From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215
Received for publication, January 11, 2001, and in revised form, April 18, 2001
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ABSTRACT |
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c-Jun is an oncoprotein that activates
transcription of many genes involved in cell growth and proliferation.
We studied the mechanism of transcriptional activation by human c-Jun
in a human RNA polymerase II transcription system composed of highly
purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein
(TBP)-associated factor (TAF) subunits of transcription factor
IID (TFIID). Protein-protein interaction assays revealed that
c-Jun binds with high specificity to the largest subunit of human
TFIID, TAFII250. The region of TAFII250
bound by c-Jun lies in the N-terminal 163 amino acids. This same region
of TAFII250 binds to TBP and represses its interaction with
TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the
TAFII250 N terminus on TFIID-driven transcription. In
support of this hypothesis, we found that c-Jun increased levels of
TFIID-driven transcription in vitro when added at high
concentrations to a DNA template lacking activator protein 1 (AP-1)
sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused
by the N terminus of TAFII250. In addition to revealing a
mechanism by which c-Jun activates transcription, our studies provide
the first evidence that an activator can bind directly to the N
terminus of TAFII250 to derepress RNA polymerase II
transcription in vitro.
Regulation of mRNA synthesis in eukaryotes is a complex
process involving gene specific activators, coactivators, and general transcription factors. Understanding how this multitude of factors sets
levels of transcription of individual genes is the goal of much
research. Emerging evidence indicates that transcriptional activators
have the potential to stimulate transcription via many different
mechanisms. For example, activators can exert their effects by binding
components of the transcription machinery, including subunits of the
TFIID1 and mediator
complexes (for review, see Refs. 1 and 2) as well as by recruiting
chromatin modifying complexes (for review, see Ref. 3).
Whereas unique combinations of activators and coactivators function at
different promoters, RNA polymerase II and the general transcription
factors (TFIIA, -B, -D, -E, -F, and -H) are thought to function at most
mRNA-encoding genes (for review, see Ref. 4). The TFIID complex
plays a central role in core promoter recognition and the assembly of
preinitiation complexes regardless of the manner by which the remaining
basal transcriptional components are recruited, either via a stepwise
pathway or as a holoenzyme (5). TFIID is composed of the TATA-binding
protein (TBP) and multiple TBP-associated factors (TAFs) whose sizes
range from 18 to 250 kDa (for review, see Ref. 6). Subunits of TFIID
contact core promoter elements including the TATA box, initiator, and downstream promoter element (7-10).
At some promoters, the recruitment of TFIID limits the overall level of
transcription in vitro. Efficient recruitment of TFIID can
be achieved by upstream activators that interact with TFIID components
(11-13). In addition, activators can elicit a conformational change in
the TFIID-TFIIA-promoter complex, as observed by DNase I footprinting
(14, 15). Although direct interactions between TBP and activators have
been noted, the primary functional targets of activators within TFIID
are the TAF subunits. TAFs were coined coactivators in recognition of
their requirement for activated transcription in some in
vitro systems (16, 17). Recently, the ability of TAFs to adopt
multifunctional roles as components of distinct complexes including
TFIID, Spt-Ada-Gen5-acetyltransferase (SAGA), p300/CBP-associated
factor (PCAF), and TBP-free TAFII complex (TFTC) has
been demonstrated (for review, see Ref. 1). The validity of the
coactivator function of TAFs has been firmly established from examples
in yeast, Drosophila, and human systems.
Although TAFs can function as coactivators, regulated transcription of
some promoters is not dependent on TAFs in vitro and in vivo (18-22). The mediator and other complexes are also
targets of transcriptional activators and serve to coactivate
transcription (for review, see Ref. 2). In the context of chromatin,
nucleosome-remodeling complexes and histone acetyltransferases such as
cAMP-response element-binding protein (CREB)-binding protein (CBP)
function to coactivate transcription (3). It is likely that activators can stimulate transcription by many different mechanisms depending upon
the conditions and the context of the promoter at which the activator binds.
One mechanism by which activators can function is by relieving the
effects of factors that repress basal transcription. Although this is
certainly true of activators in the context of chromatin, mechanisms of
transcriptional repression and derepression have also been observed in
the absence of chromatin. For example, the binding of TFIID to core
promoters is internally repressed by the largest subunit of TFIID,
TAFII250 (23). Most of the work on the mechanism of this
repression has been carried out with dTAFII230 and
yTAFII145, the Drosophila and yeast homologs of human TAFII250. Repression is mediated by a small domain
(~150 amino acids) in the N termini of these proteins that binds
directly to TBP and inhibits its ability to bind TATA boxes (23, 24). Furthermore, the N-terminal domain of dTAFII230 can repress
TBP-driven transcription when added in excess of TBP in an in
vitro transcription system (23). Mutational analysis identified
the concave DNA binding surface of TBP as a binding site of the N
terminus of dTAFII230 (25). In NMR studies, amino acids
11-77 of dTAFII230 appeared to mimic DNA by binding to the
DNA binding cleft of TBP (26). A second region of dTAFII230
(amino acids 118-143) interacts with the convex surface of TBP and
further stabilizes the dTAFII230-TBP interaction (27). The
importance of TAFII250 repression to transcriptional regulation in cells and the potential for targeting the N terminus of
TAFII250 as a means to derepress transcription are
currently under study. TFIIA, which aids TFIID in binding core
promoters, can prevent TAFII250 from inhibiting TBP-DNA
binding in vitro (28). In addition, yTAFII145
and yTFIIA bind competitively to TBP (24). The acidic activation domain
of VP16 also binds the underside of TBP and can block the interaction
of dTAFII230 with TBP (25, 29). Activators have not,
however, been shown to bind directly to the N terminus of
TAFII250 to remove its repressive effects on TBP-DNA binding.
AP-1 transcription factors of the Jun and Fos families activate many
genes including those involved in cell growth and proliferation (for
review, see Ref. 30). AP-1 proteins each contain an extended Here we have investigated the mechanism by which human c-Jun activates
transcription using a highly purified human RNA polymerase II
transcription system. Activation of transcription by recombinant c-Jun
was found to be dependent on TAFs. Interactions between c-Jun and TAFs
were identified and characterized using protein-protein affinity
chromatography, which revealed that c-Jun binds to the N terminus of
human TAFII250. The function of c-Jun in derepressing transcription driven by TFIID and the ability of c-Jun to block TAFII250 repression of TBP transcription and DNA binding
were characterized in vitro. Our results reveal a unique
interaction between c-Jun and TAFII250 that results in
derepression of TFIID-driven transcription in vitro. These
studies lead to a novel model for activation in which c-Jun interacts
with TAFII250 to relieve repression of TFIID-DNA binding,
thereby contributing to transcriptional activation.
Plasmids--
pET-Jun, a plasmid for expressing full-length
human c-Jun, was a gift from T. Hoey (Tularik, Inc.). In creating this
plasmid an NcoI site was generated that changed the fourth
base pair in the c-Jun-coding region from AT to GC, resulting in a
point mutation in the second amino acid of c-Jun (Thr to Ala). pGEX-Jun
was created by digesting pET-Jun with NcoI and
EcoRV and subcloning the c-Jun cDNA into the
NcoI and StuI sites of pGEX-2TKN (a kind gift of S. Ruppert and R. Tjian).
Plasmids for expressing TAFs (pT
Plasmid p(AP-1)5-E1b-CAT was created by inserting five
direct copies of a double-stranded oligonucleotide containing the AP-1 element from the human metallothionein IIA promoter ( Expression and Purification of Recombinant c-Jun and GST Fusion
Proteins--
Recombinant full-length human c-Jun was expressed in
Escherichia coli BL21:DE3. Cultures (500 ml) were grown in
LB containing 0.3 mM ampicillin at 37 °C until the
A600 nm was 0.4. Expression was induced
with 0.5 mM
isopropyl-1-thio-
GST-Jun was expressed in E. coli strain XA90. Cultures were
grown in LB containing 0.3 mM ampicillin at 37 °C until
the A600 nm was 0.4. Expression was induced with 0.5 mM
isopropyl-1-thio-
GST-TAFII250-(1-163) and GST were expressed in E. coli strain XA90 as described for GST-Jun above. Harvested cells
were resuspended in 1/50 culture volume of lysis buffer containing 20 mM Tris (pH 7.9), 20% glycerol, 1 mM EDTA, 0.1 M NaCl, 1 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride. Samples were sonicated 4 times for
15 s and centrifuged for 30 min in a Sorval SS34 rotor at 18,000 rpm and 4 °C. Supernatants from the centrifugation were incubated
with glutathione-Sepharose at 4 °C for 1 h with constant nutation. Resin was washed two times with 10 column volumes of buffer B
containing 1 M NaCl and 0.1% Nonidet P-40. For use in protein-protein interaction assays, the resin was washed three times
with ten column volumes of the appropriate protein-protein interaction
assay buffer (see below and figure legends). For elution, resin
was washed five times with 10 column volumes of buffer C (100 mM Tris (pH 7.9), 120 mM NaCl). Protein was
eluted by incubating resin at 4 °C for 10 min in 1 column volume of
buffer C containing 20 mM glutathione (reduced) with
constant nutation. Eluates were dialyzed overnight at 4 °C in a
buffer containing 20 mM Tris (pH 7.9), 10% glycerol, 1 mM EDTA, 0.1 M NaCl, and 1 mM DTT.
Samples were separated into aliquots and stored at Protein-Protein Interaction Assays--
Each reaction contained
1 µg of GST-Jun or GST immobilized on 10 µl of
glutathione-Sepharose beads (Amersham Pharmacia Biotech). Immobilized
proteins and extracts containing target proteins were incubated with
micrococcal nuclease at 30 °C for 10 min. Target proteins were then
incubated with GST or GST-Jun beads at 4 °C for 2 h with
constant nutation in TGEMD buffer (20 mM Tris (pH 7.9),
10% glycerol, 1 mM EDTA, 5 mM
MgCl2, and 1 mM DTT) containing 0.1% Nonidet
P-40, 0.2 mM phenylmethylsulfonyl fluoride, and 0.1 M NaCl. After incubation, beads were washed at least 3 times with 10-15 bead volumes of buffer. Protein was eluted from the
beads in SDS sample buffer and resolved by SDS-PAGE.
In Vitro Transcription--
General transcription factors (TBP,
TFII-B, -E, -F, and -H) and RNA polymerase II were expressed and
purified as previously described (40). Human TFIID was immunopurified
(39). Recombinant human TFIIA was prepared as previously described (41,
42). The amounts of general factors and buffer conditions used for transcription were as previously described (39). The amounts of the
TFIID (10 ng) and TBP (0.5 ng) used in transcription reactions contained equal moles of TBP, as determined by quantitative Western analysis with polyclonal antibody directed against TBP (39).
To study transcriptional activation (see Fig. 1B) c-Jun was
incubated with p(AP-1)5-E1b-G-less for 5 min on ice. After
the addition of general transcription factors, reactions (20 µl) were transferred to 30 °C for 25 min before adding nucleoside
triphosphates. RNA synthesis was allowed to proceed for 20 min at
30 °C. To study derepression (see Fig. 4), c-Jun was
incubated with TBP or TFIID in 13-µl reactions for 10 min on ice.
p(GAL4)5-AdMLP-G-less template DNA and the remaining
general transcription factors where then added to reactions in a final
volume of 20 µl.
To study transcriptional repression (see Fig. 5A)
GST-TAFII250-(1-163) and GST were added to transcription
reactions containing p(GAL4)5-AdMLP-G-less template DNA
before the addition of TBP, TFIIB, TFIIF, and RNA polymerase II. In
Fig. 5B, c-Jun was incubated with
GST-TAFII250-(1-163) (lanes 3 and 4)
in 3 µl for 13 min on ice. p(GAL4)5-AdMLP-G-less template
DNA was then added, followed immediately by the addition of the general
transcription factors to a final volume of 20 µl.
DNase I Footprinting--
DNase I footprinting reactions were
performed with a 183-base pair DNA fragment containing the AdMLP ( Activation of Transcription by Human c-Jun in a Highly Purified in
Vitro System Requires TAFs--
To study the mechanism of c-Jun
transcriptional activation, we developed a human RNA polymerase II
transcription system reconstituted from highly purified recombinant and
native general transcription factors. This system is useful for
studying the roles of individual subunits of the general RNA polymerase
II transcription machinery, including the TFIID TAFs, in basal and
activated transcription. Recombinant human c-Jun was expressed in
E. coli and purified to near homogeneity (Fig.
1A). To test the activity of
c-Jun for activating transcription in vitro we created a DNA
template, p(AP-1)5-E1b-G-less, containing five AP-1
elements upstream of the adenovirus E1b TATA box and a 377-base pair
G-less cassette. We detected basal transcription from this template
in vitro (Fig. 1B) in reactions reconstituted with equal molar concentrations of either immunopurified native TFIID
(lane 1) or recombinant TBP (lane 3).
Interestingly, reactions reconstituted with TBP reproducibly showed
higher levels of basal transcription than those containing TFIID even
though equivalent molar concentrations of TBP and TFIID were used (39).
This is likely to be due to activities present in the TFIID complex
that inhibit its binding to DNA, such as the N terminus of
TAFII250 (23). c-Jun (30 ng) activated transcription
~10-fold in reactions reconstituted with TFIID (Fig. 1B,
compare lanes 1 and 2) but did not stimulate
transcription in reactions reconstituted with TBP (Fig.
1B, compare lanes 3 and 4). Thus, in a
highly purified in vitro transcription system, TAFs are
required for transcriptional activation by human c-Jun.
c-Jun Binds TAFII250 with High Specificity--
The
finding that TAFs were required to mediate transcriptional activation
by c-Jun in our in vitro transcription system led us to
hypothesize that c-Jun interacted with one or more of the TAFs. To
identify interactions between c-Jun and TAFs, we performed protein-protein affinity chromatography utilizing immobilized GST-Jun
and in vitro translated 35S-labeled TAFs. TAFs
were incubated with GST-Jun or control GST resin, and after extensive
washing, proteins bound to the resins were analyzed by SDS-PAGE and
autoradiography. These studies revealed that TAFII250
interacted with immobilized GST-Jun but not control GST (Fig.
2A, lanes 1-3).
Other TAFs, for example dTAFII40, dTAFII30
To further characterize the affinity and specificity of the
c-Jun-TAFII250 interaction, we utilized extracts of insect
cells containing HA-tagged TAFII250 that was expressed from
a recombinant baculovirus. Insect cell extracts were incubated with
immobilized GST-Jun and control GST. After extensive washing, bound
protein was resolved by SDS-PAGE and visualized by staining with
Coomassie Brilliant Blue. As shown in Fig. 2B, the GST-Jun
resin specifically purified recombinant HA-TAFII250 from
the crude insect cell extract. The numerous and abundant insect cell
proteins in the extract were not retained on the GST-Jun resin.
c-Jun Binds the N-terminal Region of TAFII250 That Is
Involved in Transcriptional Repression--
To gain insight into the
role of the c-Jun-TAFII250 interaction in transcriptional
activation, we delimited the region of TAFII250 that is
bound by c-Jun. Two C-terminal deletions of TAFII250 were
expressed by in vitro translation and tested for interaction with immobilized GST-Jun: TAFII250-(1-560) and
TAFII250-(1-163). As shown in Fig.
3A,
TAFII250-(1-560) and TAFII250-(1-163) each bound GST-Jun at a level comparable with that observed for full-length TAFII250 (amino acids 1-1893).
As an additional test of the ability of the N-terminal 163 amino acids
of TAFII250 to bind c-Jun, we expressed and purified a
GST-TAFII250-(1-163) fusion protein and incubated the
immobilized GST-TAFII250-(1-163) with purified c-Jun. The
silver-stained SDS gel in Fig. 3B shows
GST-TAFII250-(1-163) bound c-Jun (lane 5). c-Jun was not retained on control GST resin (lane 4). Thus,
purified c-Jun can bind directly to
GST-TAFII250-(1-163).
These results revealed that the region of TAFII250 bound by
c-Jun is the N-terminal domain, which is known to inhibit TBP-DNA binding and transcription. Based on this finding, we hypothesized that
c-Jun binds the N terminus of TAFII250 and inhibits its
repression of TBP-DNA binding, thereby contributing to transcriptional activation.
c-Jun Derepresses Basal Transcription Directed by TFIID--
It is
possible that the lower level of basal transcription observed with
TFIID compared with recombinant TBP (see Fig. 1) was due to the
N-terminal region of TAFII250 interacting with the DNA
binding surface of TBP to repress the binding of TBP to TATA DNA. In
this case, c-Jun may bind directly to the N terminus of
TAFII250 in TFIID to derepress transcription. To test this we used a DNA plasmid that lacks known AP-1 sites,
p(GAL4)5-AdMLP-G-less, which contains the adenovirus major
late core promoter ( c-Jun Overcomes Repression of TBP-DNA Binding and Transcription
Caused by the N Terminus of TAFII250--
To demonstrate
that the N terminus of TAFII250 can repress in
vitro transcription and to directly test whether c-Jun can inhibit TAFII250-mediated transcriptional repression, we added
purified GST-TAFII250-(1-163) to transcription reactions.
The ability of TAFII250-(1-163) to repress basal
transcription was determined by comparing the effects of
GST-TAFII250-(1-163) and GST on basal transcription in a
minimal in vitro transcription system consisting of TBP,
TFIIB, TFIIF, RNA polymerase II, and negatively supercoiled p(GAL4)5-AdMLP-G-less template DNA. As shown in Fig.
5A, the addition of
GST-TAFII250-(1-163) repressed basal transcription by more than 95%, whereas GST did not affect transcription when titrated over
the same concentration range. Thus, the N terminus of
TAFII250 can repress basal transcription in a highly
purified minimal transcription system.
We next tested the ability of c-Jun to block transcriptional repression
caused by GST-TAFII250-(1-163). c-Jun was preincubated with GST-TAFII250-(1-163) before adding the
p(GAL4)5-AdMLP-G-less template (lacking known AP-1 sites)
and the remaining transcription factors. c-Jun overcame the repression
of basal transcription observed with GST-TAFII250-(1-163)
(Fig. 5B, compare lanes 3 and 4 with
lane 2). These data demonstrate that c-Jun can bind the N
terminus of TAFII250 to block its ability to repress basal transcription.
It was possible that the transcriptional effects presented above
resulted from c-Jun binding the N terminus of TAFII250 to inhibit its repression of TATA box binding by TBP. To test this, we
performed DNase I footprinting with TBP using a DNA fragment containing
the adenovirus major late core promoter but lacking AP-1 sites.
GST-TAFII250-(1-163) and c-Jun were added to determine their effects on TBP-DNA binding. Fig. 5C shows plots of
relative intensities (y axis) versus position on
the gel (x axis) for the region spanning the TATA box (left
to right represents top to bottom on the gel). TBP alone protected
bands in the TATA box region from DNase I digestion by greater than
90%. GST-TAFII250-(1-163) almost completely repressed
TBP-DNA binding. In the presence of GST-TAFII250-(1-163),
the intensities of bands in the TATA box region were decreased by less
than 10% from the control reaction performed in the absence of
transcription factors. Incubation of c-Jun with
GST-TAFII250-(1-163) blocked the repression of TBP binding, resulting in ~70% of the maximal protection in the TATA box
region that was observed with TBP alone. Since the footprinting experiments were performed with recombinant TBP,
GST-TAFII250-(1-163), and c-Jun, we can rule out a
requirement for other general transcription factors in repression by
GST-TAFII250-(1-163) and inhibition of this repression by
c-Jun. Taken together, the transcription and DNase I footprinting
experiments strongly support a model in which c-Jun binding to the N
terminus of TAFII250 inhibits repression of TBP-DNA binding
and transcription initiation.
Here we have elucidated one mechanism by which c-Jun activates
transcription. c-Jun binds the N terminus of TAFII250 to
derepress RNA polymerase II transcription. Not only did c-Jun derepress TFIID-directed transcription in vitro, but c-Jun was also
capable of blocking repression of TBP-DNA binding by the N terminus of TAFII250. Our results are the first to characterize a
mechanism by which c-Jun activates RNA polymerase II transcription in a cell-free system. In addition, we provide the first evidence that activators can directly bind the N terminus of TAFII250 to
derepress transcription in vitro.
Derepression is emerging as a general mechanism for controlling levels
of transcription in eukaryotic cells. This is not surprising since
derepression is a prevalent mechanism for controlling gene expression
in prokaryotes (43). The most widespread example of derepression in
eukaryotes is thought to be the alteration of chromatin structure by
histone acetyltransferases and ATP-dependent chromatin
remodeling factors (3). In small nuclear RNA transcription the
snRNA activating protein complex (SNAPc) has a built-in DNA binding damper that is deactivated by the transcriptional activator Oct-1 (44). Another general mechanism of transcriptional repression in
eukaryotic cells results from the interaction of the N terminus of the
largest TFIID subunit to the concave surface of TBP to prevent binding
of TFIID to TATA boxes (23, 25). The finding that c-Jun binds the N
terminus of TAFII250 to derepress TFIID-driven transcription in vitro further supports the generality of
derepression as a means of regulating eukaryotic transcription. We are
left wondering whether other transcriptional activators also bind the N
terminus of TAFII250 to derepress transcription. We note
that significant stimulation of transcription from the
p(GAL4)5-AdMLP-G-less template (which lacks known AP-1
sites) required 400 ng or more of c-Jun. These amounts are far greater
than the amounts of transcriptional activators typically used in
in vitro transcription experiments. For example, we observe
significant activation from p(AP-1)5-E1b-G-less with 15 ng
of c-Jun. With this low amount of c-Jun we do not observe activation
from any template that does not contain known AP-1 sites. Moreover, in
early c-Jun activation experiments, we did not titrate c-Jun to high
enough levels to observe AP-1 site-independent derepression. Perhaps
other transcriptional activators that bind the N terminus of
TAFII250 to derepress transcription will be revealed
through a combination of protein-protein interaction assays (with
activators and the N terminus of TAFII250) and in vitro transcription experiments aimed at testing for
site-independent transcriptional stimulation in a
TFIID-dependent transcription system.
Other models for derepression of TFIID binding have been studied. For
example, it has been shown that VP16 and dTAFII230
competitively bind the concave surface of TBP (25). A point mutation in
TBP (L114K) that disrupted the TBP-VP16 interaction prevented VP16 activation in vivo. The same point mutation in TBP also
disrupted interaction with dTAFII230. In addition, when the
N terminus of yTAFII145 was replaced with an acidic
activation domain, the fusion protein autoinhibited TFIID in yeast
(29). A two-step "hand-off" model has been proposed to explain the
function of acidic activation domains in derepressing DNA binding by
TFIID (25, 29). In the first step, the acidic activation domain
replaces the N terminus of dTAFII230/yTAFII145
by binding the underside of TBP. In the second step, TBP binds to the
TATA box when the acidic activation domain releases its hold on TBP.
This proposed mechanism of relieving the internal repression of TFIID
binding is quite different from the mechanism of derepression by c-Jun
that we have characterized here; however, both modes of derepression
serve to increase TFIID-driven transcription.
Since TFIIA has been shown to stimulate TFIID binding to the TATA box
and to compete with the largest subunit of TFIID for binding TBP (24,
28), it is possible that transcriptional activators could indirectly
derepress TAFII250 inhibition of TATA box binding by
helping TFIIA bind TBP. Although TFIIA may play a role in c-Jun
derepression, it is not required under all conditions. In our highly
purified in vitro transcription system we found that c-Jun
blocked the repression of transcription by
GST-TAFII250-(1-163) in the absence of TFIIA. Furthermore,
using DNase I footprinting we found that c-Jun blocked the ability of
GST-TAFII250-(1-163) to repress TATA box binding by TBP.
Thus, TFIIA and other general transcription factors are not required
for the function of c-Jun in derepressing TBP binding in
vitro.
TBP has also been shown to dimerize in vitro and in
vivo either alone or when part of TFIID (45, 46). Dimers form
through interactions between the concave surfaces of two molecules of TBP, resulting in a complex that is not capable of binding DNA. The
coordination of TBP dimerization and the N terminus of
TAFII250 interacting with TBP is not understood. Perhaps
these two distinct mechanisms of repressing TFIID-DNA binding function
together to ensure that the repressed state is maintained in the
absence of interaction with TFIIA and transcriptional activators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical dimerization/DNA binding domain of ~62 amino acids that consists of a leucine zipper found C-terminal of a basic region (31).
Dimers bind DNA through sequence-specific contacts between amino acids
in the basic regions and the major groove of the DNA helix (32).
Members of the Jun family can form homo- and heterodimers. Members of
the Fos family cannot form homo- or heterodimers among themselves but
can heterodimerize with Jun proteins. Although there is high sequence
homology in the basic leucine zipper domains of AP-1 proteins, there is
much less homology in other regions, which contain activation domains.
The activation domains of c-Jun and c-Fos, which are essential for
transcriptional activation in vitro, have been characterized
functionally (33-35) but not structurally and are known to bind other
proteins such as the coactivator CBP (36). Despite a wealth of studies,
the mechanisms by which c-Jun and c-Fos stimulate transcription are not
well understood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hTAF250, pT
-dTAF40,
pT
-dTAF30
, and pT
-dTAF30
) were gifts from R. Tjian.
HA-hTAFII250 was expressed in SF9 cells as previously
described (37). pT
-hTAF250-(1-560) and
pT
-hTAF250-(1-163) were created by digestion of
pT
-hTAF250 with EcoRI and SmaI, respectively,
followed by gel purification of the linearized plasmid and ligation.
pGEX-hTAF250-(1-163) was created by digestion of
pT
-hTAF250-(1-163) with NdeI and EcoRI and
insertion of the DNA fragment into pGEX-2TKN.
105 to
96) into the XbaI site of plasmid E1b-CAT (38). Plasmid
p(AP-1)5-E1b-G-less, used as template DNA in the in
vitro transcription assays, was created by inserting a 377-base
pair G-less cassette (excised from p(GAL4)5-E1b-G-less (a
kind gift from M. G. Peterson and R. Tjian) with SacI)
into the SacI site of p(AP-1)5-E1b-CAT. Plasmid p(GAL4)5-AdMLP-G-less (39), which contains five GAL4 sites
upstream of the adenovirus major late core promoter (AdMLP;
53 to
+10) and a 380-base pair G-less cassette, was generated by subcloning a
HindIII-EcoRI fragment isolated from plasmid
G5-MLP-G-less (a kind gift from Mike Carey) into the HindIII
and EcoRI site of pBluescript-KS(+).
-D-galactopyranoside for 2 h at
37 °C. Cells were harvested and resuspended in 10 ml of a solution
containing 20 mM Tris (pH 7.9), 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.2 mM
phenylmethylsulfonyl fluoride, and sonicated 4 times for 15 s.
Samples were centrifuged for 30 min in a Sorval SS34 rotor at 18,000 rpm and 4 °C. Precipitated material (inclusion bodies) containing
c-Jun was resuspended in 10 ml of 5 mM DTT and sonicated 2 times for 30 s. Samples were centrifuged for 10 min in a Sorval
SS34 rotor at 15,000 rpm and 4 °C. Insoluble material was washed
three more times by resuspending in 10 ml of 5 mM DTT, followed by centrifugation. Pellets from the final wash were
resuspended in 5 ml of buffer A (20 mM Tris (pH 7.9), 1 mM EDTA, 5 mM DTT, and 7 M urea)
containing 0.1 M NaCl. Soluble protein was loaded on an
8-ml column packed with sulfopropyl-Sepharose fast flow (Amersham Pharmacia Biotech) and washed with three column volumes of
buffer A containing 0.1 M NaCl followed by three column
volumes of buffer A containing 0.2 M NaCl. c-Jun was then
eluted with buffer A containing 0.3 M NaCl and subjected to
three sequential dialyses in buffer B (20 mM Tris (pH 7.9),
0.1 mM EDTA, 10% glycerol, 5 mM DTT)
containing the following additions: 1) 1 M urea and 1 M NaCl; 2) 1 M NaCl; 3) 0.1 M
NaCl. After dialysis, the purified c-Jun was separated into aliquots
and stored at
80 °C.
-D-galactopyranoside for 3 h
at 37 °C. Cells were harvested and resuspended in a solution containing 20 mM Tris (pH 7.9), 20% glycerol, 1 mM EDTA, 5 mM MgCl2, 0.1 M NaCl, 5 mM DTT, 0.2 mM
phenylmethylsulfonyl fluoride and sonicated 4 times for 15 s.
Insoluble material was washed with 5 mM DTT as described
for c-Jun above. Pellets from the final wash were resuspended in a
solution containing 20 mM Tris (pH 7.9), 1 mM
EDTA, 5 mM DTT, and 6 M guanidine-HCl, and
protein was extracted by overnight nutation at 4 °C. After
centrifugation to remove the remaining insoluble material, extracts
were diluted to give final concentrations of 200 ng/µl of GST-Jun.
Samples were subjected to three sequential dialyses in buffer B
containing 0.1 M NaCl and guanidine-HCl at 2, 1, and 0.5 M. The guanidine-HCl was then slowly lowered to 50 mM by pumping (over 10 h) buffer B containing 0.1 M NaCl into the third dialysis listed above. After
dialysis, samples containing GST-Jun were separated into aliquots and
stored at
80 °C. Just before use in protein-protein interaction
assays, GST-Jun was further purified by incubation with
glutathione-Sepharose for 1 h at 4 °C followed by two washes with 10 column volumes of buffer B containing 1 M NaCl and
0.1% Nonidet P-40 and three washes with the appropriate
protein-protein interaction assay buffer (see below and figure
legends). The affinity resin contained ~1 mg of protein/ml of beads.
80 °C.
53
to +33) 32P-labeled on the 5'-end of the nontemplate
strand. The buffer conditions were identical to those used for
transcription (before the addition of NTPs). When present,
GST-TAFII250-(1-163) and c-Jun were incubated together in
a reaction volume of 2 µl for 10 min on ice. These mixtures were then
added to reactions containing promoter DNA followed immediately by the
addition of TBP at a final concentration of 6 nM in a final
volume of 20 µl. After 10 min at 30 °C, 1 µl of a solution
containing 0.15 units/µl of DNase I (Promega) and 10 mM
CaCl2 was added to each reaction. Reactions were stopped,
and DNA products were analyzed as described (39).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
The TAF subunits of TFIID are required for
activation of transcription by c-Jun in a RNA polymerase II
transcription system reconstituted from highly purified transcription
factors. A, purified recombinant human c-Jun (2 µg)
was subjected to SDS-PAGE and visualized by staining with Coomassie
Brilliant Blue. B, c-Jun activates transcription in a highly
purified in vitro transcription system containing TFIID but
not TBP. Reactions in lanes 1 and 2 contained 10 ng of immunopurified TFIID. Reactions in lanes 3 and
4 contained 0.5 ng of recombinant TBP. 30 ng of recombinant
c-Jun was added to the reactions in lanes 2 and
4. The DNA template was p(AP-1)5-E1b-G-less,
which contained five AP-1 sites upstream of the adenovirus E1b TATA box
and a 377-base pair G-less cassette.
, and dTAFII30
, did not interact with GST-Jun (Fig.
2A, lanes 4-12).
View larger version (36K):
[in a new window]
Fig. 2.
c-Jun binds TAFII250 with high
specificity. A, protein-protein affinity chromatography was
used to identify TAFs that interact with GST-Jun.
35S-Labeled human TAFII250 (lanes
1-3), Drosophila TAFII40 (lanes
4-6), Drosophila TAFII30 (lanes
7-9), and Drosophila TAFII30
(lanes 10-12) were expressed by in vitro
translation and tested for interaction with immobilized GST-Jun or GST.
10% of the input 35S-labeled proteins were included in
lanes 1, 4, 7, and 10. B, recombinant human TAFII250 was specifically
purified from cell lysates using GST-Jun affinity resin.
HA-TAFII250 was expressed in insect cells using a
recombinant baculovirus. Portions of cell lysate containing
TAFII250 were incubated with GST-Jun and control GST
resins. After extensive washing, protein bound to the affinity resins
was resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. An
aliquot of insect cell lysate equal to 100% of the input was included
in lane 1.
View larger version (31K):
[in a new window]
Fig. 3.
c-Jun binds the N-terminal 163 amino acids of
TAFII250. A, 35S-Labeled
full-length TAFII250 and two C-terminal deletions were
expressed by in vitro translation and tested for interaction
with immobilized GST-Jun and GST. Bound protein was analyzed by
SDS-PAGE and autoradiography. B, immobilized
GST-TAFII250-(1-163) binds purified c-Jun. Immobilized
GST-TAFII250-(1-163) and control GST were incubated with
c-Jun. Bound protein was analyzed by SDS-PAGE and silver staining
(lanes 4 and 5). Purified GST and
GST-TAFII250-(1-163) are shown in lanes 2 and
3, respectively.
53 to +10) upstream of a 380-base pair G-less
cassette. TFIID and TBP were incubated separately with increasing
concentrations of c-Jun and then added to transcription reactions with
p(GAL4)5-AdMLP-G-less. As shown in Fig.
4, lanes 7-10, c-Jun
stimulated transcription when preincubated with TFIID. The level of
transcription with TFIID approached that observed with TBP at the
highest concentrations of c-Jun. c-Jun did not increase the level of
transcription when preincubated with TBP (Fig. 4, lanes
2-5).
View larger version (14K):
[in a new window]
Fig. 4.
c-Jun derepresses TFIID-directed
transcription from a template that lacks AP-1 sites. Recombinant
c-Jun was incubated with TBP (lanes 2-5) or TFIID
(lanes 7-10) before adding
p(GAL4)5-AdMLP-G-less (consisting of the adenovirus major
late core promoter upstream of a 380-base pair G-less cassette) and the
remaining general transcription factors. The amounts of c-Jun added to
the transcription reactions were 30 ng (lanes 2 and
7), 200 ng (lanes 3 and 8), 400 ng
(lanes 4 and 9), and 700 ng (lanes 5 and 10).
View larger version (24K):
[in a new window]
Fig. 5.
c-Jun blocks the repression of basal
transcription and TBP-DNA binding caused by the N terminus of
TAFII250. A,
GST-TAFII250-(1-163) specifically represses basal
transcription in vitro. GST-TAFII250-(1-163)
and control GST were added to transcription reactions before the
addition of the transcription factors TBP, TFIIB, TFIIF, and RNA
polymerase II. The final concentrations of GST (lanes 2-4)
and GST-TAFII250-(1-163) (lanes 6-8) in
reactions before the addition of nucleoside triphosphates were 10 nM (lanes 2 and 6), 50 nM
(lanes 3 and 7), and 100 nM
(lanes 4 and 8). B, c-Jun blocks
transcriptional repression by GST-TAFII250-(1-163).
GST-TAFII250-(1-163) was incubated alone (lane
2) or with c-Jun (lanes 3 and 4) before
being added to reactions containing p(GAL4)5-AdMLP-G-less,
followed immediately by the addition of the general transcription factors.
The amounts of c-Jun added to transcription reactions were 100 ng
(lane 3) and 200 ng (lanes 4 and 5).
C, c-Jun blocks the ability of
GST-TAFII250-(1-163) to repress DNA binding by TBP.
GST-TAFII250-(1-163) (18 ng) and c-Jun (530 ng) were
incubated together before adding TBP and AdMLP promoter DNA. After
treatment with DNase I, samples were subjected to denaturing PAGE, and
the results were analyzed and quantitated by phosphor-imaging. Plots
are shown of relative intensities of bands (y axis)
versus position on gel (x axis) for four
different reactions: no proteins, TBP alone, TBP with
GST-TAFII250-(1-163), and c-Jun with TBP and
GST-TAFII250-(1-163). Left to right in the plots
represents top to bottom of the gel. The TATA box region is indicated
with a bracket.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Natalie Ahn, Lin Chen, Jennifer Kugel, and Irene Ota for helpful discussions and comments on the manuscript. J. A. G. is grateful to Robert Tjian for generous support, especially during the early stages of this work.
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FOOTNOTES |
---|
* This research was supported by National Institutes of Health Public Health Service Grant GM-55235, a Leukemia Society of America Special Fellowship, and a Scholarship from the Pew Charitable Trusts (to J. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by a National Institutes of Health Predoctoral
Training Grant T32 GM07135 and a Graduate Student Fellowship from the
Colorado Institute for Research in Biotechnology, which is state-funded
by the Colorado Advanced Technology Institute.
§ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Colorado, Campus Box 215, Boulder, CO 80309-0215. Tel.: 303-492-3273; Fax: 303-492-5894; E-mail: james.goodrich@colorado.edu.
Published, JBC Papers in Press, April 20, 2001, DOI 10.1074/jbc.M100278200
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ABBREVIATIONS |
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The abbreviations used are: TFII-, transcription factor for RNA polymerase II; TBP, TATA-binding protein; TAF, TBP-associated factor; AP-1, activator protein 1; VP16, herpesvirus protein 16; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; AdMLP, adenovirus major late promoter; y-, yeast; d-, Drosophila; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
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