Laboratory of Biochemistry and Molecular
Biology, The Rockefeller University, New York, New York 10021
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INTRODUCTION |
The general inducible NF-
B
activity consists of a heterodimer of two subunits, p50 (NF-B) and
p65 (RelA), that both bind DNA (28, 68). p50 and p65, along
with other members of the Rel family, share a 300-residue N-terminal
Rel homology domain that contains sequences required for DNA binding,
protein dimerization, and nuclear localization. In addition to the Rel
homology domain, the p65, c-Rel, and RelB proteins contain flanking
regions involved in transactivation (for reviews, see references
2 and 69). Transient expression
assays have revealed that the p65 subunit is responsible for most of
the activation potential of Rel/NF-B (52, 59), and a
strong activation domain, which is lacking in the p50 subunit, has been
mapped to its C-terminal domain (48, 58, 60). p50 does not
usually activate transcription in transient transfection experiments,
although it has been reported to do so in vitro (17, 34).
A well-studied target promoter for NF-B activity is the proximal
enhancer of human immunodeficiency virus type 1 (HIV-1). In addition to
three Sp1 sites, this enhancer contains two NF-B sites to which
several combinations of NF-B subunits can bind and activate
transcription both in vivo and in nuclear extracts (15, 28,
34). In addition, it has been shown that these two activators
cooperate during transcription of the HIV-1 promoter in vivo (50,
51). While specific DNA-binding factors like constitutive Sp1 and
induced NF-B modulate the expression level of the HIV-1 promoter,
RNA polymerase II (pol II), and general initiation factors (TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) are required for both
activator-independent (basal) and activator-dependent transcription.
Furthermore, and in contrast to basal transcription, activator-dependent transcription in mammalian cell-free systems generally requires cofactor activities that include both the USA fraction (upstream factor stimulatory activity) and TATA-binding protein (TBP)-associated factors (TAFs) within TFIID (for reviews, see
references 5, 27, 48, 54, and
70).
The USA fraction contains both positive and negative cofactors (PCs and
NCs), and the four human PCs (PC1, PC2, PC3, and PC4) characterized so
far are distinct from components within TFIID (for a review, see
reference 27). Importantly, these USA-derived cofactors effect a large increase in promoter activity in the presence
of activators and are essential even in systems reconstituted with
homogeneous TFIID (10). Human TFIID itself is composed of at
least 13 TAFs and TBP (for a review, see reference
5). Consistent with its key role in recruiting other
basal factors to the promoter, TFIID was implicated in transcriptional
activation by the demonstration of physical and functional interactions
between TFIID and various activators, and in several cases,
activator-induced changes in TFIID binding were correlated with
increased recruitment of downstream factors (for reviews, see
references 5, 54 and 70).
Although in vivo and in vitro studies have shown that the NF-B
complex can bind NF-B sites and activate transcription, very little
is known about the cofactors required for its transcriptional activity
in vitro. Only two studies have analyzed NF-B activity in systems
reconstituted with isolated factors, and both showed a requirement for
a crude USA fraction and for TFIID (34, 60). It therefore
becomes important to determine whether individual components of the
complex USA fraction (reviewed in reference 27) can
function alone or in combination to restore the full coactivator
activity of USA. Also of key importance is an understanding of the role
of individual human TAFs (in reconstituted TFIID species) in both basal
and NF-B-mediated transcription, especially in light of core
promoter-specific effects of TAFs in both metazoans (reviewed in
reference 54) and yeast (61).
Here, we report a dissection of specific coactivator requirements for
HIV-1 promoter-driven transcription by NF-B (p50/p65) and Sp1 in a
transcription system reconstituted with highly purified factors. We
show that the synergistic action of USA-derived coactivators PC2 and
PC4 fully reconstitutes the USA coactivator function, including both
basal repression and activator-enhanced transcription. In addition, we
have used highly purified native TFIID and partially reconstituted
TFIID complexes to show that TAFs, like USA components, serve both to
repress the TBP-mediated basal transcription level and, following
activator interactions, to reverse the repression and effect a net
increase in overall activity. Thus, our data show more diverse roles
for TAFs in transcription regulation that go beyond their simple
involvement in recruiting TFIID to the promoter upon interactions with
upstream activators.
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MATERIALS AND METHODS |
Expression and purification of activators and TAFs.
cDNAs
corresponding to the coding regions of p50 and p65 (20, 46)
were used to generate, by PCR, their respective PCR fragments with
appropriate flanking restriction sites (XbaI and EcoRI or KpnI). PCR products were cloned into the
baculovirus expression vector pVLMH6 generated by insertion
of the hexahistidine (6His) oligonucleotide linker
5'-GATCCATATGAGAGGATCGCATCACCATCACCATCACT-3' into the
BamHI-XbaI sites of pVL1393 (62). Each
construct was verified by sequencing. The hexahistidine tag allows
purification through a nickel affinity column. The original initiation
methionine of each protein was mutated to valine to avoid any internal
initiation of translation. To produce the mature form of p50 without
processing of its precursor, a translational terminator was introduced
at codon position 452. For p65, a full-length cDNA was used. The p50/p65 heterodimer was made in vivo by coinfecting Sf9 cells with the
corresponding viruses for each subunit. To avoid contamination of the
heterodimer with p50 homodimers, only p65 carried the hexahistidine tag
in the coinfection. Recombinant baculoviruses were then isolated and
characterized for protein expression by Western blot analysis. For each
protein, one recombinant baculovirus was selected to infect an Sf9
suspension culture. For the coinfection experiments, the ratio between
the two viruses was 10 to 1 in favor of the virus that carries the
subunit without the hexahistidine tag (p50). The p50/p65 complex was
then purified by affinity chromatography on a nickel column, followed
by ion-exchange chromatography.
The pVL derivatives used for the expression of hemagglutinin
(HA)-hTAFII250, FLAG-hTAFII100, and
FLAG-hTAFII55 have already been described (11, 21,
66). Expression pVL plasmids for FLAG-hTAFII80,
FLAG-hTAFII80, FLAG-hTBP, His-TBP,
FLAG-hTAFII80, FLAG-hTAFII31, nontagged
hTAFII31, and FLAG-Sp1 were constructed by PCR. In each
case, an NdeI site at the N-terminal end and an appropriate
restriction enzyme site at the C-terminal end following the natural
stop codon were created. The large number of primers used in the PCRs
has precluded description of their exact sequences, but the information
is available upon request. The PCR-generated fragments were then
inserted into adaptor plasmids pFLAG(S)-7 and pFLAG(AS)-7 carrying the
appropriate epitope tag (10) and subsequently subcloned into
either pVL-1392 or pVL-1393. For each TFIID subunit or for Sp1, an
individual recombinant baculovirus was generated by cotransfecting
corresponding cDNA and BaculoGold linearized baculovirus DNA
(Pharmingen) into Sf9 cells. Each recombinant baculovirus was further
amplified by repeated infection of Sf9 cells. For production of
recombinant proteins, Sf9 cells were infected by the corresponding
recombinant viruses and harvested 48 h postinfection. Recombinant
proteins were purified from infected cells. Nuclear extracts were
prepared in buffer C (20 mM Tris [pH 7.9], 20% glycerol, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg of
leupeptin per ml, 1 µg of pepstatin per ml) containing 400 mM KCl
(BC400) and 0.1% Nonidet P-40 (NP-40) (13). Clarified
extracts were subjected to the appropriate method of purification:
affinity purification on an anti-FLAG antibody (M2 agarose; Kodak) or
anti-HA antibody (12CA5 monoclonal antibody) column or chromatography
on Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Chatsworth, Calif.).
After extensive washing, bound proteins were eluted, respectively, in
BC100 buffer containing FLAG- or HA-peptides (10, 75) or 250 mM imidazole (for Ni-NTA) and further purified by one or two steps of
ion-exchange chromatography. The recombinant proteins were more than
90% pure as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) and Coomassie blue or silver staining.
A GST (glutathione S-transferase)-p65ct construct was
created by inserting a cDNA fragment corresponding to the C-terminal 135 amino acids of p65, flanked by NdeI and EcoRI
sites, into a pGEX-2T derivative (23). The GST fusion
protein was expressed in Escherichia coli, solubilized by
sonication of cells in lysis buffer (11) and removal of
insoluble debris by centrifugation, and purified on
glutathione-Sepharose (Pharmacia). A 1-µg sample of the purified
protein was used for each binding assay.
Bacterially expressed FLAG-Gal4 fusion proteins were purified as
described previously (11, 38) and consist of the Gal4 DNA-binding domain fused either to a duplicated copy of the
transcriptional activation domain of p53 (amino acids 1 to 57) or to a
single copy of a C-terminal portion (amino acids 416 to 550) of p65
(4, 42, 55). cDNA fragments that express the corresponding
activation domains were amplified by PCR, using primers which
introduced unique XbaI and EcoRI restriction
sites, and inserted into a pGAL4 adaptor plasmid 3' of, and in frame
with, the encoded Gal4 DNA-binding domain (amino acids 1 to 94). The
corresponding recombinant pGAL4 plasmids were then digested with
XhoI and EcoRI, and Gal-p65ct and Gal-p53nt
fragments were subcloned into pFLAG-GAL4-11d (11). Constructs were verified by sequencing.
In vitro RNA pol II transcription assays.
Nuclear extracts
were prepared as previously described (13). General
transcription factors and the USA cofactor fraction were purified from
HeLa and 3-10 (FLAG-TBP cell line) nuclear extracts as previously
described (10, 41). For TFIIA, the P11 fraction in buffer C
containing 0.1 M KCl (BC100) was applied to a DEAE-cellulose (DE52)
column, eluted with BC300, dialyzed to BC100, and loaded onto a
Q-Sepharose column. The column was then washed with BC300 and eluted
with BC500. The TFIIA (BC500) fraction was further purified on an
Ni-NTA column (12). For TFIIE/F/H, the P11 0.5 M KCl
fraction was applied to a DEAE-cellulose (DE52) column, eluted with
BC300, dialyzed to BC100, and passed through a double-stranded
DNA-cellulose column to remove contaminating TFIID activity. The
flowthrough fraction from the DNA affinity column was applied to a Mono
S fast protein liquid chromatography column and eluted with BC300 after
washing of the column with BC150. For TFIID, the P11 0.85 M KCl
fraction (from 3-10 cells) was dialyzed against BC100, applied to a
DEAE-cellulose column, and eluted with BC300. After dialysis against
BC100, fTFIID was immunopurified by using an anti-FLAG antibody column
(M2 agarose; Kodak). For TFIIB, recombinant FLAG-TFIIB was expressed
in, and purified from, E. coli. To prepare the USA fraction,
the P11 0.85 M KCl fraction was dialyzed against BC100, applied twice
to a DE52 column to limit TFIID contamination, and loaded onto a
heparin-Sepharose column. The column was washed with BC300, and the USA
fraction was eluted with BC500. Finally, the TFIIE/F/H and USA
fractions were depleted of any residual TFIID by using M2 agarose and
antigen-purified antibodies against TAFII100 and
TAFII31. RNA pol II was purified essentially as previously
described (41).
By using the purified transcription factors described above, in vitro
transcription assays were carried out in 25-µl reaction mixtures
containing 40 ng of a pML
53 template and either 100 ng of
pG5HMC2AT or 50 ng of pMHIVWT or pMHIVMT.
pMHIVMT contains mutated HIV-1 NFB binding sites (34).
All transcription factors were added simultaneously to the reaction
mixtures if not indicated otherwise in the figure legends.
32P-labeled RNA was phenol-chloroform extracted, ethanol
precipitated, analyzed directly by urea-4% PAGE, and visualized by
autoradiography. Quantitation was done by PhosphorImager.
Purification of PC2 and PC4.
Recombinant PC4 was purified as
previously described (18). PC2 was purified essentially as
previously described (36). Briefly, the P11 0.85 M KCl
fraction was dialyzed to BC100 and applied to a DE52 column. The
flowthrough fraction and the BC150 step eluate were combined, dialyzed
to BC050, and applied to a second DE52 column. The column was developed
with a linear gradient of BC050 to BC250. Fractions containing the PC2
activity (peak at 90 mM KCl) were applied to a Mono S fast protein
liquid chromatography column that was developed with a linear gradient
of 0.1 to 0.5 M KCl in BC buffer. PC2 activity was eluted between 200 and 300 mM KCl and concentrated on S-Sepharose.
Partial TFIID reconstitution.
By the method of Chen et al.
(8), human hTAFII250 containing a fused
N-terminal HA epitope tag was immobilized on protein A-Sepharose
containing covalently linked monoclonal antibodies directed against the
HA epitope. After extensive washing (BC1000 with 0.1% NP-40), the
beads were incubated sequentially (at 4°C for 4 h) with molar
excesses of additional TFIID subunits. After each incubation, unbound
materials were removed by several washes with 100 volumes of BC150
(with 0.1% NP-40). Finally, the resulting complex was eluted with HA
peptide (1 mg/ml) in BC100 (with 0.1% NP-40).
In vitro protein-protein interaction assays.
The p65
activation domain interactions were carried out by using TAFs and TBP
expressed in, and purified from, Sf9 cells. In each reaction, 1 µg of
purified GST, FLAG-GAL4(1-94), GST-p65ct, or FLAG-GAL4p65ct was
immobilized on either glutathione beads or anti-FLAG M2 agarose. After
washing of the beads, purified TBP and different TAFs (100 ng of each)
were added to the appropriate binding reaction mixtures in BC150 with
100 µg of bovine serum albumin-0.1% NP-40 in a 500-µl total
volume. After incubation at 30°C for 1 h, the beads were washed
four times with 500 µl of incubation buffer. Bound proteins were
eluted with SDS loading buffer and analyzed with the inputs by SDS-PAGE
and Western blotting.
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RESULTS |
Transcription activation of the HIV-1 promoter by NF-B and
Sp1.
Full-length FLAG-tagged Sp1 (fSp1) and a hexahistidine-tagged
p65/p50 heterodimer were produced in Sf9 cells by coinfection with
corresponding baculovirus vectors and purified to over 90% homogeneity
(Fig. 1A and B) by affinity
chromatography. The function of the recombinant activators was analyzed
in a well-defined in vitro transcription system that was reconstituted
with recombinant and highly purified general transcription factors from
HeLa cells and dependent on the addition of FLAG epitope-tagged and
affinity-purified TFIID (f-TFIID) (10; see also
below). The ability of this system to support both basal and
activator-dependent transcription was tested simultaneously by using
two templates whose correctly initiated products could be
differentiated by the sizes of their transcribed G-less cassettes.
Basal transcription was assayed on a template containing only the
adenovirus (Ad) major late (AdML) core promoter sequence, whereas
activator-dependent transcription was assayed by using an
earlier-described (41) HIV-1 template that contains natural
NF-B- and Sp1-binding sites and the adjacent HIV TATA element. In
all cases, levels of transcription were quantitated by PhosphorImager
and the levels of induction cited throughout Results are based on these
values.
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FIG. 1.
Analysis of purified recombinant proteins. (A) NF-B
heterodimer 6his-p65-p50 heterodimer purified from Sf9 cells. (B)
Purified FLAG-Sp1 (f-Sp1) purified from Sf9 cells. (C) Purified
FLAG-Gal-p53 (fGal-p53nt) and FLAG-Gal-p65ct (fGal-p65ct) expressed in
and purified from bacteria. (D) GST-p65ct purified from bacteria.
Proteins were resolved by SDS-PAGE and visualized by Coomassie blue
staining. M, markers with protein molecular sizes indicated on the left
in kilodaltons.
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Both recombinant NF-B (p50/p65 heterodimer) and Sp1 activate
transcription of the HIV-1 promoter in this system (Fig.
2). The levels of induction by NF-B
(50 ng of the p50 subunit in the p50/p65 complex) and Sp1 (10 ng of
f-Sp1) were 14-fold and 8-fold, respectively. NF-B activation was
dependent on functional NF-B sites, since no activation was obtained
when the mutated template (pMHIVMT) was used (Fig. 2, lane 3 versus
lane 4).
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FIG. 2.
Site-specific transcriptional activation of the HIV-1
promoter by NFB and Sp1. Transcription activation in the highly
purified transcription system by NFB and Sp1. A p50/p65 heterodimer
containing 50 ng of the p50 subunit or 10 ng of purified f-Sp1 was
incubated with DNA templates with f-TFIID (4-ng TBP content) and 1 µl
of USA (heparin-Sepharose) at 25°C for 10 min before addition of the
other general factors and RNA pol II. The test template consisted of 50 ng of either wild-type HIV-1 pMHIVWT (WT; lanes 1, 2, 5, and 6) or
mutated HIV-1 pMHIVMT (MT; lanes 3 and 4). The control template
consisted of 40 ng of pML53, which contains the major late core
promoter sequence.
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Identification of USA-derived components required for natural
NF-B activity.
Since we have shown previously that the USA
fraction is required for full activation by natural NF-B, we wished
to determine which USA-derived PCs are responsible for the activity and
to further investigate their mechanism(s) of action. In this regard, it
was also important to determine whether there is functional redundancy
in the individual factors or whether they can act synergistically. We
focused on two USA-derived PCs, PC2, and PC4, since they are the most
potent of the USA-derived coactivators and since they together
recapitulate the function of the essential USA activity (see below). To
test for an independent effect of the well-characterized coactivator
PC4 (18, 33) on NF-B (p50/p65) activity, we replaced the
USA fraction with various amounts of purified recombinant PC4. First,
without USA or purified PCs, NF-B activated transcription by less
than 2.5-fold (Fig. 3B, lane 1 versus
lane 2). In agreement with previous observations on other promoters,
basal (activator-independent) transcription activities on both test
HIV-1 and control AdML templates were mildly increased at low PC4
concentrations (Fig. 3A, lane 1, versus Fig. 3B, lane 1) but markedly
decreased at a high concentration (Fig. 3A, lane 3). PC4 enhanced the
ability of p50/p65 to activate transcription of the HIV-1 promoter, but
the level of induction was greater at the higher level of PC4 (sixfold)
than at the lower level (fourfold) (Fig. 3A, lane 3 versus lane 4 and
lane 1 versus lane 2). The increased induction by NF-B at higher PC4
levels reflects a net decrease of basal HIV transcription that is
specifically (but only partially) reversed by NF-B.
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FIG. 3.
Effect of purified USA-derived positive cofactors PC2
and PC4 on NF-B activity. (A) Effects of various concentrations of
PC2 and PC4 on p50/p65 activity. Reaction mixtures contained the
indicated amounts of purified PC4 and/or PC2 (2 µl of the Mono S
fraction). (B) Effects of low concentrations of PC2 and PC4 in
combination on NF-B activity. Reaction mixtures contained the
indicated amounts of PC4 and/or PC2 (0.5 or 1 µl of the Mono S
fraction). Other components and reaction conditions were as described
in the legend to Fig. 2. The assays in panels A and B are directly
comparable since they were performed simultaneously with the same
reagents under identical incubation, processing, and autoradiographic
exposure conditions.
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The coactivator PC2 is less well characterized than PC4 but appears to
consist of a circa 500-kDa complex (36). As observed for
PC4, a purified PC2 fraction mildly increased the basal activities on
both the HIV-1 and AdML templates (Fig. 3A, lane 5, versus Fig. 3B,
lane 1) and also showed a significant coactivator function with NF-B
(Fig. 3A, lane 5 versus lane 6). However, the level of induction was
lower for PC2 (threefold) than for the higher level of PC4 (sixfold)
(Fig. 3A, lanes 5 and 6 and lanes 3 and 4), due primarily to more
efficient reduction of basal transcription by PC4. This also reflects,
in PC2, both the absence of an activator-reversible NC activity (as
observed for PC4) and the presence of a stronger basal stimulatory
activity that may be repressed by other NC functions in the
unfractionated USA fraction. Interestingly, the addition of high
concentrations of PC4 to reaction mixtures containing PC2 increased the
overall level of induction by NF-B from 3-fold (Fig. 3A, lanes 5 and
6) to 7.8-fold (Fig. 3A, lanes 9 and 10). However, and consistent with
the NC potential of PC4, this resulted primarily from a selective
reduction in basal activity rather than a large net increase in total
activity. In this regard, lowering the basal activity enhances
substantially the overall level of induction by an activator and more
closely mimics the in vivo situation. Taken together, these results
indicate that the combination of PC2 and PC4, with their intrinsic
positive and negative activities, can have a dual effect on
transcription
both decreasing the basal level of transcription and
potentiating higher levels of activation by natural NF-B. However,
the higher level of induction (12-fold) evident with unfractionated USA
(Fig. 3A, lane 11 versus 12) suggests that optimal induction may still
require other cofactors present in the crude USA fraction.
We next investigated whether a possible synergism between PC2 and PC4
on NF-B function could be observed at limiting concentrations of
these two coactivators. As already mentioned, NF-B activated transcription only by 2.5-fold in the absence of USA or purified PCs
(Fig. 3B, lane 1 versus lane 2). In the presence of purified PC4 (50 ng) or PC2 (1 µl) alone, NF-B activated transcription by 5.6-fold
and 5.7-fold, respectively (Fig. 3B, lane 1 versus lanes 4 and 6). A
combination of PC4 and PC2, at the same concentrations, led to an
11-fold induction of transcription by NF-B (Fig. 3B, lane 1 versus
lane 10) but had only moderate effects on basal activity in the absence
of NF-B (Fig. 3A, lane 7, versus Fig. 3B, lane 1). This indicates
that PC2 and PC4 coactivator functions can be additive for NF-B
activity.
PC4 and PC2 together can reconstitute USA functions for activation
by Sp1 and show synergistic effects.
Similar to the above
observations for activation by NF-B, Sp1 activated the HIV promoter
only about twofold in the absence of USA (Fig.
4B, lane 1 versus lane 2) and either PC2
or PC4 alone could substitute, to different degrees, for the effect of
USA on Sp1 function on the HIV promoter (Fig. 4A, lanes 1 to 6 and lanes 11 and 12). A titration of PC4 showed variable effects on the
levels of activation by Sp1 (from threefold with 50 ng of PC4 to
eightfold with 100 ng of PC4) (Fig. 4A, lane 1 versus lane 2 and lane 3 versus lane 4). The increased induction by Sp1 at higher PC4 levels
reflects a net decrease in basal HIV transcription, which is
efficiently reversed by Sp1 activity. This effect of Sp1 in reversing
the inhibitory effect of PC4 on basal transcription is specific to the
HIV promoter relative to the control AdML promoter (Fig. 4A, lanes 1 to
4).
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FIG. 4.
Combination of PC2 and PC4 can fully reconstitute USA
cofactor activity for Sp1 function. (A) Effects of various
concentrations of PC2 and PC4 on Sp1 activity. Reaction mixtures
contained the indicated amounts of purified PC4 and/or PC2 (2 µl of
the Mono S fraction). (B) Synergistic effects of low concentrations of
PC2 and PC4 on Sp1 function. Reaction mixtures contained the indicated
amounts of PC4 and/or PC2 (0.5 and 1 µl of the Mono S fraction).
Other components and reaction conditions were as described in the
legend to Fig. 2. The assays in panels A and B were performed
simultaneously with the same reagents under identical incubation,
processing, and autoradiographic exposure conditions.
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The purified PC2 fraction also showed a significant coactivator
function with Sp1 (Fig. 4A, lane 5 versus lane 6). In this case, the
overall transcription activity was higher with PC2 than with PC4 (Fig.
4A, lane 6 versus lanes 2 and 4). However, the level of induction was
lower with PC2 than with PC4 (fourfold versus eightfold) (Fig. 4A,
lanes 3 and 4 and lanes 5 and 6), similar to what was observed in the
NF-B analysis. This is due primarily to a more efficient reduction
of basal transcription by PC4 that can be reversed by Sp1.
Significantly, a combination of appropriate concentrations of PC2 and
PC4 fully reconstituted the USA effect on Sp1 function (Fig. 4A, lanes
9 and 10 versus lanes 11 and 12). The levels of induction by Sp1 were
14-fold with a combination of PC2 and PC4 and 11-fold with USA. This
combined effect of PC2 and PC4, relative to their independent effects, is due mainly to a reduction of basal transcription to the same level
observed with the USA fraction and to an effective reversal of this
inhibition in the presence of Sp1, thus leading to a higher level of
induction. These results demonstrate that while several PCs may coexist
in the USA fraction, the maintenance of a low (more physiological)
level of basal activity and the function of at least some activators
may depend, necessarily, only on the activities of very few PCs.
We next examined whether a possible synergistic effect of PC2 and PC4
on Sp1 function could be observed at otherwise limiting concentrations
of PC2 and PC4. As mentioned above, without USA or purified PCs, Sp1
activated transcription by only twofold (Fig. 4B, lane 1 versus lane
2). With lower levels of purified PC2 (0.5 µl) or PC4 (25 ng) alone,
Sp1 activated transcription by 4.5-fold and 4-fold, respectively (Fig.
4B, lane 1 versus lanes 3 and 5). A combination of the same amounts of
PC2 and PC4 led to a 13-fold induction of transcription by Sp1 (Fig.
4B, lane 1 versus lane 7), whereas basal transcription was relatively
unaffected by PC2 and PC4 alone (Fig. 4A, lane 7, versus Fig. 4B, lane
1). The greater-than-additive effect of both PC2 and PC4 indicates a
synergistic effect of these two positive coactivators on Sp1 function.
It is possible that this moderate synergism would be amplified in the
context of a more natural chromatin template, where other constraints
and cofactors may be involved.
The p65 C-terminal region is a potent activation domain in the
highly purified system.
Previous studies performed in vivo (see
the introduction) have shown that the p65 subunit has a strong
activation domain in its C-terminal region. We next examined whether
this C-terminal region (amino acids 416 to 550) can activate
transcription in a purified transcription system when fused to the Gal4
DNA-binding domain. In vitro transcription assays were performed in the
presence or absence of purified, bacterially expressed activators
consisting of the Gal4 DNA-binding domain fused either to the
C-terminal portion of the NF-B p65 subunit (fGal-p65ct) or to the
transcription activation domain from tumor suppressor protein p53
(fGal-p53nt, used as a control) (Fig. 5).
Basal transcription was assayed on the AdML template, whereas the
reporter for activator-dependent transcription for Gal fusion proteins
contains five copies of the Gal4 DNA-binding site upstream of the HIV
TATA box and the AdML initiator (Inr) element linked to a G-less
cassette of 380 bp (10). Results in Fig. 5 show that the p53
(lanes 2 and 3 versus lane 1) and p65 (lanes 6 and 7 versus lane 1)
activation domains are both potent transcription activators in the
highly purified system, whereas the Gal4 DNA-binding domain alone
showed no activation (lanes 4 and 5 versus lane 1). The strong
activation (over 20-fold) by multiply bound p65 activation domains
suggests that they most likely interact with several targets within
TFIID, general factors or cofactors to facilitate formation of the
preinitiation complex (PIC).
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FIG. 5.
C-terminal region of p65 strongly activates
transcription in vitro. The C-terminal region of p65 (the last 135 amino acids) and the N-terminal activation domain of p53 (amino acids 1 to 57, duplicated) were fused to the minimal Gal4 DNA-binding domain
(amino acids 1 to 94), expressed in bacteria as FLAG-tagged proteins,
and purified. These Gal fusion proteins were then tested for their
activation potential in transcription assays. As a control, Gal4 (amino
acids 1 to 94) alone was used. Transcription assays were performed with
no Gal proteins (lane 1) or 10 and 20 ng of either fGal-p53 (lanes 2 and 3), fGal4(1-94) (lanes 3 and 4), or fGal-p65ct (lanes 6 and 7).
The templates were pML53, as a control, and Gal4-responsive
pG5HMC2AT containing five Gal4-binding sites
upstream of the HIV TATA box core promoter.
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The p65 activation domain interacts with both TBP and several
TAFs.
TFIID is indispensable for activator-dependent transcription
(see below) under our standard in vitro assay conditions with purified
factors, and earlier studies (see Discussion) have indicated that
activator-dependent transcription can be achieved through specific
interactions between activation domains of transcription factors and
specific TFIID subunits. Therefore, we next examined potential
protein-protein interactions between the potent C-terminal activation
domain of p65 and different human TFIID subunits. A GST-p65 activation
domain fusion protein (GST-p65ct) and fGalp65ct were expressed in
bacteria, purified, and immobilized on glutathione-Sepharose or on
M2 agarose. TBP and the human TAFs were overexpressed from baculovirus
vectors in Sf9 cells, purified, and used for solution interactions with
p65ct. After extensive washing, specific complexes were analyzed by
SDS-PAGE and immunoblotting. Interactions were observed with TBP,
TAFII250, TAFII80, and TAFII28 but
not with TAFII100, TAFII55, or
TAFII31 (Fig. 6). Although
Schmitz et al. (60) reported an interaction between TBP and
p65ct, this is the first report of p65-TAF interactions. Interactions
with TAFs may be more relevant for activation than the interaction with TBP, since TBP alone does not support the high level of transcription activation by NF-B that we have observed with TFIID (see below). These results are consistent with activation domains of transcription factors interacting with basal factor (TBP) or coactivator (TAF) components of TFIID. The redundancy of interactions may be required for
synergistic activation of transcription (6).
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FIG. 6.
In vitro interactions between the activation domain of
NF-B p65 and the human TFIID subunits. Purified
HA-TAFII250 was incubated with equivalent amounts of M2
agarose-bound FLAG-Gal4(1-94) or FLAG-Gal-p65ct that was bound on M2
agarose, in BC150, 0.1% NP-40, and 100 µg of bovine serum albumin.
After extensive washing with BC500, bound protein was resolved by
SDS-PAGE and assayed by Western blotting. Input samples contained 10%
of the amounts used for binding. Similarly, purified hTBP,
hTAFII100, hTAFII80, hTAFII55,
hTAFII43, hTAFII31, and hTAFII28
were incubated with either GST alone or GST-p65ct immobilized on
glutathione beads. Reactions were then processed as for the
TAFII250 analysis, except that the washing was performed
with BC150. The arrow indicates the band corresponding to the
appropriate TFIID subunit.
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The p65 activation domain supports transcriptional activation with
partial TFIID complexes.
We first compared the abilities of TBP
and TFIID, at equimolar amounts based on quantitative Western blot
analysis, to mediate activation by fGal-p65ct on a template containing
five Gal4 sites upstream of the HIV TATA element. Our complementation
system is TAF/TFIID-free, as shown both by functional analysis (Fig.
7C, lanes 7 and 8) and by Western blot
analysis of different fractions (data not shown). Therefore, any
transcription in this system is absolutely dependent on addition of
exogenous TBP or TFIID. While transcription activation reached 17-fold
in the presence of f-TFIID, it was only 1.8-fold in the presence of an
equimolar amount TBP (Fig. 7B, lane 1 versus lane 2 and lane 3 versus
lane 4). This result shows a clear requirement for TAFs for significant transcription activation by the NF-B activation domain.
Interestingly, whereas basal transcription was comparable for TBP and
TFIID on the control AdML template, it was slightly higher for TBP than for TFIID on the test promoter (Fig. 7B, lane 1 versus lane 3). This
may reflect inhibitory effects of TAFs on TBP binding and function on
some promoters in the absence of activators or specific TAF-DNA
interactions (54).
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FIG. 7.
In vitro reconstitution and functional analysis of
partial TFIID species. (A) Partially reconstituted TFIID species were
resolved by SDS-PAGE and visualized by silver staining. The complexes
were assembled with purified subunits that were individually expressed
in Sf9 cells via baculovirus vectors (see Materials and Methods). Lane
1 contained TBP alone. Lanes 2, 3, 4, and 5 contained, respectively,
hTBP-TAFII250 (r-IID2),
hTBP-TAFII250-TAFII80 (r-IID3),
hTBP-TAFII250-TAFII80-TAFII31
(r-IID4), and
hTBP-TAFII250-TAFII100-TAFII80-TAFII55-TAFII31-TAFII28
(r-IID5) complexes. (B) Comparison of hTBP and f-TFIID transcriptional
activities. Reaction mixtures contained hTBP alone (4 ng) (lanes 1 and
2) or highly purified f-TFIID (f-IID) (lanes 3 and 4) containing the
same amount (4 ng) of hTBP. Purified recombinant fGal-p65ct (20 ng) was
added to the samples in the even-numbered lanes. Transcription was
assayed as described in the legend to Fig. 5. (C) Comparison of
transcriptional activities of intact TFIID and partial TFIID complexes.
The TFIID-dependent transcription system was supplemented with no
protein (lanes 7 and 8), hTBP-TAFII250 (r-IID2, lanes 3 and
4), hTBP-TAFII250-TAFII80 (r-IID3, lanes 5 and
6), hTBP-TAFII250-TAFII80-TAFII31
(r-IID4, lanes 1 and 2), or f-TFIID (lanes, 9 and 10). Added TFIID and
partial TFIID complexes contained the same amount of TBP (4 ng).
Transcription was tested in the absence (lanes 1, 3, 5, and 7) or
presence (lanes 2, 4, 6, and 8) of 20 ng of purified fGal-p65ct. (D)
Comparison of transcription activities of intact TFIID and a
seven-component partial TFIID complex. The TFIID-dependent
transcription system was supplemented with either the partial complex
hTBP-TAFII250-TAFII100-TAFII80-TAFII55-TAFII31-TAFII28
(r-IID5, lanes 1 and 2) or highly purified f-TFIID (lanes 3 and 4).
Added TFIID and r-IID5 complexes contained the same amount (4 ng) of
TBP. Reaction mixtures in lanes 2 and 4 contained 20 ng of
fGal-p65-ct.
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The human TFIID complex contains at least 13 TAFs in addition to TBP
(for a review, see reference 5), and studies with corresponding cDNA-encoded proteins have documented a number of TBP-TAF
and TAF-TAF interactions that may also occur within the natural TFIID
complex (5, 11, 14, 21-23, 64, 65). Based on these studies,
and others whose results are not shown here, we began the in vitro
assembly of human TFIID to study the direct requirement of specific
TAFs for both basal and activated transcription.
By use of a method similar to the one employed by Chen et al.
(8) for the assembly of Drosophila TFIID, several
combinations of human TAFs and TBP were assembled on an HA
epitope-tagged TAFII250 column, eluted with HA peptides,
and analyzed by SDS-PAGE and silver staining (Fig. 7A). Taking into
account the differential staining of different TAFs, the purified
complexes appear to contain roughly stoichiometric amounts of the
recombinant TFIID subunits.
To study the TAF requirement for activation, we utilized complexes that
contained, in addition to TBP-TAFII250, several different TAFs (TAFII80, TAFII80-TAFII31, or
TAFII100-TAFII80-TAFII55-TAFII31-TAFII28) (Fig. 7A). Complexes with either TAFII80 or
TAFII80-TAFII31 were designed to test whether
these two complexes can mediate transcription activation by the p65ct
acidic activator, as was shown for the corresponding
Drosophila TAFs in the case of the TAF-interacting p53
acidic activation domain (67). The larger complex
(containing seven subunits) was assembled to test possible effects of
other currently available human TAFs on both basal and activated
transcription.
We then compared equimolar amounts (based on TBP content) of f-TFIID
and partial, in vitro-assembled TFIID species for both basal and
fGal-p65ct-activated transcription. First, our data show that the
partial complexes containing TAFII250 have a markedly reduced capacity for basal transcription compared to natural TFIID (Fig. 7C, lanes 1, 3, and 5 versus lane 9) or to TBP (Fig. 7B, lane 3 versus lane 1). DNA footprinting experiments show that this is due to
the failure of TBP, when complexed to TAFII250 in the
absence of a complete set of TAFs, to efficiently bind DNA (data not
shown). This effect is specific for TAFII250, since a
complex containing only TBP, TAFII80, and
TAFII31 showed DNA binding quantitatively similar to that
observed with TFIID, but restricted to the TATA box on the AdML
template, and basal transcription activities comparable to those
observed with f-TFIID (47a). The inhibitory effect of
TAFII250 on TBP function is consistent with the results of
studies with Drosophila TAFII250 and TBP
(32, 45).
All of the TAFII250-containing partial TFIID species tested
were capable of mediating a moderate level (3.5-fold) of transcription activation by fGal-p65ct that was specific for the test template containing the Gal4 sites (Fig. 7C and D). Significantly, for the
partial complexes, the overall level of transcription in the presence
of the activator only reached a level comparable to the level of basal
transcription observed with complete f-TFIID alone (Fig. 7C, lanes 2, 4, and 6 versus lane 9, and Fig. 7D, lane 2 versus lane 3), which in
turn is slightly lower than the level of basal transcription obtained
with TBP (Fig. 7B, lane 3 versus lane 1). Thus, the "activation"
observed in this analysis appears to reflect primarily a reversal of
TAF-mediated repression rather than the large net increase in activity
(above the TBP level) observed with intact TFIID. Nonetheless, it is
possible that this activation represents part of the natural activation
mechanism and that it may be further enhanced by the presence of
missing human TAFs that, in this study, include TAFII135
and the putative human homolog of Drosophila
TAFII150 (8, 71). The missing TAFs also could
effect the basal transcription of the partial complexes.
The activated transcription that is observed with various
TAFII250-containing partial complexes appears to be
directly related to the interaction of the activation domain of p65
with TAFII250, rather than other TAFs. This is suggested by
the finding of comparable levels of activation with the complex that
contains only TBP and TAFII250 and with those complexes
that contain additional TAFs (Fig. 7C and D) and by the observation
that a stable TBP-TAFII80-TAFII31 complex shows
the same basal activity as TBP alone and fails to mediate any
activation by p65 (data not shown). Human TAFII250 has been
reported to interact physically with two activators (7, 19),
although functional data linking these TAFII250
interactions to transcription activation are lacking. Our study is the
first to suggest such a role for hTAFII250. It is still
possible, however, that interaction of p65ct with TBP reverses the
inhibitory effect of TAFII250-TBP interaction
(45).
Different core promoters have variable effects on both basal and
activated transcription in the presence of TFIID and partial TFIID
complexes.
In the assays described above, the core promoter of the
Gal4-p65-responsive template contained the HIV-1 TATA element and a
downstream Inr element. We next examined whether a different core
promoter within the Gal4-responsive template would alter the effect of
TAFs on either basal or fGal-p65-activated transcription when assayed
with TBP, complete f-TFIID, and partial TFIID complexes. In this case,
the template contained five Gal4 sites upstream of the adenovirus E1b
TATA and natural E1b Inr regions, and the results are as follows.
First, while TFIID and TBP supported comparable levels of basal
transcription on the AdML template (Fig.
8, lanes 9 and 11), basal activity on the
E1b template was clearly lower (circa fivefold) with TFIID than with
TBP (Fig. 8, lanes 9 and 11). This underscores a selective negative
effect of TAFs on TBP activity that is dependent on the type of core
promoter used and is consistent with the AdML versus HIV core promoter
comparisons in Fig. 7B. Second, and significantly, fGal4-p65ct
activated transcription 42-fold in the presence of f-TFIID but only
2-fold in the presence of TBP alone (Fig. 8, lane 9 versus lane 10 and
lane 11 versus lane 12), showing again the coactivator function of TAFs
but in the context of a different core promoter. It is important to
note that this high level of induction in the presence of TFIID
reflects the dual role of TAFs: an inhibitory effect on TBP-mediated
basal activity that is reversed by the activator and a coactivator
function that leads to a net increase in activity (above the TBP level) in response to the activator (Fig. 8, lane 10 versus lane 12). This
demonstrates clearly that high levels of induction with a net increase
above the intrinsic TBP-mediated basal activity can be achieved by
combined antirepression and true (net) activation mechanisms.
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FIG. 8.
Different core promoters have various effects on both
basal and activated transcription in the presence of TFIID and partial
TFIID complexes. fGal-p65 transcriptional activation was assayed with
TBP, f-TFIID, or partial TFIID complexes on a Gal4-responsive template
containing the Ad E1b TATA and Inr regions. The TFIID-dependent
transcription system was supplemented either with the partial complexes
r-IID2 (lanes 1 and 2), r-IID3 (lanes 3 and 4), r-IID4 (lanes 5 and 6),
and r-IID5 (lanes 7 and 8) or with complete f-TFIID (f-IID, lanes 9 and
10). Lanes 11 and 12 contained hTBP alone. All additions were adjusted
to the same amount of TBP (4 ng). Purified recombinant fGal-p65ct (20 ng) was added to the reaction mixtures in the even-numbered lanes.
Transcription reactions were performed as described in the legend to
Fig. 7.
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Similar to what was observed in the analysis of Fig. 7 for the
HIV-1-based template, all partial TFIID complexes showed a basal
activity lower than that observed with TBP alone and all mediated
activation by Gal4-p65ct on the E1b-based template (Fig. 8, lanes 1, 3, 5, and 7 versus lanes 2, 4, 6, and 8). However, on the E1b promoter,
the absolute levels of activated transcription observed with the
partial complexes were significantly higher than the level of basal
transcription observed with complete f-TFIID alone (Fig. 8, lanes 2, 4, 6, and 8 versus lane 9). This indicates that on the E1b core
promoter-containing template, the induction of transcription in the
presence of partial TFIID complexes represents, at least in part, a net
increase in activity rather than simply a reversal of the TAF-mediated
repression (see also below). However, consistent with the observations
on the HIV template, the failure to achieve the high absolute level of
activity observed with natural TFIID argues for additional functions of
other TAFs in achieving maximal levels of activation.
A minimal TFIID complex, TBP-TAFII250, effects a net
level of activation by fGalp65ct in the presence of PC2 and PC4.
In the assays described in Fig. 7 and 8, we used the unfractionated USA
fraction together with partial TFIID complexes to investigate the TAF
requirement for activated transcription and showed that all of the
TAFII250-containing partial TFIID species tested were
capable of mediating transcriptional activation by fGalp65ct. We next
wished to determine the effect of a minimal TBP-TAFII250
complex on activated transcription in the presence of purified USA
components PC2 and PC4 by using the Gal4-responsive template with the
E1b core promoter. First, we found that without USA or purified PCs,
Galp65ct specifically repressed transcription (by 2.5-fold) from the
test template (Fig. 9, lane 1 versus lane 2). Second, and consistent with its general inhibitory role, the addition of unfractionated USA had a repressive effect on basal transcription from both the test and the control AdML templates (Fig.
9, lane 3 versus lane 1). Consistent with the results of Fig. 8 (lanes
1 and 2), addition of Galp65ct to a reaction mixture containing the USA
fraction resulted in 5.5-fold activation (Fig. 9, lane 4 versus lane
3). This level of induction is due mainly to specific (from the test
template only) reversal of the inhibitory effect of USA and further
enhancement above the basal level of transcription. Third, purified PC2
alone mildly increased basal activity on both test and control
templates (Fig. 9, lane 5 versus lane 1) and showed a coactivator
function with fGalp65 (Fig. 9, lane 5 versus lane 6). The level of
induction for PC2 (2.5-fold) reflects a modest net increase in the
overall level of transcription. Addition of PC4 alone had no effect on
basal transcription from either template (Fig. 9, lane 7 versus lane
1), and somewhat surprisingly, the further addition of the activator
still effected a mild repression (1.5-fold) of transcription (Fig. 9,
lane 7 versus lane 8). Importantly, however, the addition of PC4 to
reaction mixtures containing PC2 increased the overall level of
induction by fGalp65ct from 2.5-fold (Fig. 9, lane 5 versus lane 6) to
7-fold (Fig. 9, lane 9 versus lane 10). The sevenfold induction
represents a clear net activation of transcription and not just an
antirepression mechanism, since the basal transcription level with a
combination of PC2 and PC4 is comparable to the basal transcription
level without any USA components (Fig. 9, lane 9 versus lane 1). Taken
together, these data show that a strong net activation (increase in
transcription above the basal level obtained without any USA
components) by the NF-B p65 activation domain can be achieved with
only a TBP-TAFII250 complex in the presence of two USA
components: PC2 and PC4. This, plus the failure to see activation with
TBP alone, provides further support for the relevance of the
p65-TAFII250 interaction.
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FIG. 9.
A minimal TFIID complex, TBP-TAFII250,
effects a net level of activation by fGalp65ct in the presence of PC2
and PC4. fGalp65 transcriptional activation was assayed with the
TBP-TAFII250 complex on a Gal-responsive template
containing the Ad E1b core promoter. All reaction mixtures contained
the TBP-TAFII250 partial complex. As indicated, the
reaction mixtures were also supplemented with either unfractionated USA
(lanes 3 and 4), 2 µl of the PC2 Mono S fraction (lanes 5, 6, 9, and
10), or purified PC4 (50 ng) (lanes 7 to 10). Purified recombinant
fGalp65ct (20 ng) was added to the reaction mixtures in the
even-numbered lanes.
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DISCUSSION |
HIV-1 and Ad promoters have been used to study cofactor
requirements and mechanisms for activation by NF-B and Sp1 through cognate binding sites. These studies have shown that recombinant NF-B and Sp1 proteins strongly activate transcription in a purified system, that activation is mediated by TAFs and USA-derived components (PC2 and PC4), that high levels of induction from a low (more physiological) basal activity result from both negative and positive functions of these two distinct classes of coactivators, and that some
of the effects are core promoter specific. These results both support
and extend previous observations regarding TAF function in metazoans
(reviewed in references 54 and
70) and are also relevant to the recent finding of a
core promoter-specific TAF function in yeast (61).
USA-derived cofactors: independent and cooperative functions in
repression of basal transcription and in activator-mediated
transcription.
Early studies have shown that USA is required for
the activity of several Gal4 fusion activators, NF-B p50 homodimers,
Sp1, and USF (18, 33-35, 41, 60). Our data for the HIV-1
promoter confirm these early reports and further show a persistent
requirement for the USA fraction for NF-B and Sp1 functions, even in
the presence of essentially homogeneous (affinity-purified) TFIID. This
argues against a conditional requirement for USA to eliminate (for
example) nonspecific inhibitory effects of other contaminating proteins. Importantly, USA effected both a large net increase in
activator-dependent transcription and a significant repression of basal
(activator-independent) transcription, leading to a very high level of
transcriptional induction that more closely mimics the in vivo
situation.
We next investigated the ability of two components (PC2 and PC4) of the
USA fraction to support transcription activation by both NF-B and
Sp1. Our results show that in both cases, either highly purified PC2,
which appears to be physically distinct from other PCs and TAFs on the
basis of molecular size (circa 500 kDa) and chromatographic properties,
or recombinant PC4 (a 15-kDa protein) is sufficient for mediation of
activator-dependent transcription. However, the basal transcription
levels observed with these positive cofactors alone are generally
higher than the basal level observed with the USA fraction. This
indicates that low levels of basal activity and the consequent high
levels of induction by activators require either the function of
negative cofactors like NC1 (40) that are usually present in
the crude USA fraction or a balance of PCs that, in some cases
(18), may also inhibit basal transcription at high
concentrations. In this regard, we have shown that a combination of PC2
and PC4, at the appropriate concentrations, can restore the total
function of the USA fraction (low basal plus high activator-dependent transcription) when assayed with Sp1. This suggests that single components of the USA fraction may act either alone or in combination with other USA components to simultaneously repress the basal activity
and enhance the activity of specific activators in an optimal fashion.
Consistent with this, and at low cofactor concentrations, we have also
observed some synergism between PC2 and PC4 in mediating activation by
Sp1. Similarly, in the case of NF-B (p50/p65), either PC2 or PC4 can
support the activator function, although a combination of both PC2 and
PC4 results in a high absolute level of activation and a higher level
of induction than that observed with either alone.
In summary, our data demonstrate that single components of the USA
fraction can function in combination to restore all or part of the
coactivator activity of USA when naked DNA is used as the template.
Other components from the USA fraction, or from other subnuclear
fractions, also may be utilized in more physiological situations, where
constraints of chromatin structure may require other types of
coactivators or chromatin-remodeling factors (reviewed in reference
30) for activator functions. In this regard, Pazin et al. (49) recently reported an in vitro synergism between Sp1 and NF-B activities that was dependent upon the use of chromatin (rather than DNA) templates. This could reflect a requirement for
additional cofactors such as those described here, although the
observed activation appeared to reflect mainly antirepression effects
and no specific (co)factor requirements were determined. Thus, in our
system, it will be important to determine whether there is a more
stringent requirement for other USA components or whether the weak
cooperativity that we have observed between PC2 and PC4 may be
amplified when activator functions are tested on chromatin templates.
Role of TAFs in activator-dependent transcription.
As we have
demonstrated, efficient activation by Sp1 and NF-B requires not only
the USA-derived cofactors, but also the TAFs that are tightly
associated with TBP in the TFIID complex. In general, the efficiency of
PIC assembly or function is controlled by the presence of transcription
factors usually bound to specific upstream sequences. Some models of
how transcription factors influence PIC assembly involve interactions
with TFIID that, through qualitative and/or quantitative effects on
TFIID binding, enhance the recruitment of downstream factors (1,
5, 9, 25, 26, 37, 73). Whereas TFIID was found to mediate both
basal and activator-dependent transcription in cell-free systems
reconstituted with purified components, TBP elicited only basal
transcription (10, 16, 24, 53, 57, 63, 75). Following the
demonstration of a number of specific activator-TAF interactions
(reviewed in reference 5), the direct involvement of
specific TAFs in the function of specific activators was shown in
studies using Drosophila TFIID complexes (or subcomplexes)
reconstituted with the recombinant TAFs in vitro (8, 67). In
addition, the synergistic function of two activators that interact with
two different TAFs was demonstrated with reconstituted
Drosophila TFIID complexes, and the mechanism was shown to
involve enhanced TFIID binding (recruitment) to the promoter
(56).
Since the C-terminal region of p65 is a very strong activator when
fused to a Gal4 DNA-binding domain and tested in the purified reconstituted transcription system, and since TFIID is a target for
many activators, we have analyzed the interactions of the p65
activation domain with different human TAFs and TBP. The activator p65
interacts directly both with TBP and with several TAFs that include
TAFII80 and TAFII250. The interaction with
TAFII80 is consistent with data published for acidic
activation domains, which seem to interact preferentially with
TAFII80 and TAFII31 and their
Drosophila homologs TAFII60 and
TAFII40 (31, 39, 67). However, as shown here,
p65ct also interacts strongly with TAFII250, and this
interaction appears to be directly relevant to transcription activation
since a complex that contains only TBP and TAFII250, but
not TBP alone, supports p65 activity in the absence of
TAFII80. These data support the conclusions of earlier
studies (see above) implicating specific TAFs in the function of other
activators (reviewed in reference 70). Although
several studies have shown that the activation domains of c-rel and p65 can both interact with TBP and TFIID (29, 60, 74), these interactions were not shown to be directly relevant to activation by
the NF-B proteins. Because we have shown directly that TFIID, rather
than TBP alone, is required for activated transcription, TAF
interactions with p65 appear to be more relevant for transcriptional activation by p65. While other studies have shown that two viral activators, E1A and ICP4, can interact with human TAFII250
(7, 19), our study is the first to suggest a direct role of
TAFII250 in activated transcription. The failure of other
p65-interacting TAFs (e.g., TAFII80) to enhance the
function, in activation, of TAFII250-TBP complexes suggests
either that these interactions do not occur in the context of the
partial TFIID complex or that they are redundant with the
p65-TAFII250 interaction. The functional consequence of
such interactions could also be that additional TAFs are required.
Dual roles of TAFs: TAF-mediated repression of TBP function and
TAF-mediated net increase in transcriptional activation.
Another
important property of TAFs that we have observed is their ability, on
certain core promoters, to inhibit TBP-mediated basal activity within
native TFIID compared to TBP alone (Fig. 7 and 8). In the presence of
an activator, there is both a reversal of TAF-mediated repression of
TBP function and a TAF-mediated net increase in overall transcription
activity. This leads to a high level of transcription induction that
more closely mimics the physiological situation. However, with
partially reconstituted TFIID complexes, and depending on the core
promoter used, the absolute levels of transcription in the presence of
the fGal4-p65 activator were only comparable to (HIV-1 promoter) or
moderately greater than (Ad5 E1b promoter) the level of basal activity
observed with natural (complete) TFIID alone and 20-fold (HIV-1
promoter) to 25-fold (E1b promoter) lower than the levels observed with TFIID in the presence of the activator. Our results on the E1b core
promoter, especially with PC2 and PC4 in place of USA, are more
consistent with previous indications from Tjian and colleagues (8,
56) that specific TAFs can serve as coactivators in partial complexes and result in levels of activator-dependent transcription greater than basal levels. Our failure to fully recapitulate the expected large net increases of activity with partial complexes could
be due to the absence of a specific subset of the TAFs in the
reconstituted system. Alternatively (see also below), human TFIID may
function optimally only as a complete entity, which may allow other DNA
interactions and conformational changes important for the efficient
recruitment and function of other general transcription factors
(references above; 9, 47). Nonetheless, by using partial TFIID complexes, we have dissociated two functions of TAFs in
transcription regulation: intrinsic repressive effects on TBP binding
and function that may be core promoter-specific and coactivator
functions, leading to reversal of the repressive effects and large net
increases in activation, that may be activator specific.
Our demonstration that TAFII250 represses the basal
transcription activity of TBP on the HIV promoter is in agreement with what was reported earlier for Drosophila
TAFII250 and TBP (32, 45). However, no apparent
repression of TBP by Drosophila TAFII250 was
observed when the latter was reconstituted in vitro by Chen et al.
(8). This may reflect differences in the functional TBP
content of various complexes (or preparations of complexes), the
specific promoters used, or the purity of the reconstituted systems. We
also have observed greater repression of basal activity with all of the
partial TAFII250-containing complexes tested compared to
native TFIID, suggesting that additional TAF-TAF and/or TAF-DNA interactions partially relieve the repression within native TFIID. In
this regard, an early study by Nakatani et al. (44) showed that downstream promoter interactions are necessary to reverse TAF-mediated repression of natural TFIID binding and function (relative
to TBP) in the gfa core promoter, and activator-TAF interactions could serve similar functions in other situations.
Finally, recent studies with yeast have shown that most TAFs are
dispensable for activated transcription from many genes in vivo but
necessary for cell cycle progression (43, 72). These results
led the investigators to propose that TAFs are only required for
transcription of a subset of genes, and Shen and Green (61) have recently identified several yeast TAFII145-dependent
genes whose TAF requirements are dictated by their (as yet undefined) core promoters rather than by specific activators. Thus, the TAF properties discussed aboveeffects on TBP binding and function that
may be core promoter specificand the presence of additional coactivators (3) for specific activators could explain, at least in part, the observations in yeast. Moreover, while the transcription of some genes in metazoans could prove to be TAF independent, it is also possible that metazoans have broader TAF requirements than yeast (65).
We thank C.-M. Chiang for the FLAG-Gal4 expression plasmid,
D. K. Lee for the E1b construct, and T. Oelgeschläger for
critical comments on the manuscript.
This work was supported by a grant from the Tebil Foundation to The
Rockefeller University and by NIH grants AI32737 and CA 42567 to R.G.R.
| 1.
|
Abmayr, S. M.,
J. L. Workman, and R. G. Roeder.
1988.
The pseudorabies immediate early protein stimulates in vitro transcription by facilitating TFIID:promoter interactions.
Genes Dev.
2:542-553[Abstract/Free Full Text].
|
| 2.
|
Baldwin, A. S.
1995.
The NF-B and IB proteins: new discoveries and insights.
Annu. Rev. Immunol.
14:649-681[Medline].
|
| 3.
|
Björklund, S., and Y.-J. Kim.
1996.
Mediator of transcriptional regulation.
Trends Biochem. Sci.
21:335-337[Medline].
|
| 4.
|
Blair, W. S.,
H. P. Bogerd,
S. J. Madore, and B. R. Cullen.
1994.
Mutational analysis of the transcriptional analysis of RelA: identification of a highly synergistic minimal acidic activation module.
Mol. Cell. Biol.
14:7226-7234[Abstract/Free Full Text].
|
| 5.
|
Burley, S. K., and R. G. Roeder.
1996.
Biochemistry and structural biology of transcription factor IID (TFIID).
Annu. Rev. Biochem.
65:769-799[Medline].
|
| 6.
|
Carey, M.,
Y.-Y. Lin,
M. R. Green, and M. Ptashne.
1990.
A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives.
Nature
345:361-364[Medline].
|
| 7.
|
Carrozza, M. J., and N. A. Deluca.
1996.
Interaction of the viral protein ICP4 with TFIID through TAF250.
Mol. Cell. Biol.
16:3085-3093[Abstract].
|
| 8.
|
Chen, J.-L.,
L. D. Attardi,
C. P. Verrijzer,
K. Yokomori, and R. Tjian.
1994.
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators.
Cell
79:93-105[Medline].
|
| 9.
|
Chi, T., and M. Carey.
1996.
Assembly of the isomerized TFIIA-TFIID-TATA ternary complex is necessary and sufficient for gene activation.
Genes Dev.
10:2540-2550[Abstract/Free Full Text].
|
| 10.
|
Chiang, C.-M.,
H. Ge,
Z. Wang,
A. Hoffmann, and R. G. Roeder.
1993.
Unique TATA-binding protein-containing complexes and cofactors involved in transcription by RNA polymerase II and III.
EMBO J.
12:2749-2762[Medline].
|
| 11.
|
Chiang, C.-M., and R. G. Roeder.
1995.
Cloning of an intrinsic human TFIID subunit that interacts with multiple transcriptional activators.
Science
267:531-536[Abstract/Free Full Text].
|
| 12.
|
Dejong, J., and R. G. Roeder.
1993.
A single cDNA, hTFIIA/ , encodes both the p35 and p19 subunits of human TFIIA.
Genes Dev.
7:2220-2234[Abstract/Free Full Text].
|
| 13.
|
Dignam, J. D.,
R. M. Lebovitz, and R. G. Roeder.
1983.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:1475-1489[Abstract/Free Full Text].
|
| 14.
|
Dubrovskaya, V.,
A.-C. Lavigne,
I. Davidson,
J. Acker,
A. Staub, and L. Tora.
1996.
Distinct domains of hTAFII100 are required for functional interaction with transcription factor TFIIF (RAP30) and incorporation into the TFIID complex.
EMBO J.
15:3702-3712[Medline].
|
| 15.
|
Duckett, C. S.,
N. D. Perkins,
T. F. Kowalil,
R. M. Schmid,
E. S. Huang,
A. S. Baldwin, Jr., and G. J. Nabel.
1993.
Dimerization of NF-B2 with RelA (p65) regulates DNA binding, transcription activation, and inhibition by IB (MAD-3).
Mol. Cell. Biol.
13:1315-1322[Abstract/Free Full Text].
|
| 16.
|
Dynlacht, B. D.,
T. Hoey, and R. Tjian.
1991.
Isolation of coactivators associated with the TATA-binding protein that mediates transcriptional activation.
Cell
66:563-576[Medline].
|
| 17.
|
Fujita, T.,
G. P. Nolan,
S. Ghosh, and D. Baltimore.
1992.
Independent modes of transcriptional activation by the p50 and p65 subunits of NF-B.
Genes Dev.
6:775-787[Abstract/Free Full Text].
|
| 18.
|
Ge, H., and R. G. Roeder.
1994.
Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes.
Cell
78:513-523[Medline].
|
| 19.
|
Geisberg, J. V.,
J.-L. Chen, and R. P. Ricciardi.
1995.
Subregions of the adenovirus E1A transactivation domain target components of the TFIID complex.
Mol. Cell. Biol.
15:6283-6290[Abstract].
|
| 20.
|
Ghosh, S.,
A. M. Gifford,
L. R. Riviere,
P. Tempst,
G. P. Nolan, and D. Baltimore.
1990.
Cloning of the p50 DNA binding subunit of NF-Bhomology to Rel and Dorsal.
Cell
62:1019-1029[Medline].
|
| 21.
|
Hisatake, K.,
T. Ohta,
R. Takada,
M. Guermah,
M. Horikoshi,
Y. Nakatani, and R. G. Roeder.
1995.
Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors.
Proc. Natl. Acad. Sci. USA
92:8195-8199[Abstract/Free Full Text].
|
| 22.
|
Hoffmann, A.,
C.-M. Chiang,
T. Oelgeschläger,
X. Xie,
S. K. Burley,
Y. Nakatani, and R. G. Roeder.
1996.
A histone octamer-like structure within TFIID.
Nature
380:356-359[Medline].
|
| 23.
|
Hoffmann, A., and R. G. Roeder.
1996.
Cloning and characterization of human TAF20/15.
J. Biol. Chem.
271:18194-18202[Abstract/Free Full Text].
|
| 24.
|
Hoffmann, A.,
E. Sinn,
T. Yamamoto,
J. Wang,
A. Roy,
M. Horikoshi, and R. G. Roeder.
1990.
Highly conserved core domain and unique N terminus with presumptive regulatory motifs in a human TATA factor TFIID.
Nature
346:387-390[Medline].
|
| 25.
|
Horikoshi, M.,
M. F. Carey,
H. Kakidani, and R. G. Roeder.
1988.
Mechanism of action of a yeast activator: direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions.
Cell
54:665-669[Medline].
|
| 26.
|
Horikoshi, M.,
T. Hai,
Y.-S. Lin,
M. R. Green, and R. G. Roeder.
1988.
Transcription factor ATF interacts with TATA factor to facilitate establishment of a preinitiation complex.
Cell
54:1033-1042[Medline].
|
| 27.
|
Kaiser, K., and M. Meisterernst.
1996.
The human general cofactors.
Trends Biochem. Sci.
21:342-345[Medline].
|
| 28.
|
Kawakami, K.,
C. Scheidereit, and R. G. Roeder.
1988.
Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-B) that activates transcription from a hum |
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B and Sp1
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Abstract
Introduction
