(Received for publication, June 24, 1996, and in revised form, February 6, 1997)
From the Wistar Institute, Philadelphia, Pennsylvania
19104 and the § Laboratory of Immunology, Internal Medicine
Clinic, University of Mainz, 55101 Mainz, Germany
Interleukin-12 (IL-12) is a proinflammatory
cytokine produced by antigen-presenting cells in response to many
microbial infections. IL-12 plays an important role in the generation
of T helper type-1 cells, which favor cell-mediated immune response.
IL-12 is composed of two different subunits, p40 and p35, whose
expression can be regulated concomitantly or differentially. Monocytic
cells, the major producers of IL-12, can be primed by interferon-
(IFN-
) to produce optimal amounts of IL-12 in response to LPS
stimulation as a consequence of bacterial infection. The priming effect
is exerted primarily at the transcriptional level on the p40 promoter in conjunction with the effects of LPS, possibly by inducing specific transcription factors, which individually have no direct effect but
which cooperatively can activate the promoter. We examined in detail
one of these DNA-protein interactions observed around an Ets-2 element
situated at
211/
207 of the p40 promoter, which is known to be a
functionally critical site. This region interacts with a nuclear
complex termed F1 that appears to be highly inducible by either IFN-
treatment for 16 h or lipopolysaccharide stimulation for 8 h.
F1 binding to the Ets-2 site requires a considerable amount of spacing
around the Ets-2 site, as revealed by gel mobility shift and in
vitro methylation assays. Supershift experiments and DNA affinity
purification indicated that both Ets-2 and a novel, antigenically
related protein with an approximate molecular mass of 109 kDa are part
of the F1 complex, together with additional components including IRF-1
and c-Rel. This novel protein is designated GLp109 for its inducibility
by IFN-
or lipopolysaccharide. Its possible role in the activation
of the IL-12 p40 promoter is discussed.
Interleukin-12 (IL-12)1 consists of a
heavy chain (p40) and a light chain (p35) linked covalently to give
rise to a heterodimeric (p70) molecule. IL-12 is produced by phagocytic
cells and other antigen-presenting cells in response to stimulation by
a variety of microorganisms as well as their products. IL-12 mediates
its biologic activities mainly through T and NK cells by inducing their
production of interferon- (IFN-
), which augments their cytotoxicity, and by enhancing their proliferative potential. Through
these functions, IL-12 plays a critical role in the early inflammatory
response to infection and in the generation of T helper type 1 Th-1
cells, which favor cell-mediated immunity.
We previously (1) reported that both the p35 and p40 genes are
regulated primarily at the transcriptional level by LPS and IFN- in
human monocytes. The human IL-12 p40 gene promoter cloned in our
laboratory as a 3.3-kb genomic fragment carries sufficient sequence
information to confer transient cell type-specific expression and can
respond to priming by IFN-
followed by LPS stimulation in monocytic
cells, in analogy to the endogenous promoter. One of the critical
regions in the human IL-12 p40 promoter resides at
222/
204, as
determined by 5
deletion coupled with transient transfection in a
murine macrophage-like cell line RAW264.7 (3). The promoter construct
deleted to
222 still retains about 50% of the 3.3-kb promoter
activity induced by the combination of LPS and IFN-
, suggesting that
additional upstream regulatory elements exist. Further deletion of the
promoter down to
204 abolishes most but not all of the activity in
response to LPS and IFN-
, indicating that a downstream element may
be responsible for this persistent inducibility. One of these potential
cis elements may be the NF
B half-site located at
117 to
107, which has recently been characterized as being responsible for
response to LPS stimulation in the murine macrophagic J774 cell line
(2). Within the
222 and
204 region, there is an Ets-like element,
TTTCCT or AGGAAA for its complement, between
212 and
207. The GGAA
motif has been established as the consensus motif for the Ets family of transcription factors (3, 4). We showed that this Ets-like element in
the p40 promoter is essential for the response of the promoter to LPS
and IFN-
stimulation, since a deletion of 5 out of 6 nucleotides
from this element in the context of the entire 3.3-kb p40 promoter
resulted in the loss of inducibility by LPS and IFN-
; the element is
also essential for activation of the promoter by Ets-2 (not Ets-1 or
PU.1), since Ets-2 expressed from a cotransfected vector failed to
activate the p40 promoter with the Ets-2 element deleted. However,
activation of the p40 promoter by exogenous Ets-2 in the transient
transfection system still depends on LPS or IFN-
stimulation,
suggesting the presence of additional inducible factors that may
account for the activation of the IL-12 p40 promoter by IFN-
and
LPS.
In the present study, we investigated DNA-protein interactions at this
element and analyzed some of the nuclear factors that interact with
this region in monocytic cells primed with IFN- followed by
stimulation with LPS. We describe here a nuclear factor, termed GLp109,
which is inducible by either IFN-
or LPS and which is part of a
complex including IRF-1 and possibly NF
B c-Rel that interacts with
the Ets-2 element in the p40 promoter.
Reagents
Murine IFN- was a generous gift from Dr. Gianni Garotta
(Human Genome Sciences, Inc., Rockville, MD). LPS was purchased from Sigma. All antibodies used in supershift and Western blot experiments were purchased from Santa Cruz Biotechnology, Inc.
Nuclear Extract Preparation
Nuclear proteins were isolated from RAW and other cell lines according to Dignam et al. (5).
Gel Electrophoretic Mobility Shift Assay (EMSA)
End-labeled DNA probes (10,000 cpm/sample) were mixed with 2 µg of crude nuclear extracts and incubated at room temperature for 20-30 min in the presence of 1 µg of poly(dI-dC) (Boehringer Mannheim) in a volume of 10 µl of 0.5 × dialysis (D) buffer (10 mM Hepes, pH 7.9, 10% glycerol, 50 mM KCl, 0.1 mM EDTA, 0.25 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A). The mix was then fractionated through a 4% polyacrylamide gel in buffer containing 6.7 mM Tris-HCl, pH 7.5, 3.3 mM sodium acetate, 0.1 mM EDTA for 1.5 h at 200 V. The gel was dried and exposed in a PhosphorImager storage screen (Molecular Dynamics) and scanned. Supershift experiments were carried out by preincubating the nuclear extract with 1-2 µg of affinity-purified polyclonal antibodies for 30 min at 4 °C before the probe was added.
Methylation Interference
In vitro methylation interference assay was performed as described (30).
Western Blot
Reducing SDS-PAGE (10%) was performed according to Laemmli (6) with 20-30 µg of nuclear proteins in each sample. The gel was then electroblotted in Tris-glycine buffer containing 40% methanol onto a nitrocellulose membrane (Trans-blot, Bio-Rad). After blocking the membrane with TBS-T buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% milk powder for 1 h at room temperature, primary antibodies (rabbit anti-mouse IgG) were added at 1:1000 dilution (1 ng/ml) in TBS-T containing 1% milk powder for 1 h at room temperature. The membrane was washed three times for 7 min each with TBS-T, and incubated with secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase, Amersham) at 1:5000 dilution for 1 h at room temperature. After washing three times in TBS-T for 5 min each, and once in TBS for 5 min, the membrane was drained briefly and subjected to the enhanced chemiluminescence (ECL) detection procedure (Amersham).
DNA Affinity Purification
Annealing of Complementary OligonucleotidesEquimolar
amounts of two complementary oligonucleotides were mixed at a final
concentration of 1 mg/ml in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, heated at
90 °C for 2 min, and brought to room temperature over a period of 30 min to 1 h. The upper strand oligonucleotide represents the human
IL-12 p40 promoter sequence 289 to
196, and the lower strand,
292
to
196. The control DNA ligand has the same upper strand, but the
lower strand spans
243 to
196.
Double-stranded synthetic
DNAs above were biotinylated at the 3 end using terminal
deoxyribonucleotide transferase and biotin-16-ddUTP following the
manufacturer's protocol (Boehringer Mannheim). To monitor efficiency
of biotinylation (generally 65-70%), 8 ng, 560,000 cpm of
[
-32P]ATP 5
-end-labeled upper strand was included in
the mix.
Streptavidin-agarose slurry (Life Technologies, Inc.) was preabsorbed with 1 mg/ml BSA for 2 h at room temperature with constant agitation. Excess BSA was removed by a brief spin followed by the removal of supernatant, and the slurry was reconstituted in 0.5 × D buffer (see above). Biotinylated DNA (30 µg) was then mixed with 20 µl of the reconstituted streptavidin-agarose slurry in 0.5 × D buffer overnight with constant mixing at 4 °C. Efficiency of the coupling reaction (generally ~95%) was monitored based on the radioactivity derived from the 32P-end-labeled DNA ligand.
Purification of Nuclear ExtractsDNA ligand (30 µg)
coupled to streptavidin-agarose in 20 µl of 0.5 × D buffer was
mixed with 20 µg of poly(dI-dC) (Boehringer Mannheim) and 60 µg of
RAW cell nuclear extract in 60 µl of D buffer, in a final volume of
140 µl for 30 min at room temperature with frequent gentle vortexing.
The mixture was spun 10 s in an Eppendorf centrifuge, and the
supernatant was collected (S1 fraction). After washing the resin with
50 µl of D buffer followed by centrifugation, supernatant was again
collected (S2 fraction). The next four washes were: 50 µl of D
buffer, 0.2 M KCl (S3); 50 µl of D buffer, 0.4 M KCl (S4); 50 µl of D buffer, 0.4 M KCl, and
0.5% SDS (S5); and 50 µl of D buffer, 0.4 M KCl, and
0.5% SDS, 1% -mercaptoethanol (S6). Each fraction (25 µl) was
analyzed by SDS-PAGE and Western blot assay. The resin was regenerated
by 10 washes with 0.5 × D buffer, reabsorbed with 1 mg/ml BSA,
reconstituted in 0.5 × D buffer, and stored at 4 °C.
The human IL-12 p40 gene promoter was recently cloned
as a 3.3-kb genomic fragment. Sequence comparison of the proximal
promoters of the human and the murine IL-12 p40 genes (Fig.
1) shows that the two promoters are quite homologous up
to about 400 with respect to the transcription initiation site, where
the homology breaks down with large gaps between them. Within the
400
proximal promoter region, several putative transcription factor binding
sites are very well conserved: Ets-2, PU.1, and an NF
B-like element,
which has been postulated to be a critical response element in the
murine p40 promoter for LPS stimulation in macrophagic J774 cells
(2).
Several Nuclear Factors Including Ets-2 Interact with the
To test whether the 222/
204 region
interacts with any nuclear factors that are induced by IFN-
and LPS,
an oligonucleotide probe was made that spanned
222 to
196. The
probe was constructed to include sequences downstream from
204
because the Ets-2-like element is quite close to
204 and thus might
require additional space for the stabilization of potential DNA-protein
interactions at this putative site. However, this probe did not yield
any significant binding with either unfractionated nuclear extracts
derived from stimulated RAW cells or with recombinant Ets-2. The only
detectable binding with this probe was observed with unstimulated RAW
nuclear extract. The complex was designated F3 (Fig. 2).
When the probe was extended upstream to
292, it bound a low mobility
nuclear complex (named F1) in RAW cells stimulated with either LPS or IFN-
(Fig. 2). The F1 binding activity was first detected around 2 h after LPS stimulation and peaked at 8 h post-stimulation, coinciding with the transient p40 promoter activity in RAW cells (data
not shown). Deletion from the 5
end to
265 or
243 abrogated F1
binding, but also resulted in the appearance of a second factor, designated F2, which was inducible by IFN-
but not by LPS, and which
was induced synergistically by the combination of IFN-
and LPS (Fig.
2). F3 binding was also detected with the
243/
196 probe in the
unstimulated RAW cell nuclear extract (Fig. 3). Deletion from the 3
end to
227 also resulted in the loss of all binding (data
not shown), suggesting that both the 5
and 3
ends of the
292/
196
region are essential for F1 binding.
We first focused on the F1 complex, reasoning that F1 might contain F2
and that F3 does not appear to be part of F1 and might be a negative
regulator of the p40 promoter. To establish the specificity of F1,
competitive EMSA was performed using a number of homologous and
heterologous oligonucleotides (Fig. 3A). The F1 complex
present in IFN-- and LPS-stimulated RAW cells was efficiently
competed off by a 20-fold molar excess of the
292/
196 DNA, but not
by the consensus Ets-2 (Ets2-CS) oligonucleotide, or by the
222/
196
oligonucleotide containing the Ets site of the p40 promoter, or by the
735/
710 oligonucleotide of the p40 promoter containing an
IRF-1-like element. The consensus IRF-1 oligonucleotide (IRF1-CS) did
compete somewhat, although the efficiency was considerably lower than
that of the
292/
196 oligonucleotides. To identify the components of
the F1 complex, a series of affinity-purified polyclonal antibodies
(
-Ets-1,
-Ets-2,
-IRF-1,
-IRF-2,
-STAT-1, and
-STAT-4) directed against a variety of transcription factors were
used in supershift experiments.
-Ets-2 caused a supershift of the F1
complex (Fig. 3B). In addition,
-IRF-1 resulted in diminished binding of F1, which correlated with the competition studies
shown in Fig. 2. The effect of
-IRF-1 on the F1 complex was
corroborated with a second antibody directed to a different epitope of
mouse origin (Santa Cruz Biotechnology, Inc.) (data not shown). The
supershift of
-Ets-2 was specific since it was completely blocked by
the peptide to which the
-Ets-2 was directed, whereas the peptide
itself had no effect on the F1 complex (Fig. 3C). These data
suggest that Ets-2 or an antigenically related molecule as well as
IRF-1 may be constituent components of the F1 complex. In addition, the
-c-Rel antibody also produced a consistent supershift of the F1
complex (see below).
To determine the relationship between
the F1 and F2 complexes and further characterize these two binding
activities, supershift and competitive EMSA were performed using the
292/
196 and
243/
196 regions as probes, respectively. As shown
in Fig. 3D, the F1 complex was specifically and strongly
supershifted by
-Ets2 and to a lesser extent by
-c-Rel, whereas
none of the four antibodies (
-Ets2,
-NF
B p50,
-NF
B p65,
and
-c-Rel) together with five other antibodies (
-Ets1,
-IRF-1,
IRF-2,
-STAT-2; data not shown) had significant
effects on the F2 complex. The F1 complex was efficiently competed off
by the
292/
196 fragment but not by the
243/
196 fragment. The F2
complex, on the other hand, was competed off efficiently by both
292/
196 and
243/
196 fragments, indicating that F1 and F2 are
distinct.
The nature of the F3 complex was
determined using RAW nuclear extract (unstimulated) and a series of
polyclonal antibodies directed against PU.1, IRF-1 (human origin, but
cross-reacts with mouse), IRF-1 (murine origin), IRF-2, Stat-1, Stat-4,
Ets-2, and Ets-1. As shown in Fig. 4, only the -PU.1
antibody had a clear effect on the F3 complex, suggesting that the
complex may contain PU.1 or a related molecule.
A Broad Region of
To examine in detail the DNA-protein interaction in the
region surrounding the Ets-2 element, DNA methylation interference was
performed with a probe that extended from 196 to
471 (Fig. 4). The
region between
268 and
196 appeared to be very active in
interacting with nuclear proteins in cells stimulated with either
IFN-
or LPS. This observation correlates well with the EMSA data,
which showed that deleting the
292/
196 probe to
265/
196 abolished F1 binding. The lack of a narrowly restricted protected sequence within this region suggests that these sequences interact with
multiple factors, consistent with the EMSA data indicating the low
mobility of the F1 complex, and with the observation that 5
sequential
deletions of the
292/
196 probe yielded multiple complexes (F1, F2,
and F3).
To
confirm the results of the supershift experiment, a DNA affinity column
was prepared using the 292/
196 region of the p40 promoter as the
target DNA. The double-stranded DNA fragment was biotinylated and bound
to streptavidin-agarose. Nuclear extracts from IFN-
- and
LPS-stimulated RAW cells were mixed with the column resin, and
flow-through and subsequent washes in increasing stringency were
collected and subjected to reducing SDS-PAGE followed by Western
blotting using
-Ets-2. The same nuclear extracts were subjected to
an identical purification scheme with a control DNA column, which is
identical to the
292/
196 probe in the sense strand, but different
in the antisense strand in that it spanned
243 to
196. This probe
was first tested for its inability to bind F1 before use. Nuclear
extracts from unstimulated, IFN-
-, LPS-, and IFN-
+ LPS-stimulated RAW cells were also directly analyzed by Western blot
without purification (Fig. 5A). In
unstimulated cells, the antibody detected only one strong band of about
54 kDa, the known size of Ets-2 (4). Upon induction with IFN-
or
LPS, two additional larger bands were detected, one of ~58 kDa, the
other ~109 kDa. Stimulation with LPS resulted in yet more bands,
notably at 52, 119, and 165 kDa. Results of bacterial alkaline
phosphatase treatment suggested that the 52-kDa protein was likely to
be the phosphorylated form of the 54-kDa Ets-2 (data not shown). The
reason for the increased mobility of the 54-kDa Ets-2 upon
phosphorylation is not clear. The 58- and 109-kDa proteins were induced
with either LPS or IFN-
, and are designated GLp58 and GLp109,
respectively. GLp58 is likely an alternative form of Ets-2, since it is
also present in recombinant murine Ets-2 translated from rabbit
reticulocyte lysate. However, it is unlikely to be a phosphorylated
form of Ets-2, since bacterial alkaline phosphatase did not change its
mobility (data not shown). The 119- and 165-kDa proteins were induced
only by LPS, and are thus designated Lp119 and Lp165, respectively.
Bacterial alkaline phosphatase treatment of these proteins also did not
result in any mobility changes (data not shown), suggesting that they
are probably not derived from phosphorylation at threonine, serine, or
tyrosine residues. In addition, the specificity of the anti-Ets-2
antibody was confirmed by including the epitope peptide with the
primary antibody in the procedure. The Ets-2 peptide to which the
anti-Ets-2 antibody was directed specifically blocked the binding of
its cognate antibody, whereas a second peptide, Ets-1/2, which
represented a separate epitope common to both Ets-1 and Ets-2, had no
effect on the action of the anti-Ets-2 antibody (Fig. 5B).
When the fractions eluted from the DNA columns were examined by
SDS-PAGE and Western blotting using
-Ets-2, most of the proteins
that reacted with the antibody, namely, the 52-, 54-, 58-, 109-, 119-, and 165-kDa proteins, were present in the flow-through of the control
DNA column, whereas the same proteins were bound tightly on the column and were eluted only by a combination of 0.4 M KCl, 1%
SDS, and
-mercaptoethanol (Fig. 6), suggesting that
these proteins specifically bound to the
292/
196 DNA.
We show here that several putative sequence motifs are highly
conserved between the mouse and the human IL-12 p40 proximal promoters,
suggesting their functional significance. We examined the physical
protein-DNA interactions at the Ets-2 site located at 212 to
207 in
the human IL-12 promoter. This site is essential for induction of the
promoter by IFN-
and LPS in RAW cells and for the response of the
promoter to activation directly by Ets-2. Although this sequence in the
form of a 27-mer oligonucleotide with 10 base pairs flanking either
side of the core motif TTTAAT (AGGAAA for the complement) failed to
bind either recombinant Ets-2 or nuclear extracts derived from IFN-
-
or LPS-stimulated RAW 264.7 cells. However, this probe did interact
with PU.1 or a related molecule in unstimulated RAW cells (F3). F3 is
diminished upon stimulation with IFN-
and LPS.
The functional role of F3 remains to be established. It has been
reported that IFN-, a cytokine that induces the expression of major
histocompatibility complex class II molecules, down-regulates the
expression of PU.1 (7), which is certainly consistent with our
observations that F3 (PU.1) binding is diminished upon stimulation of
RAW cells with IFN-
. Furthermore, Borràs et al. (8)
showed that I-A
gene expression is repressed by PU.1 binding to the PU box domain in its promoter in both bone marrow-derived macrophages and the mouse B cell line A20-2J.
Extension of the probe further upstream to 292 (the
292/
196
probe) resulted in a readily formed high molecular weight complex with
extracts from either IFN-
- or LPS-induced RAW cells, but not from
unstimulated cells. The apparent discrepancy between the ability of the
292/
196 region to bind F1 and its inability to enhance
significantly the promoter activity over that of the
222/
196 region
led us to test the possibility that F1 binding may require larger
physical space than does the
222/
196 site to stabilize the
DNA-protein complex due to its large size. We anchored the
222/
196
region in the context of the polylinker region of the PCRII plasmid
(Invitrogen). This sequence was then excised along with varying lengths
of the polylinker sequence. These fragments were used in EMSA with
nuclear extracts from IFN-
/LPS-induced RAW cells. Two requirements
for F1 binding were established. First, the length of the probe has to
be more than 66 base pairs. Second, the TTTCCT sequence is critical for
F1 binding (data not shown). This indicates that F1 binding is specific
for the Ets-2 site but requires large physical spacing around the core
motif, suggesting that F1 may be a large complex composed of multiple
factors. Indeed, when the
292/
196 probe was deleted to
243/
196,
F1 binding was diminished and F2 appeared, which responded to IFN-
stimulation more than to LPS.
In vitro methylation and footprint experiments to examine
the 292/
196 region indicated that the complex induced by either IFN-
or LPS covered quite extensive sequences. In vivo
footprint assay also revealed strong and extensive protection around
this region in either RAW cells or primary macrophages primed with IFN-
followed by LPS stimulation (data not shown). Together, the
data suggest that the region including and around the Ets-2 site
actively interacts with a series of nuclear factors that may be
involved in the activation of the IL-12 p40 promoter in response to
specific stimulation.
The first indication of the presence of Ets-2 or its derivatives in the
F1 complex came with supershift experiments in which an anti-Ets-2
polyclonal antibody was able to retard the mobility of the F1 complex.
The F1 complex can also be supershifted by two IRF-1 antibodies
directed against a very homologous epitope of human and mouse origins,
respectively, as well as by the anti-NFB C-Rel antibody, suggesting
that F1 may be a highly sophisticated complex containing members of the
Ets, IRF, and NF
B families of transcription factors. By contrast,
the F2 complex was not affected at all by any of the antibodies whose
antigens are well known IFN-
-inducible factors (IRF-1, IRF-2,
STAT-1, and STAT-4), indicating that F2 might be yet another new factor
which responds to IFN-
stimulation. When nuclear extracts containing
the F1 complex were partially purified through a DNA affinity scheme using the
292/
196 sequence as the ligand together with Western blot
analysis using the anti-Ets-2 antibody, several proteins antigenically
related to Ets-2 were revealed. First, the 54-kDa Ets-2 protein seemed
to be constitutively present in RAW cells. Upon induction with IFN-
or LPS, a 52- and a 58-kDa protein appeared, which probably represent
derivatives of Ets-2, since both of these species were also found in
recombinant Ets-2 translated in vitro in rabbit reticulocyte
extracts. In fact, the 54-kDa protein was shown to be derived from the
52-kDa protein after alkaline phosphatase treatment. In addition to the
52-, 54-, and 58-kDa proteins, which may be derived directly from Ets-2
via posttranslational modification, three other proteins were also
identified in RAW cells: GLp109, Lp119, and Lp165, which responded to
IFN-
or LPS.
The form in which Ets-2 in RAW cells interacts with the Ets-2 site at
212 and
207 is presently not clear. However, several observations
suggest that Ets-2 binds to that site only as part of a large complex.
First, recombinant Ets-2 does not bind the
222/
196 probe, but does
bind a consensus Ets-2 probe. Second, RAW cell nuclear extracts contain
some binding activities which interact with the consensus Ets-2 probe
constitutively (data not shown); this cannot account for the inducible
binding of F1 on the basis of Ets-2 alone. Third, DNA affinity
purification of the F1 complex showed that Ets-2 is indeed tightly
associated with several other Ets-2-related proteins. Finally, we have
shown also data indicating the presence of IRF-1 and NF
B c-Rel in
the F1 complex. Together, these data strongly suggest a multi-component nature of the F1 complex.
The Ets proteins contain a structural motif (winged helix-loop-helix)
that is capable of binding DNA as a monomer. However, the DNA binding
activity of several members of this family appears to be suboptimal in
the absence of a partner protein. For example, Elk-1 activates
transcription from the c-fos promoter in conjunction with
the DNA-binding protein serum response factor. A specific protein-protein interaction stabilizes the binding of Elk-1 and serum
response factor on the c-fos promoter (9-12). In the case of GA-binding protein (GABP), which is composed of an subunit with
an Ets domain and a non-DNA-binding
subunit, two
and two
subunits form a stable tetramer with augmented DNA binding activity on
the herpes simplex virus type I immediate early gene promoter (13, 14).
A domain has been mapped in Elk-1 and GABP
that is important in the
protein-protein interaction, and only Ets proteins bearing that domain
can interact with the partner (15, 16). Ets-1 and Ets-2 have also been
reported to cooperate with several different putative partners in
functional studies. Ets-1 works with Sp1 in activating transcription
from the human T-cell lymphotropic virus type 1 enhancer (17), while on
the immunoglobulin µ heavy chain enhancer, Ets-1 cooperates with PU.1 (18). Ets-1 also synergizes with AP-1 in activating the polyomavirus enhancer (19). Myb and Ets-2 cooperate in activating the mim-1 promoter
(20). In the Moloney murine leukemia virus and T cell receptor
-chain enhancers, adjacent binding sites for Ets and the
polyomavirus enhancer-binding protein-2 (PEBP2)/core-binding factor (cbf) family of proteins are both required to
constitute a phorbol ester response element (21). The B cell and
macrophage-specific transcription factor PU.1 recruits the binding of a
second B cell-restricted nuclear factor, NF-EM5, to a DNA site in the
immunoglobulin
3
enhancer. DNA binding by NF-EM5 requires a
protein-protein interaction with PU.1 and specific DNA contacts.
Phosphorylation of PU.1 at Ser148 is necessary for
interaction with NF-EM5 and for regulating transcriptional activity
(22, 23). A recent study using radiolabeled PU.1 protein as a probe to
screen a B cell cDNA expression library in search of
PU.1-interacting proteins identified three DNA-binding proteins
(NF-IL6
, HMG-I(Y), and SSRP), a chaperon protein, and a
multifunctional phosphatase (24). The physical interaction between PU.1
and NF-IL6
requires the carboxyl-terminal 28 amino acids of PU.1.
PU.1 and NF-IL6
can bind adjacent sites simultaneously. In transient
transfection assays, PU.1 and NF-IL6
can functionally cooperate to
activate transcription synergistically. A direct physical association
between Ets-1 and AP-1 has been demonstrated in normal human T cells
(25). This interaction is mediated by the binding of the basic domain
of Jun to the Ets domain of Ets proteins. The associated Jun is capable
of interacting with Fos family members to form a trimolecular protein
complex. The physical association of Ets-1 and AP-1 is required for the
transcriptional activity of enhancer elements containing adjacent Ets
and AP-1 binding sites.
Our observation of the presence of IRF-1 and NFB c-Rel in the F1
complex is not surprising. NF
B regulates a wide range of genes
involved in immune function and inflammation responses. The interferon
regulatory factor (IRF) family, on the other hand, plays a vital role
in gene regulation mediated by interferons (type I and type II).
Interactions between NF
B, HMG-I(Y), and an Ets-like protein (Elf-1)
reportedly regulate expression of the IL-2 receptor
gene (26). The
promoter of the VCAM-1 gene contains two essential NF
B sites, which
are not sufficient for activation, and may require an IRF-1 site that
is located 3
to the TATA box. Furthermore, IRF-1 physically interacts
with NF
B subunits, and its binding is stimulated by the binding of
NF
B and HMG-I(Y) (27). Based on the studies of the IFN-
gene
regulation, Maniatis and colleagues have proposed a model in which
interactions between bZip proteins (ATF-2 and c-Jun), IRF-1, HMG-I(Y),
and NF
B form a higher order complex, which is required for full
activation of the IFN-
promoter (28, 29).
In summary, our data strongly suggest that multiple nuclear factors
induced by IFN- or LPS interact with the Ets-2 element of the IL-12
p40 promoter in the form of a large complex (F1). This complex consists
of Ets-2 and some related proteins (GLp109, Lp119, and Lp165), IRF-1
and c-Rel, and probably of additional proteins yet to be identified.
The size and multi-component nature of F1 may explain its extensive
physical occupancy requirement. This interaction is necessary but may
not be sufficient to activate the promoter since neither IFN-
nor
LPS is capable of inducing the promoter significantly, despite the fact
that F1 can be stimulated by either of them. The
292/
196 region
failed to enhance the activity of a minimal thymidine kinase promoter
when placed upstream of it.2 The answer to
this question may lie in the potential interaction between F1 binding
at
212/
207 and a complex formed at
135/
99 which involves PU.1,
NF
Bp50, and c-Rel in response to LPS stimulation (our preliminary
data). It is conceivable that these factors, induced separately by
IFN-
or LPS, bind to their targets cooperatively and synergistically
to induce the p40 promoter via higher order protein-protein
interactions, which have characterized the discoveries of many recent
studies of eukaryotic gene regulation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U89323[GenBank].