2-4 h at the same
temperature. The region corresponding to the DNA-protein complex was
excised, and the proteins were eluted in 2× SDS buffer (100 mM Tris-Cl, pH 6.8, 200 mM dithiothreitol, 4%
SDS, 20% glycerol) at 37 °C overnight. They were further processed either for SDS-PAGE or for immunoprecipitation as described (26).
Methylation Interference Analysis--
Oligonucleotide 2-F (or
2a-R) was labeled with [-32P]ATP (Amersham) and used
as primer in a PCR along with 2a-R (or 2-F). The PCR fragment obtained
(5 × 105 cpm) was gel purified and partially
methylated in 200 µl of buffer containing 200 mM HEPES,
pH 7.8, 1 mM EDTA by adding 1 µl of dimethyl sulfate at
room temperature for 1 min. The reaction was stopped by addition of 40 µl of stop solution (1.5 M sodium acetate, pH 5.2, 1 M
-mercaptoethanol). DNA was precipitated and washed
several times. The conditions of recombinant protein-DNA binding
reactions were the same as for EMSA but scaled up 5-10 times. Bound
and free DNAs were eluted overnight at 37 °C in 50 mM
Tris-Cl, pH 8.0, 1 M NaCl, 2 mM EDTA. The DNA
was purified and dissolved in 90 µl of H2O, 10 µl of
piperidine was added, and the sample was incubated at 90 °C for 30 min and cooled on dry ice. Traces of piperidine were removed by three
cycles of evaporation. Cleaved DNA fragments were separated by
electrophoresis using a 13% sequencing gel.
DNase I Footprinting Assay--
Solid phase DNase I footprinting
was carried out using the methodology described previously (27). DNA
probes were generated by PCR amplification using biotinylated primer
TNF-480 (TTGAGTCCTGAGGCCTGTGT) along with
[-32P]ATP-labeled primer TNF-680
(GCATTATGAGTCTCCGGGTC) or vice versa. Following gel purification,
radiolabeled DNA probe was adsorbed onto magnetic Dynabeads M-280
Streptavidin (Dynal) according to the manufacturer's instructions.
2-4 × 104 cpm of bead-DNA probe was incubated with
5-20 µg of nuclear extracts in 160 µl of binding buffer at room
temperature for 20 min. Following addition of 160 µl of salt mixture
(10 mM MgCl2, 5 mM
CaCl2) 0.06-0.25 units DNase I (Boehringer Mannheim) was
added to the mixture and incubated at room temperature for 30 s.
An equal volume of STOP solution was added (0.5 M NaCl, 40 mM EDTA, 1% SDS), and digested DNA was recovered using the
Magnetic Particle Concentrator (Dynal). DNA was washed with 200 µl of
solution I (2 M NaCl, 20 mM EDTA) followed by
200 µl of solution II (10 mM Tris-Cl, pH 8.0, 1 mM EDTA) twice. The reaction was analyzed on 7% sequencing
gel. Where indicated autoradiograms were recorded digitally using the
UVP Image Store 5000 system (UVP Life Sciences, Cambridge, UK) and analyzed with NIH Image 1.6 software (public domain).
Expression and Purification of Recombinant Proteins--
The
protein sequences corresponding to amino acids 2-400 of human p50 and
amino acids 2-306 of human p65 were recovered by PCR using the
appropriate primers. p50 and p65 cDNAs were cloned into the
bacterial expression vector pET32a (Novagen) and used to transform
BL21(DE3)LysS bacterial strain. Cultures were grown to
A600 = 0.4-0.6, induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside (Sigma), and then
grown for an additional 3 h before harvesting. Bacterial pellets
were resuspended in buffer A (50 mM Tris-Cl, pH 8.0, 0.1 mM EDTA, 0.01% Nonidet P-40, 1 mM
dithiothreitol, 10% glycerol) containing 70 mM NaCl and
lysed by three sonication cycles. Insoluble material was removed by
centrifugation, and cleared lysate was absorbed to biotin-labeled
oligoduplex corresponding to consensus NF-
B binding sequence bound
to streptavidin-agarose (Sigma) for 30-60 min at room temperature. The
agarose beads were washed twice with buffer A containing 100 mM NaCl, and the bound protein was eluted with buffer A
containing 500 mM NaCl. To form p65
/p50 heterodimer,
cleared lysates containing equal amounts of recombinant p50 and p65
were mixed and denatured by adding urea to 8 M, followed by
step dialysis against buffer A containing 70 mM NaCl and 6, 4, 2, 1, 0.5, or 0 M urea. The heterodimer was purified
from total extract as described above. The purity and concentration of
recombinant proteins were assessed on SDS-PAGE gel.
Cell Culture, Transfections, and Luciferase Assay-- Mono Mac 6 cells were maintained as described previously (24). Transient transfections were performed on Mono Mac 6 cells by the DEAE-dextran method as described previously (15). Cells were plated in fresh medium at 5 × 105/ml 24 h prior to transfection. The concentration of DEAE-dextran (Amersham Pharmacia Biotech) used was 100 µg/ml. After transfection cells were incubated for 24 h prior to LPS activation (1 µg/ml) and for a further 6-12 h before harvesting. Luciferase assay was performed using the Luciferase assay system and Turner Luminometer model 20 (Promega) according to the protocol supplied.
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RESULTS |
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Functional Characterization of the NF-B-like Sites of the Human
TNF Promoter Region in Human Monocytes--
To examine whether
LPS-inducible specific DNA-protein complexes can be formed at
NF-
B-like sites within the human TNF promoter region in human
monocytes, oligonucleotide duplexes spanning these sites were tested in
EMSA with nuclear extracts from unstimulated and LPS-stimulated Mono
Mac 6 cells. LPS stimulation resulted in formation of
NF-
B/Rel-specific complexes at sites
B1,
B2,
B2a, and
B3, although they differed considerably in intensity (Fig.
1). Because no NF-
B/Rel-specific
complexes were formed at site CK-1 (data not shown), this site was
excluded from further analysis. The strongest binding was observed at
site
B1, which formed two NF-
B-specific complexes corresponding
to p50/p50 homodimer (I) and p65/p50 heterodimer (II), respectively, as
defined by using NF-
B/Rel-specific antibodies (data not shown). The
binding at the sites
B2 and
B2a was about three times weaker, and
the complex formed at site
B3 was at the limit of detection (>10 times weaker than that at site
B1).
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Inducible Occupancy of a Cluster of Three Sites in the Distal Part
of the Human TNF Promoter Region--
To explore DNA-protein
interactions around the B2 and
B2a sites, we performed DNase I
footprinting analysis of a segment spanning
680 to
480 nt using
nuclear extracts prepared from Mono Mac 6 cells before or after LPS
stimulation. Nuclear extracts made after LPS stimulation gave
protection from DNase I cleavage at sites
B2 and
B2a and also in
the intervening region spanning
616 to
599 nt (Fig.
3A). Nuclear extracts made
before LPS stimulation also gave protection in this region when tested
at high concentrations. A similar pattern of protection was observed
using the noncoding strand (Fig. 3B).
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Constitutive and Inducible Binding to the Novel Site, --
To
examine the binding properties of
we used an oligonucleotide duplex
matching the sequences from
619 to
597 nt as a probe for EMSA.
Nuclear extracts were obtained from Mono Mac 6 cells that had been
stimulated with LPS (Fig. 4A).
For comparison, the binding complexes observed with probes specific for
sites
B2 and
B2a are shown. Unstimulated nuclear extracts gave a
single major complex with the probe for site
. After stimulation
this constitutive complex increased in intensity, and on close
inspection, a separate sharp band appeared at its lower margin. The
latter inducible complex was at maximal intensity between 15 and 120 min after stimulation (data not shown). These data raised the question
of whether the novel site might bind two different factors, one
constitutively present and the other rapidly inducible in Mono Mac 6 cells.
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An Inducible Complex Bound to is NF-
B (p65/p50)
Heterodimer--
The rapid kinetics of the inducible complex suggested
the involvement of transcription factor(s) that do not need to be
synthesized de novo. Because NF-
B provides a classical
example of this and because
includes a sequence (GTGATTTCAC) that
has two mismatches with the consensus NF-
B site (GGGPuNNPyCCC, where
Pu is purine and Py is pyrimidine), we considered the possibility of an
inducible complex being formed by NF-
B proteins. Two items of
circumstantial evidence are given above. First, the LPS-inducible
complex at
had similar mobility to the complexes at
B2 and
B2a, which are known to bind p65/p50 (Fig. 4A). Second,
the LPS-inducible complex at
was inhibited by competition with the
p65/p50 binding sequences of sites
B2 and
B2a (data not shown).
As a more formal test of this question, we performed an EMSA using
specific antibodies against two members of the NF-
B/Rel family, p65
and p50. Both antibodies markedly diminished the inducible complex,
whereas the antibodies against another member of the family, c-Rel, and against transcription factor EGR-1 had no effect on this complex (Fig.
5). Taken together, these results
identify a major inducible complex formed at site
as p65/p50
NF-
B heterodimer.
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p50 and p65 Exhibit Different Binding Patterns across the
B2/
/
B2a Cluster--
Because the various members of the
NF-
B/Rel family possess different functional properties, we used
recombinant forms of p50 and p65 to examine the possibility that each
part of the
B2/
/
B2a cluster might preferentially recruit a
specific type of NF-
B complex. The recombinant p65 was truncated at
amino acid 306 to facilitate bacterial expression and is denoted
p65
; this truncation does not affect the DNA-binding domain.
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All Three Binding Sites Contribute to Inducible Gene Expression in
Human Monocytes--
To examine whether binding of protein complexes
at the site has any functional effect on transcriptional
up-regulation of the TNF gene, we included three different variants of
the
site whose effects on DNA-protein interactions are illustrated
in Fig. 4B, in our set of reporter constructs with 1.2 kilobase pairs of TNF promoter sequence placed in front of a luciferase
reporter gene. When expressed in Mono Mac 6 cells variations at the
site resulted in a significant decrease of luciferase activity in the range of 40-55% reduction of the wild type (Fig.
8A). When a construct with the
point mutations at the sites
B2,
B2a, and
was expressed in
Mono Mac 6 cells, LPS-induced activity of the gene-reporter dropped
even further and resulted in about 70% reduction of the wild type
(Fig. 8A).
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Characterization of the Constitutive Complex at --
UV
cross-linking experiments were carried out to investigate the nature of
the factor(s) that bind to
. Nuclear extracts from unstimulated Mono
Mac 6 cells were incubated with a bromodeoxyuridine-substituted oligonucleotide probe, and the complexes were separated by EMSA. After
UV cross-linking, the complexes were excised and eluted from the gel.
When the purified complexes were analyzed by SDS-PAGE, a major band was
seen at Mr ~50 kDa (Fig.
9A). Because the oligoduplex itself migrates at about 10 kDa in this gel system, this would suggest
that the constitutive binding factor contains a major component of
approximately 40 kDa. UV cross-linking experiments with nuclear
extracts from stimulated Mono Mac 6 cells showed the same constitutive
component plus two additional bands of ~75 and ~60 kDa consistent
with p65 and p50 (Fig. 9, A, lane 2, and B, lane 1). To provide further confirmation that
the inducible complexes contain p65/p50 heterodimer, the cross-linked
products were immunoprecipitated with monospecific antibodies and then analyzed on SDS-PAGE. Anti-p65 antibodies immunoprecipitated a complex
that would correspond to a protein of 65 kDa, whereas anti-p50
antibodies immunoprecipitated a complex corresponding to a protein of
50 kDa, and control experiments with anti-C/EBP antibodies or
anti-biotin antibodies did not yield an immunoprecipitated band (Fig.
9B, lanes 2-5). Taken together, these results
indicate that site
is constitutively occupied by a protein(s) of
about 40 kDa and that LPS stimulation causes p65/p50 NF-
B
heterodimer to bind at the same site.
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DISCUSSION |
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The aim of the present study was to examine the role of NF-B in
the regulation of TNF transcription in Mono Mac 6, a cell line chosen
because of its close similarity to well differentiated human monocytes
(24). When the functional effects of four different NF-
B-like sites
in the TNF promoter region were compared by point mutagenesis and
reporter gene expression, two neighboring sites in the distal part of
the promoter region were identified as making the greatest contribution
to LPS-induced gene expression. We show that in between these two
sites, denoted
B2 and
B2a, there is a 19-nt sequence that binds
both constitutive and inducible nuclear factors. This intervening
sequence is referred to here as
, and the cluster as a whole can be
summarized as
627
B2/
/
B2a
589,
although the functional boundaries of the novel binding sites within
remain uncertain.
The capacity of the B2/
/
B2a cluster to bind NF-
B has two
interesting features. The first is that three NF-
B complexes can
bind to such a short segment of DNA. Whether they do so simultaneously is a matter for further investigation, but DNase I footprinting patterns suggest that this might be the case. A further suggestion comes from a comparison of the EMSA behavior of the individual binding
sites with that of the full
B2/
/
B2a region. Whereas the
individual binding sites only form one complex when incubated with
recombinant p65
/p50, the full
B2/
/
B2a region forms three separate complexes that would seem to correspond to the binding of one,
two, or three heterodimers (Fig. 7B).
The second potentially important aspect is the wide variation in
binding affinity across the cluster and the fact that different members
of the NF-B/Rel family preferentially bind to different parts of the
cluster. Site
B2 has relatively high affinity for p65
homodimer
but relatively weak affinity for p50 homodimer, whereas the opposite is
true for site
B2a. Such preferential binding patterns are likely to
be explained by minor sequence differences between the sites (28), yet
it is intriguing that we find little variation in binding affinity for
p65/p50, the canonical form of NF-
B, between all three sites. As
shown in Fig. 7A, the difference in p65/p50 binding affinity
between site
B2a, which is strongest, and site
, which is
weakest, is in the region of only 3-4-fold. We do not know whether the
polarization of the cluster in respect to p65/p65 and p50/p50 binding
affinity is of functional significance. But it has been observed that
the acquisition of LPS tolerance in Mono Mac 6 cells is associated with
a shift in the predominant form of NF-
B from p65/p50 heterodimer to
p50 homodimer, suggesting that the former activates and the latter
tends to suppress TNF expression (29). Thus, it is interesting to
speculate that one function of the
B2/
/
B2a cluster within human macrophages might be to switch LPS-induced TNF expression on and
off by allowing active interaction between different forms of NF-
B
and the elements of the cluster.
Gene reporter expression was approximately halved by mutations that
interfered with NF-B binding to any of the three sites in the
B2/
/
B2a region. Artificial spacing introduced between the
sites also resulted in reduction of gene expression. These data suggest
that each element of the cluster contributes to gene transcription and
that the structural unity of this region may be important for the
maximal levels of expression. In addition, when NF-
B-specific
interactions were ablated throughout the
B2/
/
B2a region,
LPS-induced transcriptional activation was reduced by 70%. Although
the present study focuses specifically on the question of TNF
regulation in human monocytes, it raises the more general question as
to whether p65/p50 in isolation can activate TNF transcription and, if
so, to what extent this depends on the
B2/
/
B2a region. Using
COS-7 cells we find that overexpression of p65/p50 up-regulates luciferase gene expression by 50-fold in the presence of the wild type
TNF promoter but by less than 5-fold if the promoter is disabled in the
B2/
/
B2a region as depicted in Fig.
8A.3 Taken
together with the present findings, this result highlights the need for
further work to understand the context-specific factors that may
modulate the functional activity of this region of the promoter in the
natural context of human monocytes and other relevant cell types.
An important question in resolving the function of the B/
/
B2a
cluster is the nature of the constitutive
-binding factor that is
found in nuclear extracts of Mono Mac 6 cells. The UV cross-linking
data indicate that the constitutive complex includes a protein(s) of
around 40 kDa that can be clearly distinguished from the LPS-inducible
NF-
B/Rel proteins. Our EMSA analysis of mutations within
would
suggest that the inducible binding motif overlaps with, but is not
identical to, the constitutive binding motif. One possibility is that
the constitutive binding factor recognizes a
B-like motif. Examples
of this phenomenon include NF-GMa, whose recognition sequence is very
similar to the NF-
B consensus (17), and recombination signal
sequence binding protein Jk, which is associated with the
B-binding
sequence in the interleukin-6 gene promoter (30). However, these are
both unlikely candidates for the
-binding factor, because they tend
to migrate much faster than NF-
B on EMSA. A second possibility is
that the constitutive factor might recognize another motif embedded
within the
site, and this raises the further intriguing possibility
that it may occupy the site simultaneously with NF-
B. The general
principle of transcription factor cohabitation is illustrated by a site in the human interferon-
promoter, which simultaneously binds NF-
B and the high mobility group protein HMG I(Y). NF-
B contacts the outer GC-rich bases in the major groove of the DNA helix, whereas
HMG I(Y) binds to the central AT-rich section that is exposed in the
minor groove, and both factors must be present to activate
transcription (31-33). We have not formally excluded HMG I(Y) as the
-binding factor, although the DNA sequence of
and the size of
the proteins visualized by UV cross-linking would argue against this
explanation. We are currently attempting to clone the constitutive
-binding factor; however, it is clear from these observations that
whatever its molecular identity, its effects on the functional activity
of NF-
B will be of considerable interest.
Understanding the role of NF-B in human TNF gene regulation has
proven difficult, with studies using different cell types and stimuli
reaching opposite conclusions. Such context specificity may be of
particular importance if, as has been shown for other cytokine genes,
the functional activity of the promoter region under consideration
requires individual transcription factors to be assembled into higher
order enhancer complexes (32). It has been recently demonstrated that
cooperation of the
B3 site with an adjacent cAMP-responsive element
site provides an enhancement of TNF transcription in LPS-stimulated
THP-1 cells (13). Our findings support the notion that an assembly of
protein complexes at
B2/
/
B2a is required for efficient
induction of TNF transcription by LPS in Mono Mac 6 cells. Further
studies are needed to understand the functional significance of
specific interactions that may occur in this region between each of the
NF-
B binding sites and between NF-
B and other nuclear
factors.
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ACKNOWLEDGEMENTS |
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We thank Dr. H. W. L. Ziegler-Heitbrock (Institute for Immunology, Munich, Germany) for providing us with the Mono Mac 6 cell line and for helpful discussions; Dr. J. Frampton for providing us with pXP-1 plasmid; Dr. M. Shchepinov for assistance with synthesis of modified oligonucleotides; Dr. K. Rockett for help in manuscript preparation (all from Oxford University, Oxford, UK); and Dr. D. Kuprash (Engelhardt Institute of Molecular Biology, Moscow, Russia) for providing us with unpublished results and for valuable comments and advice on the manuscript.
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FOOTNOTES |
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* This work was supported by the Medical Research Council (UK).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 44-1-865-222-345; Fax: 44-1-865-222-626; E-mail: iudalova{at}worf.molbiol.ox.ac.uk.
International Research Scholar of the Howard Hughes Medical
Institute. Supported by Grant 96-04-49491 from the Russian Foundation for Basic Research.
The abbreviations used are:
LPS, lipopolysaccharide; TNF, tumor necrosis factor-; NF-
B, nuclear
factor
B; nt, nucleotide(s); PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel
electrophoresis; MOPS, 4-morpholinepropanesulfonic acid.
2 D. Kuprash, I. Udalova, and S. A. Nedospasov, manuscript in preparation.
3 I. A. Udalova, J. C. Knight, V. Vidal, S. A. Nedospasov, and D. Kwiatkowski, unpublished data.
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REFERENCES |
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