Departments of Internal Medicine and Cell
Biology and Physiology, Washington University School of Medicine,
Saint Louis, Missouri 63110
Received 14 August 1997/Returned for modification 16 October
1997/Accepted 12 February 1998
Recently, it was found that if either the TATA binding protein or
RNA polymerase II holoenzyme is artificially tethered to a promoter,
transcription is activated. This finding provided presumptive evidence
that upstream activating proteins function by recruiting components of
the preinitiation complex (PIC) to the promoter. To date, however,
there have been no studies demonstrating that upstream factors actually
recruit components of the PIC to the promoter in vivo. Therefore, we
have studied the mechanism of action of two disparate transactivating
domains. We present a series of in vivo functional assays that
demonstrate that each of these proteins targets different components of
the PIC for recruitment. We show that, by targeting different
components of the PIC for recruitment, these activating domains can
cooperate with each other to activate transcription synergistically and that, even within one protein, two different activating subdomains can
activate transcription synergistically by cooperating to recruit different components of the PIC. Finally, considering our work together
with previous studies, we propose that certain transcription factors
both recruit components of the PIC and facilitate steps in
transcriptional activation that occur subsequent to recruitment.
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INTRODUCTION |
When examined in vitro,
promoter-specific transactivating factors bind to several proteins in
the preinitiation complex (PIC), and there is much evidence that these
interactions are essential for transactivation to occur (8, 10,
18, 27, 33). However, the immediate functional consequence of the
interaction between transactivating factors and the PIC has yet to be
studied in an in vivo system. Therefore, it has been unclear whether
upstream factors activate transcription in vivo by modifying proteins
that are already assembled in the PIC or whether they do so by
recruiting proteins into the assembling PIC. Furthermore, the specific
steps in the formation of an active PIC that are regulated by upstream activating proteins have not been identified.
Several recent studies have demonstrated that artificial recruitment of
either of two components of the PIC to the promoter in vivo is
sufficient to activate transcription. Specifically, it has been shown
that artificial recruitment of TATA binding protein (TBP) or the RNA
polymerase II holoenzyme activates transcription (1, 5, 7, 13,
32). These findings suggested that promoter-bound factors might
stimulate transcription if they recruit either TBP or the holoenzyme
into an assembling PIC. It has yet to be demonstrated, however, that
promoter-specific transactivating factors actually do recruit
components of the PIC in an in vivo system, and if upstream activators
do indeed recruit components of the PIC, it is not known whether each
has a generalized recruiting function or whether different
transactivating proteins recruit different components of the PIC.
Additionally, the significance of the finding that recruitment of
either TBP or the holoenzyme is sufficient to activate transcription is
unknown: i.e., would recruitment of one of these by an upstream
activator preclude further activation by the other, or would
simultaneous recruitment of TBP and the holoenzyme result in additive
or even synergistic transcriptional activity?
We present a series of functional assays that were designed to address
these issues. The results of each are consistent with several possible
interpretations; however, when the results of all of the assays are
considered together they strongly suggest that in vivo (i) upstream
activators can recruit at least two components of the PIC in a stepwise
manner, one of which is TBP; (ii) disparate activating domains target
different components of the PIC for recruitment; (iii) activating
proteins on the same promoter that recruit different components of the
PIC activate transcription synergistically; and (iv) separate
activating subdomains within one protein that recruit different
components of the PIC activate transcription synergistically. We also
discuss the likelihood that some transcription factors activate
transcription by recruitment and then through further interaction with
certain proteins after they are assembled in the PIC.
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MATERIALS AND METHODS |
Tissue culture and transfections.
C33a cells were grown and
transfected on 60-mm-diameter plates as described elsewhere
(31). For each plate, 0.4 µg of the reporter plasmid and
0.1 µg of each expression vector were transfected, unless otherwise
indicated. Empty vector was used to bring total DNA to 5 µg for each
transfection. Cells were collected 36 h after transfection, and
chloramphenicol acetyltransferase (CAT) activity was determined as
described elsewhere (31). Each experiment was performed a
minimum of three times, and the results of representative experiments
are presented.
Plasmids.
pGal4-VP16
456 (15), pGal4-Sp1
(25), pCGNTBP (30), pCGNTBPAS
(30), and pG2E1B-CAT and pE1B-CAT
(16) have all been described previously. To construct
pGal4-TBP, p6His-T-hTBP (a gift from R. G. Roeder) was digested
with NdeI, blunted, and then digested with BamHI
to obtain the cDNA for hTBP. This was cloned between the
SmaI and BamHI sites of PM1 (23). To
construct pLexA-VP16456, pGal4-VP16456 was digested with
BamHI, blunted, and then digested with EcoRI, and
the resultant fragment was cloned between the SmaI and
EcoRI sites of pBXL1 (a gift from P. Broad). To construct pG2L2E1B-CAT, pL6EC (a gift from P. Broad) was
digested with XhoI to obtain two LexA binding sites. These
were cloned by blunt end ligation into the XbaI site of
pG2E1B-CAT. To construct LexA-TBP, Gal4-TBP was digested
with EcoRI and the resultant fragment was cloned into the
EcoRI site of pBXL1. pG2-CAT was constructed by digesting pG2E1B-CAT with XbaI and
BamHI to remove the TATA box and then closed by blunt end
ligation. pL2E1B-CAT was constructed by cloning the two
LexA sites obtained by XhoI digestion from pL6EC into the
XhoI site of pE1B-CAT. pG2RS-CAT was constructed by cloning the sequence 5'-CTAGAGGGTGTAAAGTACT-3' between
the BamHI and XbaI sites of
pG2E1B-CAT. Gal4-(Sp1-VP16456) was constructed by
digesting pGal4-Sp1 with SalI and RsaI to obtain
the coding sequence for the Sp1 activating domain. For cloning
purposes, oligonucleotides were used to add the sequence
5'-GAATTCCCGGG-3' at the SalI site
and the sequence
5'-ACTCTCAGGACAGGGTACCGAATTC-3' at
the RsaI site. The Sp1 sequence is in boldface.
EcoRI sites are underlined, and these were used to clone the
Sp1 activating domain in frame in the EcoRI site in
Gal4-VP16456. pCGNTBPAS was used to express
TBPRS.
Primer extension assays.
Transfections were performed in
exactly the same manner as they were performed for the associated CAT
assays, and total RNA was isolated 36 h after transfection with
RNeasy (Qiagen). The Primer Extension System (Promega) was used
according to the manufacturer's recommendations to assay for CAT
transcripts. Twenty micrograms of RNA was used for each assay, except
that 40 µg was used for assessing the CAT transcript induced by
Gal4-Sp1 and the combination of Gal4-Sp1 and TBP. In addition, primer
extensions were done in parallel for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) with 20 µg of the RNA from each sample. The
sequence 5'-GGGATATATCAACGGTGG-3' was used for the CAT
primer, and the sequence 5'-GACCTTCACCTTCCCCAT-3' was used
for the GAPDH primer. Each assay was performed with RNA prepared from
two different transfections, and the results were similar each time.
Immunoblotting.
pGal4-TBP (3 µg), pGal4-VP16456 (4 µg), pGal4-Sp1 (1 µg), PM1 (4 µg), pLexA-VP16456 (1 µg), and
pBLX1 (1 µg) were cotransfected with the quantity of expression
vector indicated into C33a cells as outlined above. Thirty-six hours
after transfection, immunoblotting was performed on cell lysates with
either anti-Gal4 polyclonal antiserum (Santa Cruz) or anti-LexA
polyclonal antiserum (a gift from R. Brent, Massachusetts General
Hospital) as described previously (31). Each immunoblot
assay was performed twice with protein lysates prepared from two
different transfections, and the results were similar each time.
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RESULTS |
Upstream activators differ in their abilities to recruit TBP to the
promoter in vivo.
Transcriptional activators differ in their
responses to overexpression of TBP (22). This most likely
reflects differences in the mechanisms by which activators function.
Therefore, we reasoned that overexpression of TBP might serve as a tool
for dissecting the mechanism of activator function in vivo. We first examined the effect of TBP overexpression on the activity of
promoter-bound TBP. When TBP is artificially tethered to a promoter by
the DNA-binding domain of the yeast transcription factor Gal4 (Gal4),
as a Gal4-TBP fusion protein, transcription is activated
(32) (Fig. 1a). Overexpression of TBP disrupted activation by Gal4-TBP (Fig. 1a). This effect was
specific since expression of the transcriptional activator LexA-VP16456 (a chimeric protein in which a transactivation domain corresponding to VP16 amino acids 413 to 456 [20] is
fused to the DNA-binding domain of the bacterial transcription factor
LexA) failed to inhibit the activity of Gal4-TBP, even though it
completely inhibited transactivation by Gal4-VP16456 (Fig. 1a), and
in contrast to its inhibitory effect on Gal4-TBP activity,
overexpression of TBP activated the basal promoter (Fig. 1a) and
potentiated activation by Gal4-VP16456 (see below and Fig. 1e). The
effect of overexpression of TBP on the activity of Gal4-TBP was at the mRNA level (Fig. 1b), and the effects of overexpression of TBP and
LexA-VP16456 were not due to a reduction in expression of the
Gal4-TBP or Gal4-VP16456 protein (Fig. 1c). One possible model to
explain the mechanism by which overexpression of TBP inhibits Gal4-TBP
activity is depicted in Fig. 1d: when Gal4-TBP is bound to a promoter,
the TBP moiety weakly nucleates the assembly of the remaining
components (Z) that are necessary to form an active PIC (Fig. 1d, upper
panel); when TBP is overexpressed, it competes with the promoter-bound
Gal4-TBP for binding to a protein(s) that is a component of the
assembling PIC, and so it inhibits transcription by blocking the
assembly of an active PIC (Fig. 1d, lower panel). In this model, the
effect of overexpression of TBP would be analogous to the proposed
mechanism for transcriptional squelching (9). Overexpression
of TBP also inhibited transactivation by Sp1 (Fig. 1e); in contrast,
however, TBP overexpression enhanced activation by Gal4-VP16456
(Fig. 1e). The effect of overexpression of TBP on the activity of
Gal4-Sp1 and Gal4-VP16456 was at the mRNA level (Fig. 1f), and the
effect of overexpression of TBP was not due to an alteration in the
levels of Gal4-Sp1 and Gal4-VP16456 proteins (Fig. 1g). One possible
model to explain these findings is presented in Fig. 1h: Sp1
efficiently recruits TBP to the promoter (either directly or through an
adapter, i.e., a coactivator or TBP-associated factor [TAF], and the
resulting Sp1-TBP complex acts in a manner similar to that of Gal4-TBP
(as described above) in respect to its potential to nucleate an active
PIC (Fig. 1h, left upper panel), which is disrupted by TBP
overexpression (Fig. 1h, left lower panel); VP16456 efficiently
recruits components of the PIC that assemble downstream of TBP, but TBP
only transiently or weakly associates with this complex, either
stochastically or through a weak interaction with the TATA box, other
components of the PIC, or VP16456 itself, to complete the formation
of an active PIC (Fig. 1h, right upper panel); however, if TBP is
overexpressed, the interaction of the assembling PIC and TBP is driven
by the increased concentration of TBP such that formation of a complete PIC is favored and transcription is augmented (Fig. 1h, right lower
panel). We sought further evidence in support of these models.
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FIG. 1.
Activators target different components of the PIC for
recruitment. (a) Overexpression of TBP inhibits the activity of
promoter-bound TBP. When TBP is overexpressed with a reporter that
contains Gal4 binding sites and a TATA box, transcription is activated.
However, activation of the same promoter by Gal4-TBP is inhibited by
overexpression of TBP. Gal4-TBP activity is unaffected by
overexpression of LexA-VP16456, even though LexA-VP16456
squelches Gal4-VP16456. (b) Cells were transfected in exactly the
same manner as for panel a, and primer extensions for CAT and GAPDH
mRNA were performed in parallel for each sample. The extension products
were of the expected sizes. Numbers at left show sizes of DNA
standards. (c) Overexpression of TBP does not affect the expression of
Gal4-TBP protein, and expression of LexA-VP16456 does not affect the
expression of Gal4-VP16456 protein when assessed by immunoblotting.
(d) One possible model for the mechanism of inhibition of Gal4-TBP
activity by TBP overexpression (see text for details). (e)
Overexpression of TBP inhibits the activity of Gal4-Sp1 but potentiates
the activity of Gal4-VP16456. (f) Cells were transfected in exactly
the same manner as for panel e, and primer extensions for CAT and GAPDH
mRNA were performed in parallel for each sample. The extension products
were of the expected sizes. The contrast of the image was increased to
facilitate the visualization of the CAT mRNA in the cells transfected
with the Gal4-Sp1 expression vector and the cells cotransfected with
the Gal4-Sp1 and TBP expression vector. Numbers at left show sizes of
DNA standards. (g) Overexpression of TBP does not affect the expression
of the Gal4-Sp1 or Gal4-VP16456 proteins when assessed by
immunoblotting. (h) One possible model for the mechanisms of
transcriptional inhibition and potentiation by TBP overexpression (see
text for details).
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To simplify the examination of the mechanism by which transactivating
proteins function, we eliminated a variable in the assembly of the PIC
by "clamping" TBP to the promoter by using the LexA DNA-binding
domain. We reasoned that a transcriptional activator that functions
solely by recruiting TBP would have no effect on promoter activity if
TBP was stably fixed on the promoter by LexA (as a LexA-TBP fusion
protein [5]); however, an activator that normally
functions at a step after TBP recruitment in the assembly of a
functional PIC would act in concert with LexA-TBP and further stimulate
transcription. If either Gal4-Sp1 or LexA-TBP was brought to a promoter
that contains both Gal4 and LexA operators upstream from a TATA box,
transcription was activated (Fig. 2a).
However, when both were expressed together, the Gal4-Sp1 was inert as
evidenced by the fact that the resulting promoter activity was no
higher than that induced by LexA-TBP alone (Fig. 2a). The lack of
further activation by Gal4-Sp1 was not simply due to the intrinsically low level of activity of Sp1, since the combinations of Gal4-Sp1 and
LexA-VP16456 (data not shown) and LexA-Sp1 and Gal4-VP16456 (below and Fig. 4a) activated transcription synergistically. In contrast to the lack of cooperativity between Gal4-Sp1 and LexA-TBP, Gal4-VP16456 cooperated with LexA-TBP to activate transcription synergistically (Fig. 2a). This effect was dependent upon the transactivation domains of the chimeric proteins (Fig. 2b). One possible explanation for these findings is that Sp1 normally activates transcription by recruiting TBP to the promoter; therefore, Sp1 has no
additional effect if TBP is already bound to the promoter (in this case
by LexA). Conversely, VP16456 does act cooperatively with LexA-TBP,
so it is conceivable that VP16456 normally functions in the
formation of an active PIC at a step that occurs downstream of TBP
recruitment. Although these results are consistent with several
possible mechanisms, when considered in the context of the results
presented in Fig. 1a and e they provide further evidence for the models
presented in Fig. 1d and h in which Sp1 recruits TBP and VP16456
recruits another component(s) into the PIC. Further evidence for these
models is provided by the experiments described below.
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FIG. 2.
Sp1 recruits TBP, and VP16456 targets a step that is
downstream of TBP recruitment to activate transcription. (a)
Gal4-Sp1 is inert if TBP is tethered to the promoter by LexA, but
Gal4-VP16456 cooperates with LexA-TBP to activate transcription
synergistically. (b) Synergistic activation induced by the combination
of Gal4-VP16456 and LexA-TBP is dependent upon the VP16456 and
TBP moieties in these chimeric proteins. This is evidenced by the fact
that the Gal4 and LexA DNA-binding domains are inactive in this assay
(left panel) even though the Gal4 and LexA DNA-binding domains are
readily expressed in transfection assays when assessed by
immunoblotting (right panels). NS, nonspecific bond. (c) Gal4-Sp1 and
Gal4-TBP are less dependent upon the TATA box than Gal4-VP16456 for
their transactivation function. "fold decrease" represents the
ratio of the activity of each activator in the presence to that in the
absence of the TATA box. The cell lysates for the assays depicted in
the left panel were diluted 10-fold to facilitate the comparison
between the two reporter constructs.
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We next examined the contribution of TATA box function to the assembly
of an active PIC by Sp1, VP16456, and TBP. The TATA box has a
specific affinity for TBP, and so it is likely that it participates in
the assembly of the PIC by contributing to the stabilization of TBP on
the promoter. Therefore, it might be expected that deletion of the TATA
box would have less of an effect on the activity of a transactivating
protein that efficiently recruits TBP to the promoter than on that of a
transactivating protein that cannot recruit or can only weakly recruit
TBP. In accord with this prediction and the models we have presented
for activation, we found that Gal4-Sp1 and Gal4-TBP were markedly less
dependent upon the TATA box than Gal4-VP16456. Deletion of the TATA
box resulted in a 96.5-fold decrease of the activity of Gal4-VP16456
but only 10.5- and 7.5-fold decreases of the activity of Gal4-Sp1 and
Gal4-TBP, respectively (Fig. 2c). Indeed, Gal4-VP16456 was the
strongest activator of the three when the TATA box was present, but it
had slightly less activity than Gal4-Sp1 and less than half the
activity of Gal4-TBP when the TATA box was deleted (Fig. 2c). The
simplest explanation for these findings is that Gal4-VP16456
activates transcription by targeting a step after TBP recruitment, and
so it is more dependent upon the TATA box to stabilize TBP at the
promoter than is either Gal4-Sp1 or Gal4-TBP. In accord with this
hypothesis is the finding that, if the TATA box is mutated, the
activity of LexA-Gal11, an artificial construct that activates
transcription in Saccharomyces cerevisiae by recruiting only
the RNA polymerase II holoenzyme, is attenuated to a greater degree
than that of an activator that contacts both TBP and TFIIB
(1a). Thus, these findings are consistent with and therefore
provide further corroborative evidence for the models presented in Fig.
1d and h in which Sp1 recruits TBP to the promoter and VP16456
recruits a component that assembles into the PIC after TBP.
Finally, to confirm that VP16456 transactivation truly is limited by
the amount of TATA-bound TBP, we performed an experiment in which it
was possible to regulate the TBP-TATA interaction. We took advantage of
the relaxed-specificity TBP (TBPRS) developed by Strubin
and Struhl (28). This TBP derivative contains mutations on
its DNA binding surface that allow it to recognize both the canonical
TATA box sequence and the altered TATA box sequence TGTAAA (TATARS), whereas wild-type
TBP recognizes only the canonical TATA box (28) (Fig.
3a). As has been demonstrated previously, overexpression of either TBP or TBPRS increased the basal
activity of a promoter that contains a canonical TATA box (Fig. 3b,
left panel), while only TBPRS increased activity when the
TATA box was replaced with TATARS (Fig. 3b, right panel).
As would be expected, overexpression of either TBP or TBPRS
stimulated transactivation by Gal4-VP16456 to approximately the same
extent when assessed with a reporter gene that contains a canonical
TATA box in its promoter (Fig. 3b, left panel). In marked contrast,
however, as would be predicted if the model presented in Fig. 1h is
correct, TBPRS was considerably more effective than
wild-type TBP in augmenting Gal4-VP16456 transactivation when the
canonical TATA box was replaced with TATARS (Fig. 3b, right
panel). These findings indicate that overexpressed TBP must interact
with the TATA box to effectively augment transactivation by
Gal4-VP16456. Therefore, it is most likely that overexpression of
TBP enhances activation by VP16456 because the increased
concentration of TBP drives the formation of an active PIC as depicted
in the model in Fig. 1h.
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FIG. 3.
Overexpressed TBP must be incorporated into the PIC to
cooperate with VP16456. (a) TBPRS binds to both a
canonical TATA box and a mutant TATA box (TATARS); TBP
binds only a canonical TATA box. (b) Overexpression of either
TBPRS or TBP increases the activity of a basal promoter
that contains a canonical TATA box (left panel); only TBPRS
increases the activity of the basal promoter when the TATA box is
replaced by TATARS (right panel). Both TBPRS
and TBP cooperate with VP16456 with equal effectiveness to activate
a promoter that contains a canonical TATA box (left panel);
TBPRS cooperates with VP16456 much more effectively than
does TBP to activate a promoter that contains TATARS (right
panel).
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An upstream activator can recruit a component that assembles in the
PIC downstream of TBP.
The results presented thus far are
consistent with either of two mechanisms for transactivation by
VP16456: (i) VP16456 stimulates transcription by recruiting
proteins into the assembling PIC, or (ii) VP16456 stimulates
transcription by modifying and thereby activating a protein(s) that is
already assembled in the PIC. To distinguish between these two
possibilities, we sought to determine if it is necessary for VP16456
to be tethered to the promoter to further activate promoter-bound TBP.
We reasoned that, if VP16456 functions solely by interacting with
proteins that are already assembled in the PIC and it is present at a
sufficient concentration in proximity to the PIC, it would not be
necessary for VP16456 to be promoter bound to activate
transcription. However, if VP16456 normally functions as a tether to
recruit proteins to the promoter and stabilize them in the PIC,
VP16456 itself would have to be stably bound to the promoter to
function as a transcriptional activator. When Gal4-VP16456 was
brought to the promoter, it potentiated the activity of promoter-bound
LexA-TBP in a synergistic manner (Fig.
4a, left panel), but Gal4-VP16456 that
was not bound to the promoter had no effect whatsoever (activating or
squelching) on the activity of promoter-bound LexA-TBP (Fig. 4a, right
panel). This was true even at much higher concentrations than were
necessary for synergy when Gal4-VP16456 was promoter bound (Fig. 4a,
right panel), suggesting that Gal4-VP16456 must be bound to the
promoter to recruit a component(s) into the PIC to activate
transcription. Alternatively, it was conceivable that the local
concentration of VP16456 is high enough to interact with proteins in
the PIC only if it is tethered near the PIC on the promoter, so we
examined this possibility. When 2 µg of Gal4-VP16456 was
transfected, it squelched the activity of LexA-VP16456 (Fig. 4b,
left panel); however, the same concentration of Gal4-VP16456 neither
squelched nor activated the activity of LexA-TBP when there was no Gal4
binding site on the promoter (Fig. 4a, right panel; also see analogous
results in Fig. 1a). The fact that overexpressed Gal4-VP16456
squelches activation by promoter-bound LexA-VP16456 but not
LexA-TBP-mediated activation strongly suggests that Gal4-VP16456 blocks transactivation by LexA-VP16456 because it specifically competes with the promoter-bound LexA-VP16456 for binding to its
target in the PIC and thereby interferes with the ability of
LexA-VP16456 to recruit its target protein(s) (see model in Fig.
4c). Therefore, it apparently is not sufficient for VP16456 to
simply bind to its target(s) to activate transcription. Thus, the
results presented above are most consistent with a mechanism in which
VP16456 activates transcription at least in part by recruiting
components of the PIC to the promoter. It is known that VP16 also
activates transcription at a postinitiation step. Our data, however,
suggests that recruitment is a requisite first step for transcriptional
activation by VP16456.
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FIG. 4.
VP16456 recruits a component(s) that is assembled
into the PIC downstream of TBP to activate transcription. (a) When
Gal4-VP16456 was tethered to the promoter, it potentiated the
activity of promoter-bound LexA-TBP in a synergistic manner (left
panel). Gal4-VP16456 does not cooperate with LexA-TBP unless it is
tethered to the promoter, even when high concentrations of the
Gal4-VP16456 expression vector are transfected (right panel). (b)
Even when Gal4-VP16456 is not tethered to the promoter, it can still
interact with its transactivation target(s), as indicated by the fact
that it squelches the activity of LexA-VP16456 (left panel) but not
that of LexA-TBP (a). Overexpression of Gal4-VP16456 does not affect
cellular levels of LexA-VP16456 as assessed by immunoblotting (right
panel). (c) A model for the interactions suggested by the findings that
Gal4-VP16456 squelches the activity of promoter-bound
LexA-VP16456 but not promoter-bound LexA-TBP.
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Upstream activating domains that recruit different components of
the PIC can cooperate to activate transcription synergistically.
It has been proposed that synergistic transcriptional activation might
occur when two components of the PIC, such as TBP and the RNA
polymerase holoenzyme, are recruited by different activators on the
promoter (26). Since our findings suggest that Sp1 and VP16456 recruit different components of the PIC, we sought to determine if they would activate transcription synergistically when
bound to the same promoter. We found that when LexA-Sp1 was brought to
a promoter with Gal4-Sp1, transcriptional activity was increased about
twofold over the level observed when only Gal4-Sp1 was bound to the
same promoter (Fig. 5a). However, when LexA-Sp1 was brought to a promoter with Gal4-VP16456,
transcriptional activity increased 21-fold over that induced by
Gal4-VP16456 alone and 420-fold over the level induced by LexA-Sp1
alone (Fig. 5a). These findings are consistent with a mechanism in
which Sp1 and VP16456 recruit different proteins into the PIC and
thereby cooperate when bound to the same promoter to activate
transcription synergistically.
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FIG. 5.
Transcriptional synergy. (a) Sp1 cooperates with
VP16456 but not with itself to activate transcription
synergistically. Relative promoter activity is indicated above each
assay. The cell lysate from one assay was diluted as indicated to
facilitate quantification of activity. (b) Activating subdomains within
one protein can cooperate to activate transcription synergistically. A
single chimeric protein, encoded by Gal4-(Sp1-VP16456), containing
the activation domains of Sp1 and VP16456 fused to the Gal4
DNA-binding domain is a strong transcriptional activator.
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Generally, several activators must act in concert on a eukaryotic
promoter for maximal transcriptional activity, and yet some, such as
VP16, can act alone to strongly activate promoters (reviewed in
reference 19). It is possible that the
transactivation domains of some activators contain subdomains that
independently target different components of the PIC for recruitment
and that these subdomains cooperate with each other to activate
transcription synergistically. To test this possibility, we constructed
a vector that expresses a single chimeric protein that contains both
the Sp1 activation domain and VP16456 fused to the Gal4 DNA-binding domain [Gal4-(Sp1-VP16456)]. We reasoned that if the Sp1 and VP16456 domains in this chimeric protein could act independently to
recruit their normal targets to the PIC, they would cooperate and
activate transcription strongly. The transcriptional activity induced
by Gal4-(Sp1-VP16456) was at least 200-fold greater than the
activity induced by either Gal4-Sp1 or Gal4-VP16456 (Fig. 5b)
and equivalent to that of the strong transcriptional activator Gal4-VP16 (which contains the full-length VP16 activating domain fused
to Gal4 [24]), suggesting that some transcription
factors may activate transcription efficiently because they contain
subdomains that target different components of the PIC for recruitment.
Indeed, VP16 has been shown to bind to several different proteins in
the PIC, including TBP, either directly (27) or indirectly
through a TAF (10, 14), and proteins that assemble into the
PIC after TBP (17, 18, 21, 33).
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DISCUSSION |
It has recently been demonstrated that artificial recruitment of
either of two components of the PIC, TBP and the RNA polymerase holoenzyme, is sufficient to activate transcription (1, 5, 7, 13,
32). This finding suggested that upstream transcriptional activators might function at least in part by recruiting these proteins
into an assembling PIC. Here we have used a series of functional assays
to evaluate whether upstream activators do indeed recruit components of
the PIC. The results of these assays provide evidence that Sp1 recruits
TBP to the promoter and that VP16456 functions to activate
transcription at least in part by recruiting a component(s) that
assembles into the PIC downstream of TBP.
First, we demonstrated that overexpression of TBP reduces the activity
of promoter-bound TBP and Sp1 but potentiates the activity of
VP16456. This suggests that endogenous TBP levels are not limiting
for activation by Sp1 but that they are limiting for activation by
VP16456. One possible explanation for this finding is that Sp1
efficiently recruits TBP to the promoter but VP16456 cannot.
Therefore, an increase in the cellular concentration of TBP would only
squelch activation by Sp1, but it could facilitate activation by
VP16456 (Fig. 1h). We then examined the activity of both Sp1 and
VP16456 when TBP was constitutively bound to the promoter by the
LexA DNA-binding domain. We found that, while Sp1 had no further effect
on promoter activity if TBP was bound in this manner, VP16456
potentiated the activity of the promoter-bound TBP synergistically.
These findings suggest that activation by Sp1 is solely a result of TBP
recruitment and that VP16456 activation results from a step(s)
downstream of TBP recruitment. Next, reasoning that the TATA box serves
to stabilize TBP on the promoter, we examined the effect of deletion of
the TATA box on the activity of promoter-bound TBP, Sp1, and
VP16456. Whereas Gal4-VP16456 was the strongest activator when a
TATA box was present, it became the weakest activator when the TATA box
was deleted
its activity falling to less than half that of Gal4-TBP in
the absence of the TATA box. These results suggest that Gal4-VP16456
is more dependent upon the TATA box for TBP recruitment than is either
Gal4-TBP or Gal4-Sp1 and that VP16456 activates transcription by
targeting a step that occurs downstream of TBP recruitment. This was
further demonstrated by the finding that overexpressed TBP enhances
VP16456 transactivation only when the TBP is bound to the TATA box
(Fig. 3b). We then found that, when VP16456 was expressed at a level at which it readily squelched itself, it had no effect whatsoever on
the activity of promoter-bound TBP (Fig. 1a and 4a and b). This
suggests that when expressed at this level VP16456 self-squelches because it specifically interacts with its normal target protein(s) in
the PIC and thereby interferes with the ability of promoter-bound VP16456 to recruit and/or stabilize its target protein(s) in the
PIC. Conversely, that this level of Gal4-VP16456 does not potentiate
the activity of promoter-bound TBP, even though it can squelch
promoter-bound LexA-VP16456, suggests that VP16456 must be
tethered to the promoter to recruit a protein(s) into the PIC to
activate transcription (Fig. 4c). Finally, consistent with the
hypothesis that Sp1 and VP16456 recruit different components of the
PIC was the finding that, when they were both brought to the same
promoter, either as separate proteins or together in one chimeric
protein, they activated transcription synergistically.
The protein interactions suggested by our findings have been shown to
occur either in vitro or in yeast two-hybrid systems. However, the
mechanism by which these interactions result in transactivation was not
addressed. Specifically, it has been demonstrated that Sp1 interacts
with a protein that is tightly associated with TBP in the TFIID
complex, hTAFII130, and its Drosophila homolog, dTAFII110 (6, 8, 12, 29), and that VP16456 can bind to the mediator subcomplex in a yeast RNA polymerase II holoenzyme (11). Our data advances these findings by providing evidence that these interactions result in recruitment and that this is, at least in part,
the mechanism by which Sp1 and VP16456 activate transcription. It is
notable that VP16 has also been shown to interact with TAFII40 (10), suggesting that VP16 should also recruit TFIID to the promoter. However, it was shown that the C-terminal 39 amino acids of
VP16 are necessary for this interaction (10). These amino acids are deleted from VP16456; hence, VP16456 is deficient in
this function. It is possible that the Sp1-VP16456 construct we
studied was as efficient an activator as intact VP16 because the Sp1
portion of the chimeric protein compensated for the loss of this
function by binding to hTAFII130.
Several models have been proposed for the mechanism by which
transactivating proteins function together in a synergistic fashion. Indeed, it is likely that there are several mechanisms by which they
can act in a synergistic fashion. We have provided evidence that some
transcription factors may cooperate to activate transcription synergistically when they simultaneously but independently recruit different components of the PIC. Carey and coworkers originally proposed a model for synergy in which multiple activator molecules simultaneously contact a single target in the PIC and thereby stabilize
it at the promoter (4). In this model, an arithmetic increase in the number of activating molecules on the promoter should
lead to an exponential increase in stability, which would result in a
synergistic increase in transcription. Alternately, a kinetic model in
which multiple activator molecules contact a target in a sequential
manner has been proposed (3). Increasing the number of
activator molecules on the promoter would increase the frequency of
activator-target functional contacts. Once a threshold frequency of
activator-target interactions is surpassed, transcription would occur.
Thus, increasing the number of activators would result in synergy once
the threshold is exceeded.
Several transcription factors have been shown to activate transcription
by targeting steps that occur postinitiation. Indeed, VP16 has been
shown to facilitate elongation, and this is thought to be a result of
the interaction of VP16 with TFIIH (2, 34). Therefore,
considering these findings together with our results, it is likely that
VP16 and other transcription factors function as transcriptional
activators both by recruiting components of the PIC and by activating a
postinitiation function of the assembled PIC.
We thank J. T. Lis for critical review of the manuscript. We
thank A. Berk for pGal4-VP16456, W. Schaffner for pGal4-Sp1, W. Herr
for pCGNTBP and pCGNTBPAS, M. R. Green for
pG2E1B-CAT and pE1B-CAT, R. G. Roeder for
p6His-T-hTBP, I. Sadowski for PM1, and P. Broad for pBXL1
and pL6EC.
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Abstract
Introduction
