(Received for publication, July 25, 1994; and in revised form, November 3, 1994)
From the
Distinct NF-B subunit combinations contribute to the
specificity of NF-
B-mediated transcriptional activation and to the
induction of multiple cytokine genes including interferon-
(IFN-
). To evaluate the regulatory influence of different homo-
and heterodimers, NF-
B subunits were analyzed for transcriptional
activity in vitro using test templates containing two types of
NF-
B recognition elements (the human immunodeficiency virus type 1
enhancer and the IFN-
-positive regulatory domain II (PRDII)) as
well as IFN-
PRDIII-PRDI-PRDII linked to the -56 minimal
promoter of rabbit
-globin. Recombinant NF-
B subunits (p50,
p65, c-Rel, p52, and I
B
) and interferon regulatory factor 1
were produced from either Escherichia coli or baculovirus
expression systems. Transcriptional analysis in vitro demonstrated that 1) various dimeric complexes of NF-
B
differentially stimulated transcription through the human
immunodeficiency virus enhancer or PRDII up to 20-fold; 2) recombinant
I
B
specifically inhibited NF-
B-dependent transcription in vitro; and 3) different NF-
B complexes and interferon
regulatory factor 1 cooperated to stimulate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible regulatory domains
of the IFN-
promoter. These results demonstrate the role of
NF-
B protein dimerization in differential transcriptional
activation in vitro and emphasize the role of cooperativity
between transcription factor families as an additional regulatory level
to maintain transcriptional specificity.
Transcriptional activation of eukaryotic gene expression in response to developmental or environmental signals requires the assembly of multiple DNA-binding proteins with specific DNA regulatory domains in a stereospecific nucleoprotein complex (reviewed in Refs. 1 and 2). Changes in the specific combination of transcription factors associated with a particular gene by modification, de novo synthesis, or protein-protein interaction may account for developmental or temporal regulation of transcription. Protein-protein interactions between transcription factors also confer a level of transcriptional specificity that would not be achieved by individual proteins(1, 2) .
Two broad groups of transcription
factors, general transcription factors and upstream activators, are
involved in the accurate transcription of mRNA by the enzyme RNA
polymerase II. The general transcription factors including the entire
TFIID ()complex, which consists of TBP and the
TBP-associated factors, and RNA polymerase II are required for
transcriptional regulation by upstream activators(1) . TBP and
TFIIB are targets for the direct interaction with upstream activator
proteins (3, 4, 5) . Mutations in basic
residues of TFIIB that crucially affect the interaction with acidic
activators fail to bind activator proteins such as VP16 and do not
respond to activation, but still function in basal transcription. These
observations suggest that interaction between an acidic activator and
TFIIB is required for transcriptional activation (6) .
Modulation of cellular transcription can also be produced as a
consequence of virus infection. The type 1 interferon genes (IFN-
and IFN-
) have served as a paradigm to examine the transcriptional
mechanisms controlling virus-inducible gene
expression(2, 7, 8) . The IFN-
promoter
contains multiple regulatory domains that are targets for transcription
factors involved in inducible expression of the IFN-
gene(2, 7, 9, 10) . The positive
regulatory domains I and III (PRDI and PRDIII) interact with interferon
regulatory factors 1 and 2 (IRF-1 and
IRF-2)(8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) ,
while PRDII interacts with subunits of the NF-
B/Rel family of
transcription
factors(23, 24, 25, 26, 27, 28) .
PRDIV interacts with ATF-2 and c-jun; in vivo synergism between PRDIV and PRDII is provided by the binding of
the high mobility group I/Y proteins to the minor groove of DNA within
AT-rich sites in PRDIV and PRDII(29, 30) .
NF-B/Rel proteins participate in the activation of numerous
genes involved in inflammatory reactions and immune regulatory
functions (reviewed in (31, 32, 33) ). The
DNA-binding NF-
B family members share a Rel homology domain that
is responsible for DNA binding, nuclear localization, and protein
dimerization. DNA-binding members of NF-
B/Rel include p50
(NFKB1)(34, 35, 36) , p65
(RelA)(37, 38) , c-Rel (39, 40) , p52
(NFKB2, lyt-10)(41, 42, 43) , RelB
(I-Rel)(44, 45) , and dorsal(46) . p50 and p52
are synthesized as precursors of p105 and p100, respectively, that are
proteolytically processed to generate active DNA-binding p50 and
p52(34, 35, 36, 41, 42, 43) .
Recently, the acidic activation domains of both c-Rel and v-Rel were
shown to interact with TBP and TFIIB in vitro and in
vivo(5) ; p65 also interacted with TBP and TFIIB in
vitro, demonstrating that NF-
B/Rel activators stimulate
transcription via direct interaction with basal factors.
The
intracellular localization and post-translational activity of
NF-B/Rel proteins are regulated by the ankyrin repeat-containing
I
B proteins (I
B
, I
B
, bcl-3, p105, and p100)
(reviewed in (47, 48, 49, 50) ). The in vitro DNA binding activity of NF-
B complexes can be
inhibited or dissociated by I
B
addition(51, 52) ; in some cases, I
B
addition can enhance DNA binding activity, depending on the NF-
B
subunits(53) .
Different NF-B dimers bind to variant
NF-
B sites (consensus sequence, 5`-GGGANNYYCC-3`) (54) present in the promoter regions of many genes (reviewed in (31) and (33) ). Virtually every homo- and heterodimer
combination has been identified: p50 and p52 homodimers were detected
in unstimulated T cells and macrophages(55, 56) ;
p50/c-Rel and p50/p65 heterodimers were constitutively present in B
cells(33) ; p50/p65, p50/c-Rel, p65/p65, and p52/c-Rel
complexes were present in stimulated T cells(57, 58) ;
and p65/c-Rel heterodimers were purified from HeLa cells(59) .
These observations indicate that various homo- or heterodimeric
NF-
B complexes exist in different cell types and contribute to
differential regulation of NF-
B-dependent gene
expression(33) .
Qualitative changes in NF-B
heterodimer formation also occur as a function of time after virus
induction or cellular
differentiation(17, 57, 58, 60) .
Previously, we demonstrated a temporal shift in the composition of
NF-
B subunits in association with PRDII that correlated with a
virus-induced degradation and de novo resynthesis of
I
B
(17) . In this study, recombinant NF-
B
subunits and IRF-1 proteins were produced from either Escherichia
coli or baculovirus expression systems and were used to examine
NF-
B-dependent and IFN-
promoter-mediated transcription in vitro. These experiments demonstrated that 1) various
dimeric complexes of NF-
B differentially stimulated transcription
through two distinct NF-
B elements; 2) recombinant I
B
specifically inhibited NF-
B-dependent activation; and 3) different
NF-
B heterodimers and IRF-1 cooperated to activate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible
regulatory domains of interferon-
.
Vectors for
production of recombinant baculoviruses were generated by subcloning
different cDNAs into the pAcH6N1 vector. For p65, a 2232-bp BamHI fragment (38) from pGEX3X/p65 was inserted into
the BglII site of pAcH6N1. For p50/p65 chimera, a 2286-bp StuI fragment from pSVK3/p50-p65 was subcloned into the BglII site (filled in with Klenow enzyme) of pAcH6N1. For
c-Rel, a 2169-bp BamHI-EcoRI fragment (the EcoRI site was filled in with Klenow enzyme) (39) from
pGEX2T/c-Rel was subcloned into the BamHI-BglII
fragment (the BglII site was filled in with Klenow enzyme) of
pAcH6N1. For IRF-2, a 1.4-kilobase XbaI-XhoI
fragment from CMV-BL/IRF-2 (18) was inserted into the XbaI-XhoI fragment of pSVK3, and a
1.3-kilobase Cfr10I-KpnI fragment (the Cfr10I site was
filled in with Klenow enzyme) from the resulting plasmid was subcloned
into the BglII-KpnI fragment (the BglII site
was filled in with Klenow enzyme) of pAcH6N1. IRF-1 (22) was
similarly subcloned into the pAcH6N1 vector.
In vitro transcription templates HS-RG, PRDII
2, and PRDI
2 were a kind gift from N. MacDonald and C. Weissmann and were
described previously(8) ; the IFN-
reference template p901
was also described previously ( (12) and (23) ; refer
also to Fig. 3). Test transcription templates containing the
insert sequences of the HIV-1 enhancer, the HIV-1 enhancer mutant, and
the PRDIII-PRDI-PRDII elements (P512) (see Fig. 3) were
synthesized chemically and flanked by a 5`-ClaI-compatible
overhang (CG) on the top strand and a 5`-HindIII-compatible
overhang (TCGA) on the bottom strand. These sequences were inserted
into the ClaI-HindIII fragment at position -56
of PRDII-R
G to replace the PRDII sequence. For construction of the
reference template
R
G, HS-R
G was digested with SalI, filled in with Klenow enzyme, and self-ligated to block
one of two AccI sites. The resulting plasmid was digested with AccI, treated with exonuclease III, and ligated, yielding an
internal reference plasmid with a 22-bp deletion (residues
704-725). An upstream SV40 enhancer sequence was removed by
digestion with HindIII and EcoRI, fill in with Klenow
enzyme, and self-ligated. All constructions were verified by
sequencing.
Figure 3:
DNA templates for in vitro transcription analysis. The construction of HIV-1 enhancer, HIV-1
enhancer mutant, and PRDIII-PRDI-PRDII templates and the internal
reference template R
G is described under ``Materials and
Methods.'' The insert sequences were cloned upstream of the
minimal rabbit
-globin promoter (position -56) and
structural sequences of the rabbit
-globin gene(8) . A
5`-end-labeled 452-bp BamHI-PstI fragment derived
from the R
G cDNA was used as probe for S1 nuclease mapping
analysis; the 408-bp readthrough transcript, the 352-bp correctly
initiated transcript from the test gene, and the 180-bp transcript from
the reference gene are illustrated schematically. Hatchedboxes, TATA sequence; shaded boxes, R
G exon
sequences; arrows, transcription start site. To eliminate
readthrough from the reference template,
R
G was linearized
with BglII. The IFN-
cDNA deleted to position -56
(p901) was used as an internal reference gene in some
experiments(11, 12) .
For the production of polyhistidine-tagged
protein, recombinant baculoviruses were prepared by using a
BaculoGold transfection kit as recommended by the
manufacturer (Pharmingen). Sf9 cells were infected with recombinant
baculoviruses and cultured for 4 days at 28 °C. Infected cells were
harvested, washed with phosphate-buffered saline, and lysed in binding
buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)). Recombinant proteins were purified by using
rapid affinity purification with His-Bind metal chelation resin under
nondenaturing conditions as recommended by the manufacturer (Novagen,
pET system manual) (76).
Figure 1:
Expression
of recombinant NF-B and IRF proteins. A, schematic
representation of recombinant p50, p52, and I
B
proteins
expressed as glutathione S-transferase (GST) fusion
proteins in the E. coli glutathione S-transferase
gene fusion system. p50 and I
B
were enzymatically cleaved
with thrombin to release glutathione S-transferase. The
p50/p65 fusion protein (see below) was also produced a a glutathione S-transferase-linked protein (data not shown). B,
p65, c-Rel, p50/p65 fusion, IRF-1, and IRF-2 recombinant proteins
expressed as polyhistidine-tagged proteins in the baculovirus system.
All proteins contain 12-21 polyhistidine-linked amino acids at
their N-terminal ends (illustrated as checkeredboxes). The boxes represent different protein domains: dark grayboxes, Rel homology domain; boxes with diagonal widewhite stripes,
transactivation domain; boxes with wavy lines,
ankyrin domain; ( ANK ); box with verticalstripes, glutathione S-transferase domain; boxes with diagonalnarrowblackstripes, IRF-binding domain; light gray boxes,
IRF-1 transactivation domain; NLS, nuclear localization
sequence. C, Coomassie-stained SDS-polyacrylamide gel showing
the purified recombinant proteins (indicated above the lanes). The
molecular masses of the size markers are indicated in lane9. aa, amino acids.
The p65, c-Rel, p50/p65 fusion, IRF-1, and IRF-2 proteins were expressed in baculovirus as polyhistidine-tagged proteins (Fig. 1B). These polyhistidine-containing recombinant proteins were isolated from insect cell lysates under native elution conditions by a rapid affinity purification using His-Bind metal chelation resin. The purified recombinant proteins were analyzed by SDS-polyacrylamide gel electrophoresis, and their apparent molecular masses were slightly higher than predicted: 85, 85, 75, 50, and 48 kDa for p50/p65, c-Rel, p65, IRF-1, and IRF-2, respectively. The purity was estimated at >80% for p50/p65, p65, and IRF-1; 60% for IRF-2; and only 10% for c-Rel (Fig. 1C, lanes 4-8).
The DNA
binding activity of the recombinant NF-B proteins was assessed in vitro by electrophoretic mobility shift assay using the
HIV-1 enhancer and P2 probes. All recombinant NF-
B subunits were
able to bind to either the HIV-1 enhancer or P2 probe (Fig. 2, lanes1, 5, 13, and 17;
data for the P2 probe not shown). The binding of p65 generated one
major and one minor DNA-protein complex (Fig. 2, lane5) due to a truncated N-terminal p65 (as shown in Fig. 2, p65 contained
25-30% degraded product). When
p50 and p65 were preincubated to form heterodimers, two distinct
DNA-protein complexes were formed, reflecting the involvement of
full-length and truncated p65 (Fig. 2, lane9). Heterodimers consisting of p50/c-Rel, p65/c-Rel,
p52/p65, and p52/c-Rel also formed DNA-protein complexes in binding
assays; the binding of recombinant NF-
B protein to both probes was
specific since the DNA binding activity of recombinant NF-
B
subunits could be competed with excess HIV-1
B probe, but not with
the HIV-1 mutant probe (data not shown).
Figure 2:
Binding of NF-B subunits to HIV-1
enhancer probe and effect of I
B
on DNA binding. Recombinant
proteins of p50 (1 ng), p65 (1.3 ng), c-Rel (1.5 ng), and p52 (20 ng)
were individually assayed for specific binding to the HIV probe (lanes1, 5, 13, and 17).
Heterodimers of p50 (0.5 ng) and p65 (0.65 ng), prepared as described
under ``Materials and Methods,'' were also analyzed for DNA
binding (lane9). Binding assays with recombinant
proteins were performed in the presence of increasing amounts of
recombinant I
B
(1, 3.3, and 10 ng, as indicated by the triangles above the lanes).
Recombinant IB
,
preincubated with the NF-
B proteins before probe addition,
affected the DNA binding activity of all recombinant NF-
B
subunits. I
B
completely inhibited the binding activity of p65
and the p50/p65 heterodimer (Fig. 2, lanes6-8 and 10-12); after I
B
inhibition of p50/p65 complex formation, a complex comigrating with the
p50 dimer was formed, indicating the specificity of I
B
inhibition for p50/p65 (lanes 10-12). I
B
also
inhibited formation of the c-Rel
DNA complex (Fig. 2, lanes 14-16). Surprisingly, the DNA binding activity of
p50 and p52 was stimulated by recombinant I
B
(Fig. 2, lanes 2-4 and 18-20); DNA binding of the
p50/p65 chimeric protein, like p50, was also enhanced by the addition
of I
B
to the reaction (data not shown).
The HIV enhancer template was stimulated 13- or
15-fold by homodimers of p50 or p65, respectively (Fig. 4, lanes8 and 9), whereas the same proteins
were unable to stimulate transcription in vitro using the HIV
enhancer mutant template (lanes 1-3). The p50/p65 fusion
protein links the N-terminal DNA-binding region of p50 (amino acids
10-502) to the C-terminal transactivation domain of p65 (amino
acids 397-550). This fusion protein also stimulated
NF-B-dependent transcription in vitro
15-fold from
the wild type, but not from the HIV-1 enhancer mutant template (Fig. 4, lanes4, 5, 10, and 11). The p50/p65 fusion proteins were purified either from E. coli (Fig. 4, lanes5 and 11) or from baculovirus (lanes4 and 10). Preincubation of p50 and p65 to permit heterodimer
formation also resulted in a dramatic 17-fold increase in
NF-
B-dependent transcription in vitro (Fig. 4,
compare lanes6 and 12). In contrast to the
results obtained with the HIV-1 enhancer template, the same template
containing two copies of the PRDII element was not stimulated by p50
homodimers (Fig. 4, lane14), in agreement
with previous results(23, 24) . Homodimers of p65 or
the p50/p65 fusion protein resulted in 3.4- and 4.4-fold increases,
respectively, in PRDII-dependent transcription (Fig. 4, lanes 15-17), which is significantly lower than the
induction observed with the HIV enhancer test template (Fig. 4, lanes 9-11). Formation of p50/p65 heterodimers
stimulated PRDII-dependent transcription by 4.2-fold (Fig. 4, lane18).
Figure 4:
Transcriptional activation by recombinant
NF-B in vitro. The transcriptional activity of
recombinant NF-
B subunits was measured using HeLa cell nuclear
extracts in vitro. Transcription reactions (25 µl)
contained 250 ng of HIV-1 enhancer mutant-R
G (lanes
1-6), HIV-1 enhancer-R
G (lanes 7-12), or
PRDII-R
G (lanes 13-18) test genes and 250 ng of
p901 internal reference gene(11, 12) . Homo- or
heterodimers of NF-
B (200 fmol) were added to the reactions as
indicated below the lanes (+). Bands corresponding to the S1
nuclease mapping product for readthrough (R.T.) transcripts
and correctly initiated (C.I.) transcripts are indicated. LaneP, probes; laneM, size
markers (pAT153 digested by HaeIII). RTL, relative
transcription level, which was calculated as the ratio of the test
correctly initiated transcript level divided by the reference correctly
initiated transcript level, as determined by laser densitometric
scanning of appropriately exposed autoradiographic films. The
transcript ratio obtained in the control reaction (without the addition
of protein) was taken as a value of 1. All other relative transcription
levels were expressed relative to the
control.
The effects of other NF-B subunit
combinations on transcriptional activation in vitro were also
examined; in some reactions, recombinant I
B
was included to
evaluate the effect of the inhibitor on transcriptional activity (Fig. 5). The NF-
B subunits p52 and c-Rel were unable to
stimulate transcription from the HIV-1 enhancer in vitro (Fig. 5, lanes5 and 7), in
contrast to the inducibility by p50 and p65 (Fig. 4, lanes8 and 9). Interestingly, the p50/p65 fusion
protein stimulated transcription
10-fold (Fig. 5, lane3); the addition of I
B
to the reaction
containing the p50/p65 fusion protein reduced transcriptional induction
to <2-fold. The effect of I
B
on relative transcript levels
was specific to NF-
B-dependent transcription since I
B
addition did not affect the basal level of transcription from the HIV-1
test template or from the reference template (Fig. 5, lanes1 and 2). Differential transcriptional induction
by NF-
B heterodimer combinations was also sensitive to
I
B
-mediated inhibition. For example, p50/p65 and p50/c-Rel
heterodimeric complexes enhanced NF-
B-dependent transcription 15-
and 17-fold, respectively (Fig. 5, lanes9 and 11), and the strong induction by both heterodimers was blocked
by coincubation with I
B
(lanes10 and 12). Heterodimer combinations consisting of p52/p65,
p52/c-Rel, and p65/c-Rel also differentially stimulated
NF-
B-dependent transcription 7-, 6.5-, and 3.3-fold, respectively (Fig. 5, lanes13, 15, and 17). Coincubation with I
B
inhibited transcriptional
induction in each case (Fig. 5, lanes14, 16, and 18). Differential stimulation of
transcription in vitro by NF-
B heterodimer combinations
was completely dependent upon a functional NF-
B site since the
heterodimer combinations had no stimulatory effect on transcription in vitro from the HIV-1 enhancer mutant template (data not
shown).
Figure 5:
Transcription in vitro by
NF-B combinations and I
B
inhibition. In vitro transcription reactions were performed as described for Fig. 4, except that 250 ng of linearized
R
G internal
reference template was used to replace the p901 template. The test
template was the HIV-1 enhancer template. Recombinant I
B
(50
fmol) and different NF-
B complexes (200 fmol) were added as
indicated below the lanes (+). See Fig. 4legend for
definitions of abbreviations.
The capacity of NF-B heterodimer combinations to
modulate PRDII-dependent transcriptional activation was also evaluated in vitro, and the cumulative results of these studies are
summarized graphically in Fig. 6. The inhibitory effects of
I
B
on HIV-1 enhancer-mediated transcriptional activity are
also summarized. Heterodimers of p50/c-Rel resulted in a 10-fold
stimulation of PRDII-dependent transcription, whereas p52/c-Rel,
p65/c-Rel, and p52/p65 heterodimers stimulated transcription to a
lesser extent: 6.0-, 7-, and 3-fold, respectively (Fig. 6B). The addition of I
B
to the
transcription reactions containing either homodimers (Fig. 6A) or heterodimers (Fig. 6B)
inhibited NF-
B-specific stimulation of transcription in all cases.
These results demonstrate the differential inducibility of PRDII and
the HIV-1 enhancer element by distinct NF-
B heterodimer
combinations in an in vitro transcription assay and also
illustrate the sensitivity of transcriptional activation in vitro to the presence of I
B
.
Figure 6:
Transcriptional activity by NF-B
subunit combinations. The cumulative results of in vitro transcription reactions using different NF-
B combinations are
summarized for the HIV-1 enhancer template (black bars) and
the PRDII template (shaded bars). The effect of I
B
addition (50 fmol) on HIV-1 enhancer-dependent transcription is also
shown (cross-hatched bars). The bar graphs show the relative
transcript levels normalized to the internal reference template and
represent the average of duplicate measurement with <20% variation. A, transcriptional effect of NF-
B homodimers and
inhibition by I
B
; B, transcriptional effect of
NF-
B heterodimer combinations and inhibition by
I
B
.
Figure 7:
Cooperation between NF-B and IRF-1 in
PRDIII-PRDI-PRDII-dependent transcription in vitro. The test
template contained one copy of PRDIII-PRDI-PRDII (positions -94
to -55) of the IFN-
promoter linked to the -56
promoter of R
G. Homo- or heterodimers of NF-
B (200 fmol)
and/or IRF-1 (200 fmol) was used in the transcription reactions as
indicated below the lanes. The
R
G internal reference template
was used as control in the in vitro transcription assay. See Fig. 4legend for definitions of
abbreviations.
Differential stimulation of the PRDIII-PRDI-PRDII-dependent template
was observed when distinct NF-B complexes were used together with
IRF-1 in vitro. p50, p65, or p50/p65 fusion homodimers
together with IRF-1 increased test gene transcription 4.2-, 6.5-, and
6.9-fold, respectively (Fig. 7, lanes 13-15),
whereas the p52 homodimer alone slightly inhibited IRF-1 stimulation (lane16). c-Rel completely inhibited the
transcriptional activation in vitro, including the stimulation
by IRF-1 (Fig. 7, lane17). Strikingly, the
combination of the NF-
B heterodimeric complex p50/p65 and IRF-1
stimulated PRDIII-PRDI-PRDII-dependent transcription 10-fold; other
heterodimer combinations (p50/c-Rel, p52/p65, p52/c-Rel, or p65/c-Rel)
incubated together with IRF-1 stimulated transcriptional activation
4-8-fold (Fig. 7, lanes 18-22). In all
cases, heterodimer combinations stimulated transcription in vitro more strongly in the presence of IRF-1 protein than in its absence (Fig. 7, compare, for example, lanes7 and 18, 8 and 19, 9 and 20, 10 and 21, and 11 and 22). These
experiments demonstrate transcriptional cooperation between NF-
B
heterodimeric complexes and IRF-1 in the in vitro activation
of the IFN-
PRDII-PRDI-PRDII virus-inducible enhancer element.
In this study, we used recombinant NF-B and IRF proteins
to evaluate transcriptional specificity and cooperativity in vitro in the activation of interferon-
regulatory domains or the
HIV-1 enhancer. We demonstrate that 1) distinct combinations of
NF-
B subunits contributed to the specificity of transcriptional
activation in vitro either through the HIV-1 enhancer or
IFN-
PRDII; 2) I
B
protein specifically inhibited
NF-
B-dependent DNA transcription in vitro; and 3) homo-
and heterodimeric NF-
B combinations together with IRF-1 cooperated
to stimulate interferon-
PRDIII-PRDI-PRDII-dependent transcription in vitro.
Transcriptional induction of IB
mRNA by NF-
B itself,
leading to increased I
B
levels and subsequent sequestration
of NF-
B
I
B
complexes, clearly represents an
important autoregulatory mechanism for down-regulation of
NF-
B-induced gene
expression(60, 67, 68, 69, 70, 71) .
The ability of I
B
to dissociate NF-
B
DNA complexes
under gel shift conditions (52) and the presence of
I
B
in the nucleus when overexpressed (72) suggest
that another function of newly produced I
B
may be to enter
the nucleus and dissociate NF-
B
DNA transcriptional
complexes(47, 48) . Since the concentration of
I
B
required to completely inhibit NF-
B DNA binding or
transcription was at least four times lower than the concentration of
NF-
B, it would appear that inhibition by I
B
is not due
solely to a stoichiometric formation of NF-
B
I
B
complexes.
Transcriptional cooperativity between
NF-B complexes and IRF-1 was also examined using the
PRDIII-PRDI-PRDII template. Both NF-
B heterodimers and IRF-1
activated the transcription from the test promoter to differing
degrees; in all cases, however, transcriptional activity in vitro from the PRDIII-PRDI-PRDII template was increased by the
simultaneous incubation with NF-
B complexes and IRF-1. In
cotransfection experiments, overexpression of IRF-1 with p50 and p65
activated transcription from the intact IFN-
promoter in a
synergistic manner(17) . The in vitro transcription
assay demonstrated additive but not synergistic activation by NF-
B
proteins and IRF-1, suggesting that other proteins required for proper
regulation of IFN-
transcription in vivo are absent from in vitro systems. While the exact nature of these factors
remains unclear, previous studies showed that HIV-1 promoter activation
by p50 or by p50 + p65 was absolutely dependent on the cofactor
fraction USA(61, 73) . Another cofactor, the
chromatin-associated high mobility group I/Y proteins, was also shown
to be involved in the activation of NF-
B-dependent IFN-
gene
expression. High mobility group I/Y protein does not act as an
activator of transcription, but interacts with AT-rich DNA sequences
through the minor groove to facilitate the activity and/or binding of
NF-
B to PRDII(29, 30) . While it may be expected
that high mobility group I/Y proteins are abundant in nuclear extracts,
it is possible that the proper stereospecific arrangement of
transcription factors and cofactors may not be achieved in
vitro. Transcription from a target promoter may also be regulated
by histone H1-mediated repression. Transcriptional activators in part
modulate transcription by counteracting histone H1-mediated repression
(antirepression); for example, the GAL4-VP16 fusion protein stimulated
transcription 2-fold in the absence of histone H1 (true activation) and
20-fold in the presence of histone H1, i.e. 10-fold
antirepression(74, 75) . These limitations
notwithstanding, these in vitro transcription studies
emphasize the role of protein-protein dimerization as a distinct level
of control that permits functional diversification of a limited number
of components. Cooperativity between transcription factors such as
NF-
B and IRF-1 maintains an additional level of specificity that
would not be achieved by individual proteins.