From the Department of Microbiology, Goethe
University, 60439 Frankfurt am Main, Germany, and
Department of Molecular Physiology and Biophysics, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232
Received for publication, December 9, 2002
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ABSTRACT |
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An important goal is to identify the direct
activation domain (AD)-interacting components of the transcriptional
machinery within the context of native complexes. Toward this end, we
first demonstrate that the multisubunit TFIID, SAGA, mediator, and
Swi/Snf coactivator complexes from transcriptionally competent
whole-cell yeast extracts were all capable of specifically
interacting with the prototypic acidic ADs of Gal4 and VP16. We then
used hexahistidine tags as genetically introduced activation
domain-localized cross-linking receptors. In combination with
immunological reagents against all subunits of TFIID and SAGA, we
systematically identified the direct AD-interacting subunits within the
AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell
extracts and confirmed these results using purified TFIID and partially
purified SAGA. Both ADs directly cross-linked to TBP and to a subset of
TFIID and SAGA subunits that carry histone-fold motifs.
Most proteins function within the context of large complexes (1).
Affinity and specificity of interactions involving large multiprotein
complexes often depend on the sum of cooperative interactions,
interactions that individually can be rather weak and only moderately
specific. For this reason it is desirable to identify directly
interacting polypeptides in the context of their native complexes. A
particularly clear case for this need is exemplified in the
difficulties inherent in identifying the direct targets of
transcriptional activation domains
(ADs)1 within transcription complexes.
The ADs of transcriptional activators are thought to recruit large
coactivator complexes to promoters by direct protein-protein interactions (2). Two types of coactivator complexes can be distinguished: (i) those that are directly associated with the transcriptional machinery like the RNA polymerase II-associated mediator and the TAF components of the general transcription factor TFIID and (ii) chromatin-associated complexes like the
chromatin-remodeling complex Swi/Snf and the histone acetyltransferase
(HAT) complex SAGA.
Most attempts to identify the AD-interacting subunits within these
coactivator complexes have been based on binding experiments using
isolated polypeptides outside of their native holocomplexes. Interactions observed in these analyses were weak, with apparent dissociation constants of An elegant approach that avoids the formation of large and therefore
difficult-to-analyze cross-linking complexes is the use of
photoactivatable label transfer cross-linking reagents. This method has
been successfully employed to identify several activator targets within
intact complexes, including mediator (14), SAGA (15), and Swi/Snf (16)
complexes, as well as in bacterial RNA polymerase (17). However, as
powerful as this approach is, it does have limitations. First, the
label has to be at or immediately abutting the site that directly
contacts its target, while at the same time the 15-21 Å photoactivatable group must not interfere with complex formation.
Second, labile, light-sensitive radioactive protein-photoactive
variants of ADs must be prepared. Finally, radioactively labeled target
proteins are identified by size, a requirement that potentially limits
the use of this approach for the analysis of large polypeptides or to
analyze subunits with similar sizes relative to each other or to the
labeled probe molecule (for instance, see Ref. 14).
Here we describe a variation on protein-protein cross-linking wherein
we used immunodetection coupled with electrophoretic mobility changes
to score protein-protein interactions. With this approach, we can
clearly distinguish all of the subunits of the relevant, multisubunit
complexes. We employed a newly developed site-specific cross-linking
technique to identify protein-protein interactions termed
hexahistidine-mediated cross-linking (18, 19) that uses a common
nickel-activated hexahistidine tag as receptor for a nondiffusible
cross-linking reagent. With this method, there is no need to chemically
modify the activator with photoprobes or radioactivity, and the
hexahistidine tag can be genetically introduced at any inert position
of the molecule. Finally, the reagent only cross-links residues that
are very closely apposed (18, 19).
In this analysis, we concentrated on the interactions of ADs with the
TFIID and the SAGA complexes. These complexes partially overlap in
subunit composition (20) and function (21), a fact further complicating
interpretations based on the analysis of the interactions of ADs with
isolated, individual subunits.
TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated
factors (TAFIIs) (22). Five TAFIIs are present
in both TFIID and in the 17-subunit SAGA complex (20, 23), and each complex contains a subunit with intrinsic HAT activity (20, 24).
Although all but one of the yeast TAFIIs are essential for
viability, only certain TAFIIs, particularly the ones
shared by TFIID and SAGA with similarity to histones, are needed for the transcription of a broad set of genes (25). Using the hexahistidine cross-linking method, we have identified AD interacting subunits in the
context of intact TFIID and SAGA and also show that although other
proposed targets do bind and cross-link to ADs as isolated polypeptides, they are not accessible in the native complexes.
Yeast Strains and Genomic Tagging--
Yeast strains with
genomically triple-HA tagged TBP, TAF1, TAF5, TAF6, and TAF10 were
described previously (26). All other factors were genomically triple-HA
tagged for this work in strain 21R (27) using standard procedures
(28).
Antibodies--
Immunopurified rabbit polyclonal antibodies
directed against TAF1, TAF2, TAF4, TAF5, TAF6, TAF11, TAF12, TAF13,
TBP, Ada2, and Gcn5 were described previously (26). The antibody
directed against Tra1p was a kind gift from J. L. Workman. Anti-HA
mouse monoclonal antibody (clone 12CA5) was purchased from Roche.
Recombinant Proteins--
Recombinant GST and
GST-His6 fusion proteins were expressed in
Escherichia coli from pGexCS (29) variants in which an
oligonucleotide encoding His6 was inserted into the
NcoI site and from pKM vectors (30). GST-His6
fusion proteins contain a cleavage site for TEV protease, which
increases cross-linking efficiency by providing tyrosine residues as
electron donors in the vicinity of the His6 tag (18).
Fusion proteins were bound and purified on glutathione-Sepharose (Amersham Biosciences) using standard protocols (30).
TAF9-HA2 open reading frame was amplified by PCR from the
TAF9-HA3 strain and cloned into pGexCS for expression.
Preparation of WCE--
Transcriptionally competent WCE were
prepared from 800-ml cultures of each genomically tagged yeast strain.
Cells were grown to a density of OD600 Pull-down and Cross-linking--
5-10 µg (0.3 µM, or concentrations as indicated in Fig. 1) of GST or
GST-activator fusion protein bound to glutathione-Sepharose beads were
incubated with 500 µg of WCE protein (~20 µl), each, for 1 h
at 4 °C on a tiltboard in 1 ml of transcription buffer (S. Hahn:
www.fhcrc.org/science/basic/labs/hahn/): 10 mM
Hepes-KOH pH 7.5, 100 mM K-glutamate, 10 mM
Mg-acetate, 2.5 mM EGTA, 3.5% glycerol, 0.01% Nonidet
P-40). Beads were then washed 3× for 5-10 min in 500 µl of
transcription buffer, each, on ice.
Washed beads were resuspended in 500 µl of transcription buffer. 100 µl of the suspension were removed to control GST-fusion protein input
(Coomassie stained gel) and 150 µl as control for the pull-down
reaction. The remaining beads with bound complexes were pelleted and
resuspended in 100 µl of buffer. Hexahistidine tags were complexed
with Ni2+ by addition of 25 µl of 60 mM
Ni-acetate and incubation for 10 min at room temperature. The
cross-linking reaction was started by addition of 25 µl of 60 to 120 mM magnesium monoperoxyphtaleic acid (MMPP) and allowed to
proceed for 6 min. Reactions were terminated by centrifugation, removal
of supernatants, and resuspension of beads in 10 µl of 2× SDS sample
buffer. Each cross-linking reaction was repeated at least once with
independently prepared extracts. Further information on the
His6-mediated cross-linking method can be found at
www.uni-frankfurt.de/fb15/mikro/melcher.html.
Tobacco Etch Virus (TEV) Protease Cleavage of Cross-linking
Products--
Beads from half of the cross-linking reactions were
washed 3 times with 500 µl of transcription buffer. Washed beads were resuspended in 10 µl of the same buffer supplemented with 0.1 mM EDTA and 1 mM dithiothrietol plus 1 µl of
TEV protease (2.5 µg/µl) and incubated for 30 min at room
temperature. Supernatants were made 1× SDS sample buffer, separated
by SDS-PAGE, and analyzed by immunoblotting.
SAGA Enrichment and TFIID Purification--
To separate SAGA
from TFIID, WCE was prepared from 4 liters of culture of yeast cells
and loaded onto a nickel-nitrilotriacetic acid agarose column as
described (20). Whereas essentially all of TFIID was found in the flow
through, SAGA bound to the nickel-nitrilotriacetic acid agarose and was
eluted with 300 mM imidazole. TFIID was purified as
described (22).
An Acidic Activation Domain Interacts with TFIID, Holopolymerase,
Swi/Snf, and SAGA Complexes--
To immunologically identify
individual subunits of yeast transcription complexes, we genomically
tagged and/or raised antibodies against all subunits of TFIID and SAGA
as well as against the mediator component Med6 and the Swi/Snf
component Swi2 (Fig. 1). Genomically
tagged strains were created by introducing a triple influenza virus
hemagglutinin (HA)-tag by homologous recombination at the 3' end of the
respective open reading frames. Thus, tagged proteins were expressed
from these genes under their native promoters and in their endogenous
chromosomal environment. WCEs were prepared from these strains and used
for GST-AD pull-down experiments.
We incubated glutathione-Sepharose-bound GST-VP16 AD with
transcriptionally competent yeast WCE and determined whether the four
coactivator complexes bound to the VP16 AD. As shown in Fig. 2A, all four components were
retained by saturating amounts of GST-VP16 AD but not by GST alone. To
test the specificity of these interactions, transcription extracts were
incubated with the wild-type Gal4 and VP16 ADs as well as with
transcriptionally inactive variants of the two ADs. Both SAGA (as
scored by Ada1p) and TFIID (as scored by TAF4) were retained by the two
functional ADs but even under saturating conditions of immobilized ADs,
these coactivator complexes did not bind inactive derivatives (Fig.
2B). To determine the relative affinities of interactions,
we titrated the concentration of immobilized ADs in 3-fold increments
down from the saturating concentrations used in panels A and
B. The minimal concentration of Gal4p AD needed to
efficiently retain SAGA from WCE was at least 10-fold lower than the
concentrations of any of the other coactivator complexes, consistent
with SAGA being the predominant complex recruited in vivo by
Gal4 AD in the absence of a core promoter and other coactivator
complexes (32). Surprisingly, TFIID, which is only recruited at
sub-stoichiometric levels to the GAL1 promoter in
vivo (33), is actually more efficiently retained by Gal4 AD than
the mediator and Swi/Snf complexes (Fig. 2C), suggesting
that the efficient recruitment of mediator (34) likely depends on
additional interactions. Importantly, a series of coimmunoprecipitation
experiments indicated that TFIID and SAGA remained stable in the WCE
during the course of these experiments (data not shown, and see below)
indicating that these experiments truly scored the interactions of the
coactivator complexes with ADs.
Cross-linking of ADs to TFIID and SAGA Complexes in WCE--
We
used hexahistidine-mediated cross-linking (19) to probe for direct
interactions within the context of intact TFIID and SAGA. Briefly,
complexes were collected from WCE by fusion proteins consisting of a
GST tag, a hexahistidine tag, and the Gal4 or VP16 AD bound to
glutathione-Sepharose beads as described above. The His6
tag was then complexed with Ni(II) by short incubation with nickel
acetate followed by incubation with the peracid MMPP, which activates
the complexed Ni(II) to Ni(III). It has been proposed that this Ni(III)
extracts an electron from an aromatic side chain, resulting in the
formation of highly reactive radicals that lead to fast and efficient
0Å cross-linking (i.e. direct cross-linking of side chains,
without a linker in between). Importantly, it has been documented that
even in crude cellular extracts, only those interacting proteins that
are complexed with a hexahistidine-tagged protein become cross-linked
(19). Fig. 3A shows an outline
of the experiment, and Fig. 3B shows an example of
MMPP-dependent, His6-mediated cross-linking of
the VP16 and Gal4p ADs to TAF12 within TFIID and/or SAGA in WCE.
SAGA and TFIID complexes derived from WCE were bound to the Gal4 and
VP16 ADs, cross-linked to the AD with MMPP, and denatured. Cross-linking products were identified by immunoblotting. The top
panel of Fig. 3B shows the blot incubated with
anti-TAF12 antibody. In the lane containing the cross-linking reaction
with Gal4 AD, the anti-TAF12 antibody recognized three bands, one band of around 61 kDa corresponding to monomeric TAF12, one band at around
120 kDa corresponding to the 60-kDa GST-His6-Gal4AD dimer cross-linked to TAF12, and one band that did not enter the gel corresponding to a high molecular weight complex (see below). In the
cross-linking reaction with VP16 AD qualitatively the same 61 kDa and
high molecular weight bands were visible. However, instead of the
120-kDa band (i.e. the
TAF12(GST-His6-Gal4AD)2 adduct), a band of
about 130 kDa was recognized by the anti-TAF12 antibody corresponding
to the 70-kDa GST-His6-VP16AD dimer cross-linked to TAF12.
The characteristic shift in mobility of the cross-linking complexes,
due to the size difference of the Gal4 and VP16 ADs, serves to indicate
that the cross-linking complexes recognized by the antibody do indeed
contain the two different ADs. Radicals created in the cross-linking
reaction can migrate through more than one protein in a complex and can
thus cause high molecular weight complexes as well (19). The protein
complexes not entering the gel are likely such high molecular weight
complexes and were not analyzed further. Only cross-linked complexes
consisting solely of the GST-His6-AD and the subunit
detected by the antibody are indicative of direct interaction.
The anti-TAF12 blot shown at the top of Fig. 2B
was then stripped and reprobed with anti-Ada2 antibody to determine
whether cross-linked AD-Ada2p adducts had also been formed in the same samples and experiment. Like TAF12, Ada2p is also part of the SAGA
complex and was retained by the Gal4 and VP16 ADs to the same degree as
TAF12 (cf. lanes 2-4, top and bottom
blots of Fig. 2B). However, no cross-linking product of the
correct size was observed between Ada2p and either AD (Fig.
2B, lower panel), suggesting that the ADs bind
the SAGA complex without directly contacting Ada2p.
Untagged SAGA specifically binds to Ni-agarose and can be efficiently
separated from TFIID by metal affinity chromatography (20).
Hexahistidine-mediated cross-linking might therefore also occur within
SAGA in the absence of the His6-tagged AD. To test this
possibility, we repeated the above experiment with GST-Gal4AD lacking
the His6 tag. As expected, GST-Gal4AD retained TAF12 just as efficiently as GST-His6-Gal4AD, however, no TAF12-AD
adducts were observed when the GST-AD-SAGA beads were treated with
cross-linker (Fig. 3C). We conclude that the SAGA component
TAF12 does not form cross-linking products in the absence of an
introduced His6 tag. These data provide additional support
for the specificity of the observed AD-TAF12 adducts described above.
Having demonstrated the utility of this chemical cross-linking
approach, we next performed identical binding and cross-linking reactions with extracts that allowed analysis of each subunit of TFIID
and SAGA in turn. Only TAF2 was not examined as we were neither able to
genomically tag TAF2 nor reproducibly detect this TAF with our
polyclonal anti-TAF2 IgG. Another limitation of our technique applies
to the Tra1p subunit of SAGA. The Tra1 monomer has a size of 430 kDa,
close to the exclusion limit of the SDS-PAGE gels used. We were
therefore not able to separate a potential cross-linked product
(i.e. Tra1 and the ADs, Mr 430 + 60 kDa) from monomeric Tra1. Each experiment was repeated several times with independently prepared extracts and with different concentrations of cross-linking reagents, with equivalent results. In this extensive series of experiments, in addition to TAF12, we found that TBP, TAF4,
TAF6, and Ada1p directly cross-linked to both Gal4 and VP16 ADs (Fig.
2B). In no case have we observed differential interaction of
either AD with the five target proteins. Importantly, although all
other subunits were retained specifically and to a very similar degree
by GST-AD beads, consistent with TFIID and SAGA being intact, we did
not detect any specific cross-linking complexes with any of the other
TFIID and SAGA subunits (Fig. 4). With
the exception of TBP, the proteins cross-linked contained histone-fold
motifs and were the ones with homology to histones H2A (TAF4 and Ada1), H2B (TAF12), and H4 (TAF6) in both complexes.
Confirmation that Cross-linked Complexes Contain ADs Bound to TFIID
and SAGA Subunits--
The characteristic mobility shift of
cross-linking complexes from Gal4 versus VP16 reactions
argued strongly that these ADs were present in the cross-linked
products. To further confirm this conclusion, we engineered a TEV
protease cleavage site between the N-terminal tags and the VP16 AD. We
reasoned that if the putative AD-protein complexes truly contain the
VP16 AD, TEV cleavage should induce a significant mobility shift upon
SDS-PAGE fractionation of the digested complexes (see schematic in Fig.
5A). We used this TEV-cleavage
approach to examine all five of the AD-protein complexes that we had
positively scored for direct interactions in our standard cross-linking
protocol (i.e. TBP, TAF4, TAF6, TAF12, and Ada1).
After pull-down and cross-linking, half of the reactions were incubated
with TEV protease before denaturation (Fig. 5A). In every
case, TEV cleavage induced the predicted dramatic mobility change in
these complexes (Fig. 5B), demonstrating that the
cross-linking complexes did indeed contain the AD. The two major TEV
cleavage products correspond to cross-linked adducts in which one or
both of the TEV protease sites were cleaved (cf. Fig. 5A:
TAF4, TAF6, TAF12, Ada1). Note that the cross-linking and cleavage
experiment with the TBP-HA3-tagged strain was performed
with a different preparation of AD-fusion protein, which, due to a
read-through product, migrates as two distinct monomer bands on
SDS-PAGE and consequently gives rise to three different forms of
dimers. Like the dimeric fusion protein by itself, both the uncleaved
cross-linking product as well as the singly and doubly cleaved
complexes migrate as characteristic triplet bands, providing additional
proof for the presence of the VP16 AD in the cross-linking complex and
in the proteolytic cleavage products.
Ada2p and TAF9 Cross-link to the Gal4 and VP16 ADs When Outside of
Their Physiological Context--
Ada2p and TAF9 did not cross-link to
the Gal4 and VP16 ADs when probed within the context of intact
complexes in WCE (Figs. 3B and 4). However, it has been
reported that recombinant Ada2p (35) and the recombinant
Drosophila and human TAF9 (d TAFII40 and h
TAFII32) directly bind to the VP16 AD (36, 37). One possible explanation for this apparent discrepancy is that the AD-interacting surfaces of these factors are not accessible in the
native complexes but are only (artificially) exposed when in the
absence of their normal binding partners. Alternatively, these proteins
could also bind ADs in the context of their multiprotein complexes, but
our cross-linking method fails to work efficiently with these two
proteins. Although the proposed mechanism of hexahistidine-mediated cross-linking does not predict such a selectivity for the target proteins, it was important to experimentally address this possibility. We therefore isolated the ADA2 and
TAF9-HA2 genes and expressed and purified the
corresponding proteins from E. coli. To avoid any potential
driving of cross-linking efficiencies by mass action, the
concentrations of recombinant Ada2p and TAF9-HA2 in binding and cross-linking reactions were chosen to be in the same concentration range (about 1 nM) used in the cross-linking experiments
with WCE (Figs. 3 and 4). As shown in Fig.
6, both recombinant Ada2p and
TAF9-HA2, as well as TBP as a positive control, bound and cross-linked to the Gal4 and VP16 ADs. We conclude that the inability of Ada2p and TAF9 to cross-link ADs when in the context of SAGA and
TFIID is not caused by an inherent incompatibility with the cross-linking reagents, but is most likely due to the inaccessibility of the AD-binding surfaces within Ada2 and TAF9 when these two proteins
are present within the context of their native complexes. Of course, we
can not rule out the possibility that these surfaces could become
accessible upon potential conformational changes when present within
TFIID and SAGA.
TAF6 and TAF12 Can Interact with ADs in the Context of Purified
TFIID and SAGA--
TAF6 and TAF12 are components of both TFIID and
SAGA. Thus, the TAF6-and TAF12 AD-cross-linking we observed could have
been in the context of TFIID, of SAGA, or of both. Similarly, TBP is also a component of several distinct TBP-TAF complexes, including Pol
I- and Pol III-specific TBP-TAF complexes (SL1 and TFIIIB respectively)
and due to the fact that WCEs were used in the experiments of Figs.
3-5, we do not know in which context (SL1, TFIID, or TFIIIB) TBP
cross-linked with the Gal4 and VP16 ADs. To distinguish between these
possibilities and to further support our contention that we are scoring
direct AD-target interactions, we examined the cross-linking behavior
of ADs to purified TFIID and partially purified SAGA. Separation of
TFIID and SAGA was confirmed by immunoblotting with antibodies against
the SAGA-specific Ada2p and the TFIID-specific TAF4 subunits (Fig.
7A). As predicted from the
data presented above (Figs. 3 and 5) Gal4 AD and VP16 AD cross-linked
to TAF6 and TAF12 in the context of both TFIID and SAGA (Fig.
7B). For reasons that we do not presently understand,
cross-linking to TAF6 varied significantly with different preparations
of TFIID and SAGA (data not shown). The ADs also cross-linked to TBP
within the purified TFIID complex (Fig. 7C), which
demonstrates that TBP can interact with ADs in the context of TFIID.
AD-target interactions appear to be inherently weak and, because
of their hydrophobic nature and limited specificity, direct measurements of these interactions using purified, isolated ADs and
(putative) target proteins can be problematic. Here, we describe an
approach to analyze AD interactions in the context of native complexes
and report the first identification of AD-binding subunits within
intact TFIID, as well as the identification of previously unrecognized
direct AD contacts within intact SAGA. Our finding that Ada2 and TAF9
can directly interact and cross-link with the Gal4 and VP16 ADs as
isolated polypeptides, but apparently not in the context of their
native complexes, underscores the utility of our approach.
Our use of HA3-tagged strains in combination with
antibodies raised against specific subunits enabled us to unambiguously test the presence of all subunits except TAF2 and Tra1p in
cross-linking products. Because, for most experiments, the same anti-HA
antibody was used for detection of differently sized
HA3-tagged proteins, we could exclude immunoreactive
signals being due to antibody cross-reactive proteins within the
indicated cross-linking products. Three different lines of evidence
demonstrate the presence of ADs within cross-linking products. First,
the size of cross-linking products is consistent with monomers and
dimers of AD fusion proteins cross-linked to the immunologically
detected factors. Second, the size difference between the Gal4 and VP16
ADs can be seen in all cross-linking products. Third, incubation with
the highly selective TEV protease resulted in the production of the
expected specifically cleaved complexes. In addition, the cross-linking products between GST-His6-VP16AD and TBP-HA3 as
well as their cleavage products migrate as characteristic triplet
bands, indicative of the presence of the VP16 AD.
An important feature of His6-mediated cross-linking is that
it functions without a linker between reactive groups. Cross-linking is
therefore likely to result from proteins that directly touch each other
rather than just being in close proximity. This conclusion is supported
by our TEV cleavage analysis (Fig. 5), where in all cases the sizes of
cleavage products are consistent with direct cross-linking of the AD
rather than cross-linking with other parts of the fusion protein. In
contrast, in control experiments using dithiobis(succinimidyl
propionate) (DSP), a conventional cross-linker consisting of two
reactive groups separated by a 12-Å linker, recombinant TBP
cross-linked predominantly to GST, the largest moiety of the
His6-GST-VP16 fusion protein (data not shown). Direct interactions of the ADs with TBP, TAF6, and TAF12 is further supported by binding experiments with recombinant TBP (Fig. 6) and in
vitro-translated TAF6 and TAF12 (data not shown) and by an
in vivo interaction between Gal4 and TAF6 (38).
Cross-linking initiates solely at the His6-tagged protein
(19) (see also Fig. 3C), which favors the formation of
direct cross-linking products between ADs and interacting subunits. All indicated cross-linking complexes with Gal4 and VP16 AD monomers and
dimers as well as their corresponding TEV cleavage products migrated
with the mobilities expected for direct cross-linking products (Figs.
3, 5, 6, and 7). The only three AD-cross-linking subunits within SAGA
(TAF6, TAF12, and Ada1) that we have identified are all of very similar
size (54-61 kDa) and consequently produce cross-linking products of
similar mobilities. If cross-linking of any of these three proteins
would have occurred indirectly via one of the two other subunits, then
mobility of the resulting complex would be expected to be significantly
retarded relative to the two other complexes (e.g. compare
the doubly TEV-cleaved cross-linking products of TAF6, TAF12, and Ada1
in Fig. 5, all of which migrate with their calculated sizes between 72 and 79 kDa as opposed to sizes between 132 and 139 kDa expected for any of the combinations of indirect cross-linkings).
The fact that all 14 TFIID subunits detectable and all 14 SAGA subunits
were retained by the AD fusion proteins strongly supports the
conclusion from co-immunoprecipitation experiments that TFIID and SAGA
were intact in our transcriptionally competent WCE preparations. Cross-linking of TBP, TAF6, and TAF12 to the Gal4p and VP16 ADs in
reactions with purified TFIID further confirms that interaction occurred in the context of complexes and not with dissociated subunits.
Although a lack of a cross-linking signal may not unambiguously exclude
a direct interaction, the presence of a cross-linking product
consisting solely of the AD and a specific subunit demonstrates a
physical AD interaction in the context of native complexes.
The Workman (15) and Struhl (39) labs have described two different
approaches to probe for AD targets within coactivator complexes. Brown
et al. (15) used label transfer cross-linking to identify
targets of the VP16 AD within two HAT complexes, SAGA and NuA4. They
demonstrated that Tra1p, which is the only common subunit of SAGA and
NuA4, directly interacts with acidic ADs in the context of both
purified complexes (15). Very recently, Hall and Struhl (39) pioneered
a method that combines in vivo formaldehyde cross-linking
with the immunoprecipitation of activator proteins. Using this
approach, they identified TBP, TFIIB, and 9 of the 14 subunits of SAGA,
including Tra1, TAF6, TAF12, and Ada1 in Gal4-VP16 immunoprecipitates.
Although truly in vivo, the formaldehyde-based approach is
not site-specific and requires reversal of cross-linking after
immunoprecipitation. Thus, this method cannot distinguish which of the
9 subunits of SAGA cross-linked directly to the VP16 AD and which were
indirectly cross-linked. However, we note that in this study in
vivo cross-linking of Ada1 to VP16 AD was not reduced in a
These identifications of AD-interacting subunits clearly show the
limitations of each assay. First, Tra1p is the only SAGA component
that, because of its large size, cannot be analyzed by
His6-mediated cross-linking and conversely, Ada1p, TAF6, or TAF12 could not be analyzed by the label-transfer technique because their protein sizes (54-61 kDa) directly overlap with the broad signal
from the labeled activator (15). Second, in vitro, both NuA4
with its accessible Tra1 subunit and TFIID with its accessible TAF4,
TAF6, and TAF12 subunits bind Gal4 and VP16 ADs with specificity and
can mediate Gal4-VP16-dependent transcriptional activation in vitro (Refs. 42 and 43; Fig. 2B). In
vivo, however, only SAGA, containing both Tra1p and TAF6/12/Ada1,
is efficiently recruited by Gal4 AD to Gal4 target genes (32, 44),
whereas NuA4 is not detectably recruited at all (45) and TFIID only at
substochiometric levels (33) to the same target genes (TFIID is
recruited to the GAL1 promoter at about 20% of the level of
TBP and other general transcription factors and inactivation of a
TFIID-specific TAF compromises GAL1 transcription only
partially (33, 46, 47)). Moreover, we have shown that TFIID binds Gal4
AD with at least 10-fold lower affinity than SAGA does (Fig.
2C). This raises the possibility that Tra1p and TAF6/12/Ada1
as the direct AD-interacting subunits need to cooperate to achieve high
affinity binding to acidic ADs and that this high affinity binding is
the necessary prerequisite for a stoichiometric recruitment in
vivo.
What, then, is the physiological role of the interaction between TFIID
and acidic activators in yeast? We suggest that (i) TFIID may not be a
relevant in vivo target for Gal4 at all, (ii) TFIID may be
quantitatively recruited to GAL1 and be essential for its
transcription only under certain conditions, comparable with the
conditional requirement of Swi/Snf for GAL1 transcription (48), and/or (iii) the TFIID-AD interaction may not result in recruitment but in the release of an inhibitory interaction between TAF1 and the DNA-binding surface of TBP (49, 50). In support of the
last possibility, activators like Gal4 overcome the inhibitory TAF1-TBP
interaction, although this interaction poses a severe obstacle in
vivo to recruitment of the transcriptional machinery by artificial
recruitment, i.e. in the absence of an AD (51). Given the
functional redundancy of TFIID and SAGA (21) and the presence of
multiple AD-interacting subunits within these complexes, challenging
molecular genetic analyses will be required to determine the
contribution of each of the identified interactions for recruitment and
transcriptional activation in vivo. Analyses involving the shared HF-TAFs is further hampered by the fact that they are necessary for SAGA (52) and TFIID (53, 54) integrity and that changes in the
expression level of a histone-fold TAF can dramatically affect the
TFIID/SAGA ratio in vivo as well as the retention of both
TFIID and SAGA by Gal4 and VP16 AD from
WCE.2
In summary, we have demonstrated that His6-mediated
cross-linking in combination with affinity enrichment and genomic
tagging can be used as a powerful technique to study protein-protein
interactions. We have applied this technique to identify interaction
sites of ADs in native coactivator complexes and have complemented and extended the results obtained by in vitro label transfer and
in vivo formaldehyde cross-linking.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
10
7 M (3-7).
Moreover, many of these AD-target interactions display only moderate
specificity. For example, certain nontranscriptional proteins bind ADs
with strengths comparable with physiological targets. Furthermore,
there is a remarkable correlation between apparent AD "strength"
in vivo and binding to transcriptional and
nontranscriptional proteins in vitro, suggesting that
"stickiness" is an inherent and important property conferring
function to ADs (5). The most critical amino acids in ADs for both
in vivo function and in vitro binding are bulky
hydrophobic amino acids, which in the acidic ADs are exposed by their
vicinity to negatively charged amino acids (8-13). Hydrophobic
surfaces are typically interaction sites between polypeptides within
complexes, and it is likely that these surfaces are artificially
exposed when these polypeptides are taken out of their native
complexes. Based on the common negative-hydrophobic-negative residue
distribution pattern of acidic ADs, hydrophobic surfaces exposed in the
vicinity of positively charged amino acids are likely to interact, at
least to some degree, with acidic ADs. Therefore, in vitro
binding studies with individual subunits are potentially prone to
artifacts. Thus, it is an important challenge to determine the
interaction sites of ADs in the context of native complexes, for
instance by chemical cross-linking. However, this effort is hampered by
the weakness of AD-target interactions relative to the much stronger
interactions between the subunits of AD-interacting complexes.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1, and extracts
were prepared according to Woontner et al. (31).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
Immunological reagents for the detection of
specific subunits of transcription complexes.
Rectangles represent the indicated 15 subunits of yeast
TFIID and 14 subunits of SAGA as well as Med6 and Swi2. Note that after
completion of this work, three additional SAGA subunits were identified
by a proteomic approach (23). TFIID-specific subunits are shown in
medium gray, SAGA-specific subunits in
dark gray, and shared subunits as
light gray rectangles. Symbols
indicate antibodies raised and/or genomically triple-HA3
tagged strains constructed. The antibody against Tra1 was a gift of J. Workman. The old nomenclatures of yeast TAF subunits and their
corresponding human homologs are listed to the right of
yeast TFIID and SAGA subunits. HAT activity and homology to the core
histones H2A, H2B, H3, and H4 have been indicated for subunits of both
complexes at the very left.
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Fig. 2.
Binding of transcription complexes to acidic
ADs. A, extracts from four isogenic strains with
different factors genomically tagged were incubated with 900 nM GST or GST-VP16AD. HA3-tagged factors were
Med6p (mediator), TAF4 (TFIID), Ada1p (SAGA), and Snf2p
(Swi/Snf). Input, pooled extracts representing 10% of the
binding reactions. Extracts used for pull-downs are indicated on
top. B, TFIID and SAGA do not bind to
transcriptionally inactive derivatives of the Gal4p and VP16 ADs. Gal4
AD, Gal4 (841-875); Gal4 , Gal4 (841-854); VP16 AD, VP16
(420-490); VP16
, VP16(420-456/F442P). The top panel
shows pull-downs from the Ada1-HA3 extract, the
lower panel from the TAF4-HA3 extract.
C, Gal4 AD binds coactivator complexes with variable
affinities. Pull-down reactions contained varying concentrations of
Gal4 AD (30, 100, 300, 900 nM) to accentuate the relative
strength of AD-coactivator interactions. All reactions were separated
by SDS-PAGE, blotted to polyvinylidene difluoride membrane, and
incubated with anti-HA antibody.
View larger version (68K):
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Fig. 3.
The ADs of Gal4 and VP16 cross-link to TBP,
TAF12/yTAFII61,
TAF4/yTAFII48, Ada1p, and
TAF6/yTAFII60 from transcriptionally competent
extract (WCE). A, cartoon of binding and cross-linking
reaction. The x represents cross-links. B,
specific binding and cross-linking to TAF12. Top, immunoblot
with anti-TAF12 antibody. Lane 1, extract (10% of input).
Lanes 2-4, pull-down of a TAF12-containing complex with
GST-His6, GST-His6-Gal4pAD, and
GST-His6-VP16AD. Lanes 5-7,
His6-mediated cross-linking of bound complexes.
TAF12xVP162 and TAF12xGal42 represent
cross-linking complexes consisting of one TAF12 monomer and one
GST-His6-AD dimer. Below the anti-TAF12 blot is
the same blot stripped and reprobed with anti-Ada2 antibody. *,
residual TAF12 signal after stripping. C, same as top
panel of B, but including GST-Gal4 AD lacking a
His6 tag (Gal4). D, detection of
TAF4-HA3, TBP-HA3, Ada1-HA3,
and TAF6 cross-linking complexes.
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[in a new window]
Fig. 4.
Immunological detection of remaining TFIID
and SAGA subunits in cross-linking reactions. Complexes from
extracts were bound and cross-linked to His6-GST-ADs as
described in Fig. 3. The left lane of each
panel contains the cross-linking reaction with
Gal4 AD, the right panel with VP16 AD. The factor
immunologically detected is indicated below each panel. Each
factor was tested in cross-linking reactions from at least two
independently prepared extracts. *, Tra1p is shown together with the
subunits that do not cross-link to the ADs, although potential Tra1p-AD
cross-linking products may not separate from the monomeric 433-kDa
Tra1p.
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Fig. 5.
Analytical proteolysis of cross-linking
products. A, schematic outline of the reaction with the
major cross-linking and cleavage products. Immobilized
GST-His6-VP16AD with a cleavage site for the highly
specific TEV protease (indicated by the thick bar) were
bound and cross-linked to complexes from extract. After cross-linking,
part of the immobilized fusion proteins with covalently cross-linked
TFIID and SAGA subunits were treated with TEV protease before
denaturation. B, immunoblots of cross-linking and cleavage
reactions. All blots were probed with anti-HA antibody. As indicated in
panel A, A represents the uncleaved complex,
B the complex with one site cleaved, and C the
doubly cleaved complex. Note that the VP16 preparation used for
cross-linking of complexes from the TBP-HA3 tagged strain
differed from the preparation used for the other figures.
GST-His6-VP16AD dimers from this preparation migrate as
characteristic triplet bands and serve as additional control.
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Fig. 6.
Recombinant TAF9 and Ada2p bind and
cross-link to ADs. Recombinant TBP, TAF9-HA2, and
Ada2p were bound and cross-linked to the indicated GST-His6
fusion proteins. Reactions were performed and analyzed by
immunoblotting as described in Fig. 3. Top, reactions with
TBP as positive control, detected with anti-TBP antibody.
Middle, reactions with TAF9-HA2, detected with
anti-HA antibody. Bottom, reaction with Ada2p, detected with
anti-Ada2p antibody.
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Fig. 7.
Cross-linking reactions with purified TFIID
and SAGA. A, purified TFIID and partially purified SAGA
were tested for the presence of the TFIID-specific TAF4 and the
SAGA-specific Ada2p by immunoblotting. B, TAF6 and TAF12
cross-link to ADs in the context of both TFIID and SAGA. TFIID and SAGA
preparations used in A were bound and cross-linked to the
indicated GST fusion proteins. Cross-linked complexes were analyzed by
immunoblotting with anti-TAF6 and anti-TAF12 antibodies. C,
TBP directly binds ADs in the context of purified TFIID. TFIID was
bound and cross-linked to the indicated GST fusion proteins.
Cross-linked products were analyzed by anti- TBP immunoblotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
spt3 mutant, a condition in which AD cross-linkings of
both Tra1 and TAF12 were strongly reduced (19). This result implies
that Ada1 was not indirectly cross-linked to the AD via Tra1 or TAF12,
data consistent with our own. Here we have shown that Gal4 and VP16 ADs
cross-link with TAF6, TAF12, and Ada1 within native SAGA and with TAF6,
TAF12, and TAF4 (homologous to Ada1) subunits of native TFIID. Putative
TAF4-TAF12 and TAF6-TAF9 HF heterodimers have been mapped to two
different locations each within TFIID. At one of these locations, both
heterodimers colocalize and potentially could form a histone
octamer-like substructure (40), a structure that can also be generated
from recombinant proteins (41).
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ACKNOWLEDGEMENTS |
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We thank Christine Brown and Jerry Workman for providing antibodies against Tra1p and a strain expressing FLAG-tagged Tra1p.
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FOOTNOTES |
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* This work was supported by Grants DFG Me 1575 and SFB 474/A8 from the Deutsche Forschungsgemeinschaft (to K. M.) and GM52461 from the National Institutes of Health to (P. A. W.)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.
§ Present address: RiNA, Takustrasse 3, 14195 Berlin, Germany.
¶ Present address: Dept. of Genetics, University of Cologne, 50674 Köln, Germany.
** Present address: Wellcome/CR United Kingdom Institute, Dept. of Pathology, University of Cambridge, Cambridge CB2 1QR, UK.
To whom correspondence should be addressed: Dept. of
Microbiology, Goethe University, Marie-Curie-Stir. 9, N250, 60439 Frankfurt am Main, Germany. Tel.: 49-69-798-29522; Fax:
49-69-798-29527; E-mail: K.Melcher@em.uni-frankfurt.de.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M212514200
2 K. Melcher, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AD, activation domain; GST, glutathione S-transferase; HA, influenza hemagglutinin; HAT, histone acetyl transferase; HF, histone fold; MMPP, magnesium monoperoxyphtaleic acid; SAGA, Spt-Ada-Gcn5-acetyltransferase; TAF, TBP associated factor; TBP, TATA-binding protein; TEV, tobacco etch virus; WCE, whole-cell extract; TFIID, transcription factor IID.
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