(Received for publication, October 21, 1994; and in revised form, December 27, 1994)
From the
We show that the transactivating COOH terminus of the p65
subunit of human transcription factor NF-B directly binds the
general transcription factors TFIIB and TATA-binding protein (TBP) in vitro. Interaction of p65 with TFIIB required the most
COOH-terminal sequence repeat within TFIIB. A functional interaction of
TFIIB with p65 was evident from assays in yeast cells. Cotransfection
experiments in COS cells revealed that only overexpression of TBP was
able to further stimulate p65-dependent transactivation of a reporter
gene. The coexpression of neither TBP nor TFIIB was able to relieve
squelching, indicating the involvement of additional factors in
transactivation by p65. A cell-free assay using highly purified factors
revealed a specific transcriptional stimulation through the
COOH-terminal activation domain of NF-
B by at least one cofactor,
PC1, isolated from HeLa cells. These data show that the potent acidic
transactivation domains in the COOH terminus of p65 are able to
functionally recruit various components of the basic transcription
machinery as well as coactivators.
The accurate and regulated transcription by RNA polymerase II
requires dynamic interactions between several classes of
transcriptional regulatory proteins. One class is constituted by
sequence-specific DNA-binding proteins. These are typically composed of
several discrete domains minimally mediating DNA binding, nuclear
translocation, and transactivation (reviewed by Ptashne and Gann
(1990)). In the physiological situation, the template for transcription
is contained in the condensed chromatin structure. Some transcription
factors are found to counteract the chromatin-mediated repression
(``antirepression''), whereas all transcription factors are
able to facilitate the inherent transcription reaction, which is
designated as ``true activation'' (reviewed by Croston and
Kadonaga(1993)). This process requires a second class of proteins,
which are the general transcription factors. Those include TFIIA, ()TFIIB, TFIID, TFIIE, TFIIF, TFIIH, TFIIG/TFIIJ, and RNA
polymerase II (reviewed by Roeder(1991)). A key step in transcription
initiation is the binding of the multisubunit complex TFIID to the TATA
element close to the transcription start site. A component of the
TFIID complex is the TATA-binding protein (TBP), which is also required
for transcription of RNA polymerase I and III promoters (reviewed by
White and Jackson(1992)). TFIID contains numerous additional factors
referred to as TBP-associated factors (TAFs), from which many of the
corresponding cDNAs from Drosophila and human were recently
cloned (reviewed by Goodrich and Tjian(1994)). Recombinant TBP protein
can substitute for TFIID fractions for basal RNA polymerase II
transcription from TATA-containing promoters. However, recombinant TBP
fails to mediate activated transcription induced by transcriptional
activators (Pugh and Tjian, 1990; Hoffmann et al., 1990).
Activated transcription requires a third class of transcriptional regulatory proteins, the so-called coactivators. Many of the TAFs in TFIID were found to have this coactivating function (reviewed by Goodrich and Tjian(1994)). But also non-TAF proteins were found to have such an activity. Examples are ADA2, ADA,3, SW1, SW2, and SW3, which have been identified in yeast by biochemical and genetic approaches (Berger et al., 1992; Peterson and Herskowitz, 1992; Piña et al., 1993; Kim et al., 1994). Among coactivators isolated from mammalian cells are the B cell-derived factor Oca-B (Luo et al., 1992) as well as the positive cofactors (PCs) PC1, PC2, PC3/Dr2, and p15/PC4 (Meisterernst et al., 1991; Kretzschmar et al., 1993, 1994a, 1994b; Merino et al., 1993). All of these coactivators stimulate activator-dependent transcription. The specific protein/protein interactions required for activated transcription involve interactions between transcription activation domains of sequence-specific activators with coactivators (Goodrich et al., 1993; Ferreri et al., 1994; Ge and Roeder, 1994) and general transcription factors (reviewed by Tjian and Maniatis(1994)).
A well studied
paradigm for an inducible transcriptional activator is nuclear factor
B (NF-
B) (reviewed by Grilli et al. (1993) and
Baeuerle and Henkel(1994)). NF-
B is retained in the cytoplasm by
the inhibitory I
B subunits in an inactive form (Baeuerle and
Baltimore(1988); reviewed by Schmitz et al. (1991)). The
factor becomes readily activated when cells are treated with
inflammatory cytokines, tumor promoters, viruses, and
lipopolysaccharides (reviewed by Baeuerle and Henkel(1994)). All these
agents lead to phosphorylation and subsequent proteolytic degradation
of I
B, thereby allowing a DNA-binding NF-
B dimer to enter the
cell nucleus. The DNA-binding form of NF-
B activates transcription
of numerous target genes encoding proteins involved in inflammatory,
immune, and acute phase response (reviewed by Grilli et
al.(1993)). NF-
B DNA-binding subunits comprise p50 (Kieran et al., 1990; Ghosh et al., 1990; Meyer et
al., 1990), p65 (Baeuerle and Baltimore, 1989; Ruben et
al., 1991; Nolan et al., 1991; Ballard et al.,
1992), p52 (Schmid et al., 1991), RelB (Rysek et al.,
1992; Ruben et al., 1992b), and c-Rel (Wilhelmsen et
al., 1984). These proteins share a conserved 300-amino acid
sequence in the NH
-terminal portion, which is also present
in the oncogene v-rel from the avian retrovirus REV-T
(Stephens et al., 1983) and the morphogen dorsal from Drosophila (Steward, 1987). We refer to this conserved region
as the NRD (NF-
B/Rel/dorsal) domain, which contains the
subdomains important for DNA-binding, dimerization, nuclear
localization, and protein/protein interactions. The p65 subunit is
frequently detected in NF-
B complexes and has a strong
transcription activation potential (Schmitz and Baeuerle, 1991; Ruben et al., 1992a; Fujita et al., 1992; Ballard et
al., 1992; Schmitz et al., 1994). It contains at least
two independent transcription activation domains (TADs) in its
COOH-terminal 120 amino acids. One of these domains, TA
, is
contained in the COOH-terminal 30 amino acids (amino acids
521-551) of p65. Both TADs of p65 belong to the class of acidic
activation domains and are very similar to the TAD of herpes simplex
virus protein VP16 (Schmitz et al., 1994).
In this study we
have addressed the question of which proteins interact with the
transactivating COOH terminus of human p65 to mediate its activating
potential. The general transcription factors TBP and TFIIB were found
to specifically bind to the p65 TADs. The binding of TFIIB to NF-B
p65 required the most COOH-terminal sequence repeat of TFIIB. TBP was
able to stimulate p65-dependent expression in intact cells. Under
cell-free conditions, activation of transcription by the p65 COOH
terminus was found to depend on both the TFIID complex and on at least
one coactivator fraction, called PC1 (Meisterernst et al.,
1991).
Proteins tested in
immunoprecipitation assays were labeled with
[S]methionine (Amersham Corp.) by in vitro transcription/translation. The respective proteins were incubated
in a total volume of 50 µl in IP buffer consisting of 12 mM Hepes/KOH, pH 8, 12% (v/v) glycerol, 400 mM KCl, 5 mM MgCl
,1 mM phenylmethylsulfonyl fluoride, 10
µM ZnCl
, 1 mM spermidine, and 0.5%
(v/v) Tween 20 for 30 min at room temperature. Subsequently, 0.5 µl
of a polyclonal antibody (Santa Cruz Biotechnology) directed against
the NRD region from p65 was added and the sample was incubated on a
rotating wheel for 3 h at 4 °C. After addition of 50 µl of
Protein A-Sepharose beads (Pharmacia Biotech Inc.) preswollen for 20
min in IP buffer and 10 µg of BSA (Sigma), the mix was incubated
again on a rotating wheel for 1 h at 4 °C. The beads were washed
six times with IP buffer. Finally, the beads were boiled for 5 min in
20 µl of 1
SDS sample buffer and proteins separated on a
reducing 10% SDS-polyacrylamide gel. The gel was then dried and exposed
to x-ray film at -80 °C.
Figure 1:
Specific association of TBP and TFIIB
with the transactivation domain of human NF-B p65 in
vitro. A, TBP and TFIIB bind to the transactivating COOH
terminus of human p65. Equal amounts of in vitro translated
[
S]methionine-labeled TBP (lanes
1-3) and TFIIB (lanes4-6) were
incubated with Gal4(1-147), Gal-p65
, and
BSA, which were covalently coupled to Sepharose beads as indicated at
top of the figure. The beads were extensively washed and eluted with
SDS, and proteins were separated by 12% SDS-PAGE. An autoradiogram of a
representative experiment is shown. The position of prestained
molecular weight markers is indicated. The arrows point to the
positions of TFIIB and TBP. B, the COOH terminus of p65 is
required for binding of TBP and TFIIB. Rabbit reticulocyte lysates were
programmed in the presence of [
S]methionine with
cDNAs for the p65, TBP, TFIIB, and p65
C (lacking the 109
COOH-terminal amino acids of p65) proteins. The labeled proteins were
incubated in the indicated combinations and subsequently precipitated
with an anti-p65 antibody. An autoradiogram of a 12% SDS-polyacrylamide
gel is shown and the positions of the marker proteins indicated at the left. The arrows mark the positions of the respective
proteins. C, the COOH-terminal sequence repeat of TFIIB is
necessary for the specific interaction with the p65 COOH terminus. The
structure of the wild-type (TFIIB) and the COOH-terminally truncated
TFIIB (TFIIB
C) molecule is drawn schematically at the top. The
location of the repeated domains is shown by arrows, and their
amino acid positions within the molecule are given. +, positions
of two regions that are highly enriched in basic amino acids. Lanes1 and 2 show the input proteins TFIIB
C and
TFIIB, respectively. Each of the proteins was incubated with
Gal-p65
protein coupled to Sepharose, and the
experiment was further conducted as described for A. Lanes3 and 4 show an autoradiogram of the eluates
after SDS-PAGE. Details of the figure legend are as explained in A.
TFIIB carries in its COOH-terminal half two imperfect direct
repeated sequences, the first of which contains a potential positively
charged amphipathic -helix at its COOH terminus (see Fig. 1B). Binding experiments with a TFIIB mutant
retaining the complete first repeat but lacking the more COOH-terminal
repeat were performed in order to assess the role of this conserved
structure. In contrast to the full-length TFIIB protein, the
COOH-terminally truncated version of TFIIB was not able to efficiently
bind to the Gal-p65
protein (Fig. 1B, compare lanes3 and 4). This shows that the first repeat structure within TFIIB,
including the positively charged amphipathic
-helix, is not
sufficient to mediate the specific contact to p65. Alternatively, the
truncation might have altered the structure of TFIIB to preclude p65
binding. In conclusion, the transactivating p65 COOH terminus can
directly interact with the basal transcription factors TFIIB and TBP,
as was found for various other acidic domains.
Figure 2:
TFIIB and p65 interact in vivo.
The experiment is displayed in a schematic drawing at left.
Yeast cells bearing an integrated lacZ reporter gene
controlled by LexA binding sites were cotransformed with two plasmids:
a plasmid constitutively expressing a fusion protein between the
DNA-binding domain of LexA and TFIIB, and a plasmid encoding NF-B
p65
controlled by a Gal1 promoter, which is shown as an arrow. Induction of p65
expression with galactose leads
to intracellular association of p65
with the TFIIB portion. The
resulting transcriptional activation of the lacZ reporter gene
is symbolized by an arrow. The right part of this figure shows
yeast colonies grown on Ura
, Leu
,
His
plates containing the LacZ substrate X-Gal either
in the presence of glucose (top) or galactose (lower).
Figure 3:
Functional interaction of p65 and TBP in
transcription activation. COS7 cells were cotransfected with the
B-dependent CAT reporter plasmid J16 (Pierce et al.,
1988) and the expression vectors for p65, TBP, and TFIIB as indicated.
Gene activation was measured as percent acetylation of
[
C]chloramphenicol, and the activity of the
reporter plasmid alone was set to 1. The standard deviations are
indicated by bars and were obtained from four independent
experiments.
Figure 4:
Activated transcription by p65 requires
additional proteins. COS7 cells were transfected with a Gal4-dependent
CAT reporter gene and the indicated expression vectors. The negative
and positive controls in lanes 1 and 2 received the
same amount of DNA as ``empty'' expression vector (RcCMV).
The squelching plasmid relADNA is a p65 mutant incapable of
binding to DNA (Schmitz et al., 1994). relA
DNA
TA is
a derivative thereof lacking both transactivation domains. The figure
shows the result from a representative CAT assay in which the
squelching construct was present at 12-fold molar excess over the Gal
activator plasmid. The presence (+) or absence(-) of the
respective expression plasmids is indicated in the lower part of the
figure. In the typical experiment shown, 2 pmol of expression vectors
for TBP and TFIIB were used. The weak increase in lane7 was not reproducible in three independent
experiments.
Figure 5:
In vitro transcription activity of the
Gal4-TA protein in crude nuclear extracts. The indicated
amounts of purified Gal4(1-147) and Gal4-p65
proteins were tested for their influence on the activity of the
Gal4-dependent Ad2ML promoter. An arrow points to the specific
transcript from the G-less cassette. The autoradiogram shows the
typical result from a transcription reaction performed for 1 h at 28
°C.
In contrast to crude transcription systems, both Gal4-p65 and a Gal4-AH (amphipathic helix) protein were completely inactive (Fig. 6A, lanes2 and 4) in transcription systems reconstituted of highly purified general transcription factors (Kretzschmar et al., 1994a). Both proteins were transcriptionally active only in the presence of coactivators, as exemplified here for the coactivator fraction PC1 (Fig. 6A, lanes6 and 8).
Figure 6:
Activated transcription by
Gal4-p65 is specifically stimulated by the PC1
coactivator fraction. A, effects of the PC1 fraction on
transcriptional activation by Gal4 derivatives as indicated in a
transcription system consisting of general transcription factors TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II. B,
comparison of PC1 and PC2 effects on Gal4 derivatives as indicated. An
autoradiogram is shown with the upperarrow (Gal) pointing to the Gal-dependent transcript and the lowerarrow (ML
) pointing to the
internal control template without Gal4 binding
sites.
Several positive cofactors, originating from the upstream
stimulatory activity fraction (Meisterernst et al., 1991),
which had been shown previously to stimulate activator-dependent
transcription in vitro, were subsequently analyzed for their
ability to allow activation of transcription by Gal4 derivatives. All
four cofactors tested (PC1 to p15/PC4; reviewed by Kretzschmar et
al. (1994b)) stimulated transcriptional activation by the
Gal4-p65 protein (data not shown). Remarkably,
as exemplified for PC2 (Fig. 6B, lane8versuslane9), the coactivating
potential of cofactor fractions PC2, PC3/Dr2, and p15/PC4 was mostly
dependent on the Gal4(1-147) protein, while the domain
responsible for transcriptional activation in vivo (the
TA
domain) had very little or no additional effect on
transcription. However, in the presence of a partially purified PC1
fraction, the Gal4p65
protein could stimulate
transcription above levels observed with a Gal4(1-147) protein (Fig. 6B, lane2versuslane1). Conversely, and in agreement with
previous studies (Kretzschmar et al., 1994a), Gal4-AH did
stimulate transcription above levels observed with a Gal4(1-147)
protein in the presence of a partially purified PC2 fraction (Fig. 6B, lane6versuslane8) but not in the presence of a PC1
fraction (lane2versuslane4). Although specific effects on transcription of
activation domains AH and TA
were weak (approximately
2-fold above levels observed with Gal4(1-147)), these
differential effects of activators and cofactors, if analyzed in
parallel (Fig. 6B), were reproducibly observed in
experiments that were conducted with optimal activator concentrations
(Kretzschmar et al., 1994a). These experiments suggested that
PC1, in contrast to other presently defined cofactors (PC2, PC3/Dr2,
and p15/PC4), displays specificity for the COOH-terminal
transcriptional activation domain of p65, while the cofactor PC2 acts
preferentially through the AH domain of Gal4-AH. They further indicate
structural differences in the activation domains AH and TA
,
although both have been classified previously as acidic activators
(Ptashne and Gann, 1990; Schmitz et al., 1994). It therefore
seems possible that distinct cofactors bind activation domains with
different affinity.
In this study, we demonstrate a direct interaction of the
acidic activation domain of NF-B p65 with two components of the
basal transcription machinery: TBP and TFIIB. The interaction with
TFIIB was of sufficient avidity to allow assembly of p65 with a
LexA-TFIIB fusion protein and subsequent LexA-dependent transcriptional
activation in yeast cells. The overexpression of TBP did significantly
stimulate p65-dependent gene expression. Similar enhancing effects of
TBP were detected for gene activation by the NRD protein family member
c-Rel (Kerr et al., 1993; Xu et al., 1993) and the
human T-cell leukemia virus type 1 transactivator Tax (Caron et
al., 1993). The stimulatory effect of TBP may be explained by the
fact that it can be rate-limiting in cells (Colgan and Manley, 1992).
This could be caused by the absorption of most cellular TBP to the
associated TAF proteins, which may not allow a direct interaction of
TBP with the acidic domain of p65. Alternatively, TBP overexpression
could titrate negative regulators of RNA polymerase II transcription
such as NC2/DR1, which directly contact TBP (Meisterernst and Roeder,
1991; Yeung et al., 1994). The failure of TFIIB to enhance
p65-dependent transactivation can be explained by squelching effects
mediated by TFIIB or, alternatively, by the fact that it is not present
in limiting concentrations. TFIIB and TBP contact next to each other
the promoter DNA, and binding of p65 to them is likely to occur
simultaneously in the cell. This redundant binding to both general
transcription factors would have the advantage of increasing the
overall affinity of p65 to the transcription initiation complex. The
stimulatory effect of TBP did never exceed a factor of 3, indicating
that transactivation by p65 requires additional coactivators. This is
consistent with squelching and in vitro transcription
experiments, showing that TFIID consisting of TBP and TAFs, but not TBP
alone, mediates the response of regulatory factors.
The finding that
transcriptional activation domains can directly bind general
transcription factors is not without precedent. The general
transcription factors TFIIB, TBP, TFIIJ, and TFIIH were all found to
have the potential to interact with certain activation domains (Xiao et al., 1994) (reviewed by Tjian and Maniatis(1994)). General
transcription factor TFIIB was found to specifically interact with the
acidic activation domain of VP16 (Lin et al., 1991; Lin and
Green, 1991), the glutamine-rich activation domain of Fushi tarazu
(Colgan et al., 1993), and the human thyroid hormone receptor
(Baniahmad et al., 1993). The TBP protein is also
specifically contacted by various types of transactivation domains,
such as that of p53 (Seto et al., 1992), E2F-1 (Hagemeier et al., 1993a), c-Fos and c-Jun (Ransone et al.,
1993), PU-1 (Hagemeier et al., 1993b), Sp1 (Emili et
al., 1994), and the c-Myc transcription factor (Maheswaran et
al., 1994). The TATA box-binding protein TBP is also contacting
viral activator proteins that are devoid of intrinsic DNA binding
activity such as VP16 (Stringer et al., 1990), the Tat protein
of human immunodeficiency virus (Kashanchi et al., 1994), the
human T-cell leukemia virus type 1 activator protein Tax1 (Caron et
al., 1993), the adenovirus activator E1A (Horikoshi et
al., 1991; Lee et al., 1991), and the Epstein-Barr virus
proteins Zta (Lieberman and Berk, 1991) and R (Manet et al.,
1993). Transcription activation domains from some transcription factors
bind to both TFIIB and TBP, as seen for ICP4, a transcriptional
regulatory protein from herpes simplex virus (Smith et al.,
1993), the immediate early protein 2 from human cytomegalovirus
(Caswell et al., 1993), v-Rel, chicken and mouse c-Rel (Kerr et al., 1993; Xu et al., 1993), as well as p65 (this
study).
One study (Kerr et al., 1993) mapped the region within mouse c-Rel that is responsible for the interaction with TBP to the first 50 amino acids within the NRD domain. Contradictory to that, Xu et al.(1993) mapped the COOH-terminal transactivation domain of c-Rel as TBP- and TFIIB-binding domain (Xu et al., 1993). Our data are consistent with a COOH-terminal location of this domain in the c-Rel-related protein p65/RelA. The interaction of general transcription factors with transactivating domains rather than DNA binding domains is in agreement with the notion that transactivating domains are used to contact the basal transcription machinery (reviewed by Hahn(1993)).
Within TFIIB the more
COOH-terminal repeat was found to be required for the interaction with
p65. Accordingly, deletion of this repeat also abrogated binding to
VP16 (Roberts et al., 1993) and to the human thyroid hormone
receptor (Baniahmad et al., 1993). The functional
importance of this repeat structure was also seen in experiments where
cotransfection of a COOH-terminally truncated TFIIB inhibited
activation by a Gal4-Fushi tarazu protein in Drosophila Schneider cells (Colgan et al., 1993).
The direct
interactions between a transactivating domain and components of the
basic transcription machinery are thought to bring an activation domain
over large distances into close proximity of the initiation complex
close to the transcription start site. Here an activation domain may
serve multiple functions. It can stabilize the interaction of TFIID
with promoter DNA, as is seen in the case of the Zta protein (Lieberman
and Berk, 1991). After binding of TBP to the promoter DNA, TFIIB enters
the complex through interaction with TBP. Using an immobilized DNA
template assay, it was possible to show that this recruitment of TFIIB
to the initiation complex is enhanced by the acidic activation domain
of VP16 (Roberts et al., 1993), which is highly related to
that of p65 (Schmitz et al., 1994). In a subsequent
TAF-dependent step, the activation domain helps the general
transcription factors TFIIF, TFIIE, and RNA polymerase II to enter the
transcription initiation complex (Choy and Green, 1993). Activation
domains were further proposed to stabilize conformation-specific
TBP TFIIB
DNA complexes, thus reducing the amount of
non-productive initiation complexes (reviewed by Hahn (1993)). Finally,
activation domains were found to exert their effect in enhancing the
rate of transcription elongation.
The cell-free reconstitution of
transcription activation with the TA domain of p65 revealed
a requirement for cofactors in addition to general transcription
factors and TAFs. The activity of Gal4-TA
was not
stimulated by the coactivator fractions PC2, PC3/Dr2, and p15/PC4 above
levels seen with Gal4(1-147) alone. It has been shown previously
that the DNA-binding portion of Gal4 (amino acids 1-147),
although almost inactive in intact cells, exerts transcriptional
activity in in vitro assays (Lin et al., 1988; Ge and
Roeder, 1994; Kretzschmar et al., 1994a), while effects by the
acidic activation domains of VP16 were moderate. This discrepancy may
be caused by the absence of a bona fide chromatin structure in the test
tube (and, e.g. the failure of Gal4(1-147) to mediate
antirepression). Alternatively or in addition it may indicate the
absence of additional functional cofactors in the in
vitro-reconstituted transcription systems. Specific stimulation of
transcription by the Gal4-TA
above levels obtained with
Gal4(1-147) was seen only in the presence of the PC1 coactivator
fraction. Moreover, at least one additional cofactor fraction has been
identified in fractionated HeLa nuclear extracts, which also
specifically supported transcriptional activation through the acidic
activation domains of both VP16 and NF-
B. (
)However, it is presently not clear by which
mechanism PC1 supports p65-dependent activation of transcription. It
might be possible that PC1 works in a manner similar to that of PC2,
which was proposed to act by increasing the activity of the
preinitiation complex in a TAF-dependent process (Kretzschmar et
al., 1994a). Coactivators were also found to act as
``bridging factors,'' such as the recently cloned p15/PC4,
which binds to TFIIA as well as to VP16 (Kretzschmar et al.,
1994b; Ge and Roeder, 1994). Here we have presented evidence that PC1
preferentially activates through the acidic activation domain of p65,
while a second acidic activation domain, the AH peptide, is more
efficient with PC2. The distinct responsiveness of these two activation
domains to coactivators may add a further level of regulation to the
transcription process. Future studies are necessary to study the
interplay of activators, coactivators, and general transcription
factors to elucidate the mechanism by which acidic activation domains
influence the activity of the basal transcription machinery.