Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch Cédex, C.U. de Strasbourg, France
Received 1 February 1999/Returned for modification 1 March
1999/Accepted 12 April 1999
 |
INTRODUCTION |
The RNA polymerase II
transcription factor TFIID is a multiprotein complex composed of the
TATA-binding protein (TBP) and a series of TBP-associated factors
(TAFIIs) (3, 7). Not only are TAFIIs
components of transcription factor TFIID, but distinct subsets of
TAFIIs are also components of the SAGA, PCAF, and
TFTC complexes (13, 14, 21, 31, 40). For human TFIID (hTFIID), cDNAs for 11 hTAFIIs have been characterized
(10, 16, 18, 24, 25).
Genetic and biochemical experiments show that some
TAFIIs are important for promoter recognition and
expression of a subset of promoters (15, 38, 39), while
others are more generally required for transcription in
Saccharomyces cerevisiae (2, 26, 27, 29). An
increasing body of results also shows that hTAFII28,
hTAFII135, and hTAFII105 can act as specific
transcriptional coactivators in mammalian cells. Expression of
hTAFII135 specifically potentiates activation by the
ligand-dependent activation function 2 (AF-2) of the nuclear
receptors (NRs) for all-trans retinoic acid (retinoic acid
receptor), thyroid hormone (thyroid hormone receptor), and
vitamin D3 (vitamin D3 receptor [VDR])
(24). Distinct domains of hTAFII135
interact specifically with Sp1, CREB, and E1A, and coexpression of the
TAFII135 domains with which these activators interact has a
dominant negative effect on their activity (23, 28, 33, 36).
Similar experiments have shown that hTAFII105 interacts
specifically with the p65 subunit of NF-
B and that
TAFII105 expression strongly potentiates activation by
NF-B in mammalian cells (42). Coexpression of
hTAFII28 and/or TBP strongly
potentiates activation by the viral Tax protein, and Tax interacts
directly with hTAFII28 and TBP to form a ternary
complex (8).
Expression of hTAFII28 also potentiates ligand-dependent
activation by the AF-2s of many NRs, the most dramatic effects being seen with the receptors for 9-cis retinoic acid (retinoid X
receptor), estrogen (estrogen receptor [ER]), and the VDR
(22). Deletion analysis showed that coactivator activity
required amino acids 150 to 179 in the C-terminal domain of
hTAFII28. Subsequent determination of the three-dimensional
structure of the hTAFII28/hTAFII18 heterodimer at 2.6-Å resolution by X-ray crystallography indicated that these two
proteins interact via a histone fold motif present in the C-terminal
domain of hTAFII28 and in the central region of
hTAFII18 (4). Amino acids 150 to 179 required
for coactivator activity form the amphipathic 
2-helix of the
hTAFII28 histone fold. In the
hTAFII28/hTAFII18 heterodimer, residues
on the hydrophobic face of the 2-helix make intermolecular
contacts with hTAFII18, while the residues on the mainly
hydrophilic solvent-exposed face are available for mediating
interactions with other proteins.
Although the ability of hTAFII28 to act as a
transcriptional coactivator did not require direct interactions with
the NRs, it apparently required interactions with TBP.
hTAFII28 interacts directly with TBP both in vitro and in
transfected mammalian cells (22, 25). This interaction
requires amino acids 150 to 179 of hTAFII28, since deletion
of this region dramatically reduced interactions with TBP. However, as
this region is also required for interaction with hTAFII18,
the possible roles of these different interactions in
hTAFII28 coactivator activity could not be determined.
We have used the structural information to better characterize the
amino acids required for hTAFII28 coactivator
activity. We show that coexpression of hTAFII28 with
the altered-specificity mutant TBP spm3 results in a
synergistic enhancement of NR AF-2-activated transcription from a
reporter plasmid with a mutated TGTA element. Mutation of several
residues on the solvent-exposed surface and one residue on the
hydrophobic surface of the 2-helix of the hTAFII28
histone fold abolishes this synergy. The amino acids on the
solvent-exposed surface are also required for hTAFII28 to
interact with coexpressed TBP.
We further show that mutations in the -helix H1' of TBP affect
interactions between TAFII28 and TBP. Several of these TBP mutations strongly reduce interaction with hTAFII28, while
one mutation results in increased interaction. Surprisingly, however, TBP mutations which reduce interactions with hTAFII28 do
not impair the functional synergy. In contrast, no synergy is observed
with the TBP mutant which shows increased interaction with
hTAFII28, although this mutant does support activation by
other activators.
| |
MATERIALS AND METHODS |
Construction of recombinant plasmids.
Mutations in
hTAFII28 were generated by PCR amplification with the
appropriate oligonucleotides and cloning of the resulting fragments in
expression vector pXJ41 (41). Mutations in TBP were
constructed in the same way in the TBP spm3 background and cloned in
expression vector pSG5. The previously described E271R and L275R
mutants (a kind gift from A. Berk) were recloned into the pSG5
expression vector. All plasmids were verified by automated DNA
sequencing, and further details of constructions are available on
request. The G4-NR, G4-VP16, and G4-AP2 expression vectors are also as
described previously (22, 25).
Transfection of Cos cells and immunoprecipitations.
Cos
cells were transfected by the calcium phosphate coprecipitation
technique, and immunoprecipitations were performed as previously
described (22, 25). Forty-eight hours following transfection, the cells were harvested by three cycles of
freezing-thawing in buffer A (50 mM Tris-HCl [pH 7.9], 20% glycerol,
1 mM dithiothreitol, and 0.1% Nonidet P-40) containing 0.5 M KCl. The
expression of the transfected proteins was verified on Western blots.
For immunoprecipitations, cell extracts were incubated for 1 h at
4°C with 1 to 2 µg of the indicated monoclonal antibodies (MAbs),
after which time 50 µl of protein G-Sepharose was added and
incubation was continued for another 2 h. The protein G-Sepharose
was then washed four times for 10 min each at room temperature with
buffer A containing 1.0 M KCl and once with buffer A containing 0.1 M
KCl. The resin was resuspended in sodium dodecyl sulfate-polyacrylamide
gel electrophoresis loading buffer, boiled for 5 min, and subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound
proteins were detected on Western blots with the indicated antibodies
with an ECL kit (Amersham). For functional assays, in addition to the expression vectors described for each figure, all transfections contained 2 µg of pXJ-LacZ as internal standard for luciferase assays, 5 µg of the TGTA-Fos-Luc reporter, and
pBSK
DNA as carrier. Transfections were performed in
dextran-charcoal-treated medium, and ligands were added [50 nM
all-trans-retinoic acid, 100 nM
1,25(OH)2D3, and 15 nM E2] at the same time as
the DNA-calcium phosphate coprecipitate. Cells were harvested 48 h
after transfection, and 
-galactosidase and luciferase assays were
performed by standard procedures. In all cases, similar results
(±10%) were obtained in at least three independent transfections, and
the results of typical experiments are shown in the figures.
Antibody preparation.
MAbs against hTAFII28
(15TA and 1C9), TBP (3G3), and the B10 epitope of the ER were as
previously described (1, 5, 18, 25).
| |
RESULTS |
Amino acids in the hTAFII28 histone fold required for
enhancement of NR activity in transfected mammalian cells.
Coexpression of hTAFII28 potentiates ligand-dependent
transcriptional activation by the ER and VDR AF-2s in transfected Cos cells (22, 24). To better understand the molecular mechanism of this effect, we tested the ability of hTAFII28 to
potentiate activation by the VDR and ER AF-2s in the presence of the
altered-specificity mutant TBP spm3 (35) with a promoter
with a TGTA rather than a TATA element.
Expression of a chimera containing the DNA binding domain of the yeast
activator GAL4 and the ligand binding domains (containing the AF-2) of
the hER [G4-ER(EF)] or the hVDR [G4-VDR(DE)] led to only a small
increase in the activity of a cotransfected luciferase reporter gene
driven by a G4-responsive promoter with a TGTA element (6)
(see Materials and Methods) (Fig.
1A and B, lanes 1 and 2).
Expression of either TBP spm3 or TAFII28(1-179)
significantly increased activation by G4-ER(EF) or G4-VDR(DE) (Fig. 1A
and B, lanes 3 and 4), while coexpression of both led to a much
stronger synergistic activation (lanes 3 to 5). All of the effects
described were ligand dependent (data not shown). Thus, coexpressed TBP and TAFII28 synergistically enhance activation by the AF-2s
of these NRs.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 1.
Functional analysis of hTAFII28 mutants. (A
and B) Graphic representation of luciferase assays (arbitrary units).
Cells were transfected with the vectors shown below each lane.
Transfections contained 250 ng of the G4-ER or G4-VDR expression
vectors, 1 µg of the TBP expression vector, 4 µg of the
hTAFII28 expression vectors, 5 µg of the luciferase
reporter vector, and 2 µg of the pXJ--galactosidase reporter as an
internal control. All transfections contained the appropriate ligands.
The structures of the G4-NR activator plasmids and the luciferase
reporter are schematized. The numbers represent the amino acid
coordinates in the natural receptors. The mutants used in panels A and
B are shown below the graph in panel A. (C) Transfections were as
described for panels A and B. The mutants used are schematized below
the graph. (D) Transfections contained 2 (lane 9) or 4 (lanes 5, 6, and
10) µg of the hTAFII18 expression vector as indicated
along with the amounts of the other expression vectors described above.
WT, wild type.
|
|
We have previously shown that mutation of three of the exposed glutamic
acid residues (E164P, E167P, and E168R) in the hTAFII28 2-helix abolishes hTAFII28 coactivator activity
(22). However, since two of these substitutions
introduced prolines which would disrupt the -helical structure,
this did not permit identification of precise amino acids within the
2-helix involved in activation.
To better define the hTAFII28 amino acids required for
coactivator activity, residues located on the hydrophobic and/or
hydrophilic faces of the 2-helix were mutated either
individually or in groups. Hydrophobic residues were replaced by
charged residues, and charged residues, predominantly E and D, were
replaced by alanine (see Materials and Methods) (Fig. 1A and B and
2A).
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 2.
hTAFII28-TBP and
hTAFII28-hTAFII18 interactions. (A) The
sequence of the hTAFII28 2-helix is shown. The numbers
indicate the amino acid coordinates. Amino acids exposed in the
hTAFII28/hTAFII18 heterodimer are underlined.
The amino acid substitutions in each mutant are shown below the
wild-type sequence. M25 contains a truncated 2-helix. (B)
Coexpression of TBP and hTAFII28 mutants. Extracts from
cells transfected with the vectors shown above each lane were analyzed
by immunoblotting with the MAbs shown below the panel. The locations of
TBP and the TAFII28 mutants are indicated. (C and D)
Coimmunoprecipitation of hTAFII28 mutants with TBP.
Extracts from cells transfected with the vectors shown above each lane
were immunoprecipitated with the anti-TBP MAb 3G3 or the
anti-hTAFII28 antibody 15TA as indicated above the panel,
and the immunoprecipitated proteins were analyzed on immunoblots with
the antibodies indicated below the panel. The positions of TBP and
hTAFII28 are indicated along with the heavy chain of the
antibody used in the immunoprecipitations revealed by the
peroxidase-conjugated secondary antibody used in the immunoblots. (E)
Coprecipitation of hTAFII28 mutants with
B10-hTAFII18. The layout is as described for panels C and
D. Lane 1 shows the precipitation of extracts from cells transfected
with only B10-hTAFII18. IgG(H), immunoglobulin G heavy
chain; IP, immunoprecipitation; WT, wild type.
|
|
The ability of hTAFII28 mutants with substitutions in the
2-helix to synergistically enhance activation by the ER or
VDR AF-2s in the presence of TBP spm3 was evaluated. Close to
wild-type activity was seen when mutant m21 (G156K-I157E) or m22
(V160E-V162K) was coexpressed with TBP spm3 (Fig. 1A and B, lanes 11 and 13). Similarly, the double substitutions in m8 (E167A-D171A) and
m10 (C173K-W176A) or the triple mutation m11 (G163E-L170K-E174A) also did not significantly affect the ability of hTAFII28 to
synergize with TBP spm3 (Fig. 1C, lanes 12 to 17). In contrast,
coexpression of mutants m19 (V151D-I152K), m20 (M154D), and m25, in
which the C-terminal portion of the helix is deleted, along with
TBP spm3 did not result in the strong increase in activation seen with wild-type TAFII28 with either activator (Fig. 1A and B,
lanes 5, 7, 9, and 19). These mutations have therefore completely
abrogated the synergy with TBP.
The single substitutions in m23 (E164A) and m24 (E168A) had only a
minor effect on activation by G4-ER and no significant effect on
activation by G4-VDR (Fig. 1A and B, lanes 14 to 17). The corresponding
double mutant m5, in which both residues were mutated (E164A-E168A),
was, however, completely inactive (Fig. 1A and B, lanes 20 and 21, and
1C, lanes 8 and 9). Similarly, mutant m6, in which K175 is additionally
mutated, was also inactive while mutation m4 (K175A alone) had no
effect (Fig. 1C, lanes 10 and 11 and 6 and 7). Therefore, synergy
between hTAFII28 and TBP is abolished by mutation (m5) of
two residues (E164 and E168) on the solvent-exposed surface and residue
M154 (m20) on the hydrophobic surface. Synergy is also abolished by
m19, in which residues on both surfaces are mutated.
The above results show that TAFII28 is functionally
limiting in the Cos cells, since the increase in its intracellular
concentration brought about during transfection increases activation by
the NR AF-2s. This would imply that its heterodimeric partner is not functionally limiting for these activators, since hTAFII18
was not overexpressed in these experiments. To test this idea,
hTAFII18 was coexpressed either in the absence or in the
presence of both TBP spm3 and hTAFII28.
As described above, coexpression of hTAFII28 and TBP spm3
strongly increased activation by the G4-VDR chimera (Fig. 1D, lanes 1, 3, 7, and 8). In contrast, coexpression of hTAFII18 did not potentiate VDR AF-2 activity either in the presence or in the absence
of TBP spm3 (lanes 5 and 6). Similarly, coexpression of hTAFII18 did not further potentiate the activation seen in
the presence of hTAFII28 and TBP spm3 (lanes 9 and 10).
Thus, in contrast to TAFII28, TAFII18 is not
functionally limiting, since its overexpression does not potentiate
activation by the VDR AF-2.
Residues in the 2-helix of the hTAFII28 histone fold
required for interaction with TBP.
To determine whether the above
mutations in hTAFII28 were defective in their interactions
with TBP and/or hTAFII18, they were coexpressed in Cos
cells, where we have previously shown interactions between these
overexpressed proteins (18, 22). In this way, interaction
assays are performed under the same conditions as those for the
functional assays, thus facilitating a comparison of the two.
The mutant TAFII28 proteins were expressed in Cos cells
along with TBP or hTAFII18, and the recombinant proteins
were detected in the transfected cell extracts by immunoblotting with
the appropriate MAbs (see Materials and Methods). All of the mutant
hTAFII28 proteins were expressed at comparable levels (Fig.
2B and data not shown). The complexes formed between the overexpressed
proteins were then precipitated with MAbs against TBP or
TAFII28.
As previously shown, hTAFII28(1-179) can be coprecipitated
by the anti-TBP MAb 3G3 following coexpression of both proteins in Cos
cells (Fig. 2C, lane 1). Compared to hTAFII28(1-179) wild type, interaction with TBP was not affected by hTAFII28
mutations m20, which abolishes coactivator activity, m21, and m22 (Fig. 2C, lanes 3 and 4, and data not shown, summarized in Fig.
3). Similarly, the double mutations m8
and m10 or the triple mutation m11 had no effect on interaction with
TBP (summarized in Fig. 3).
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 3.
The effects of mutations in hTAFII28 on
physical and functional interactions with TBP are summarized. WT, wild
type.
|
|
In contrast, coprecipitation with TBP was strongly reduced by mutations
m19 and m25 (Fig. 2C, lanes 2 and 5). Mutants m23 and m24 interacted
with TBP (Fig. 2C, lanes 6 and 7, and 2D, lanes 1 to 4), whereas with
m5, in which both residues were mutated, strongly reduced interaction
was observed (Fig. 2D, lanes 5 and 6). These results show that
hTAFII28 residues V151 and/or I152, E164, and E168 play
critical roles in the interaction with TBP.
The ability of these mutations to affect heterodimerization with
hTAFII18 was also determined by coexpression of the mutants with a B10 epitope-tagged derivative of hTAFII18 and
immunoprecipitation with the anti-B10 MAb (22, 25). When
compared with the wild-type proteins (Fig. 2E, lane 2), comparable
coprecipitation of all the mutants was observed (lanes 3 to 7 and data
not shown). Therefore, while some of the above hTAFII28
mutations significantly diminished interactions with TBP, they did not
abolish interactions between coexpressed hTAFII28 and
hTAFII18. This is true even for alleles, for example, m20
and m22, harboring radical mutations on the hydrophobic face of the
2-helix. Similarly, none of the mutations affected the
coprecipitation of hTAFII28 with hTAFII55 or
hTAFII135 (data not shown).
Amino acids in -helix H1' of TBP required for interaction with
hTAFII28.
The histone fold region of
hTAFII28 shows marked sequence homology with the C-terminal
region of the SAGA and PCAF subunit SPT3 (4). SPT3 in yeast
plays a role in transcription from a subset of promoters with
nonconsensus TATA elements (9). Genetic experiments have
shown a reciprocal allele-specific suppression of mutants in the
putative 2-helix of yeast SPT3 (ySPT3) (E240K) and the H1' helix of
yTBP (allele spt15-2; G174E) (9, 11, 20). This suggests that
these two helices mediate the functional and perhaps also physical
interactions between the respective proteins. The homology with SPT3
suggested to us that the H1' helix of hTBP may also be involved in
physical and functional interactions with hTAFII28.
Furthermore, mutation F237V in the yTBP C terminus is an intragenic
suppressor of the G174E mutation in the H1' helix, and yTBP mutation
K239E is an extragenic suppressor of the E240K mutation in the putative
2-helix of ySPT3 (11). This suggests that residues at the
C terminus of the TBP H2' -helix may also contribute to SPT3-TBP
interactions in accordance with the fact that this region and the H1'
helix are in close proximity in the folded TBP molecule (17,
30).
To test the possible contribution of both of these regions in
interactions with hTAFII28, TBP spm3 derivatives with
additional substitutions in the H1' helix or deletion of the C-terminal
region (Materials and Methods and Fig.
4A) were coexpressed along with wild-type
hTAFII28 in Cos cells. Each TBP mutant accumulated to similar levels in the transfected Cos cells when coexpressed along with
hTAFII28 (Fig. 4B). The coexpressed proteins were then
immunoprecipitated with MAb 3G3 directed against the extreme N
terminus of TBP (19).
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 4.
Mutations in TBP which affect interactions with
hTAFII28. (A) The sequences of the H1' and C-terminal
regions of hTBP are shown along with the amino acid coordinates. The
amino acid substitutions are shown below the wild-type sequence. The
asterisk indicates the stop codon in the C-terminal deletion. (B)
Coexpression of TBP mutants and hTAFII28. The layout is as
described for Fig. 1B. (C) Coprecipitation of hTAFII28 with
TBP mutants. The layout is as described for Fig. 1C. The lower panel
shows a longer exposure of the region of the gel containing
hTAFII28. IgG(H), immunoglobulin G heavy chain; IP,
immunoprecipitation; WT, wild type.
|
|
Compared with TBP spm3, interaction with hTAFII28 was not
affected by the mutation E271R in the N-terminal portion of H1' (Fig.
4C, lanes 1 and 2). In contrast, interaction with hTAFII28 was strongly reduced by mutations in the C-terminal portion of this
helix, L275R and L275E-T276A (lanes 3 and 6). Interaction with
TAFII28 was also strongly reduced by the mutation G272E
(lane 5), equivalent to mutation G174E of allele spt15-21 in yTBP.
Interestingly, interaction of hTAFII28 with mutant
L270A-E271A and with the 
C mutant was actually increased (Fig. 4C,
lanes 1, 4, and 7). Therefore, specific residues in the TBP H1' helix
are required for efficient interactions with hTAFII28.
A mutation in the H1' helix of TBP which specifically impairs
synergy with hTAFII28.
Mutations in -helix H1' of
TBP do not affect interaction with any of the identified basal factors
and have only a minor effect on the ability of TBP to support
activation by the VP16 and E1A activation domains in transfected cells
(6). We tested the ability of several of the above mutants
in this helix to support activation by the G4-NR chimeras. As described
above, coexpression of hTAFII28 and TBP spm3
synergistically enhanced activation by the ER AF-2 (Fig.
5A, lanes 3 to 6) TBP spm3 mutants E271R,
G272E, L275R, and L275E-T276A also synergized with hTAFII28
to enhance activation by the ER AF-2 (lanes 7 to 9 and 13 to 18 and
data not shown, summarized in Fig. 6C).
In contrast, expression of mutant L270A-E271A did not enhance
activation by the ER AF-2 activity in the presence of
hTAFII28 (lanes 10 to 12). Furthermore, no synergy was seen
with this TBP mutant in titration experiments over a 20-fold range of
the corresponding expression vectors (0.5 to 10 µg), nor in the
presence of coexpressed hTAFII18 (data not shown).
Similarly, the C mutant also failed to synergize with coexpressed
hTAFII28 (lanes 19 to 21).
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 5.
Functional analysis of TBP mutants. The layout is as
described for Fig. 2. Transfections contained 1 or 2 µg of the TBP
expression vectors along with 4 µg of the hTAFII28
expression vector as indicated. WT, wild type.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 6.
Effect of TBP mutations on activation by VP16 and AP2.
(A and B) Transfections contained 1 µg of the TBP expression vectors
and 100 ng of the G4-VP16 or G4-AP2 expression vector. (C) The effects
of mutations in TBP on physical and functional interactions with
hTAFII28 are summarized. WT, wild type. Exposed residues
are underlined.
|
|
Wild-type TBP spm3 and mutant L275R also synergized with
hTAFII28 to enhance activation by the VDR AF-2 (Fig. 5B,
lanes 3 and 4 and 6 to 8). However, as observed with the ER AF-2,
mutants L270A-E271A and C did not synergize with
hTAFII28 to enhance activation by the VDR AF-2 (lanes 9 to
14). Similar results were obtained with a G4-retinoic acid receptor
chimera (data not shown).
The ability of these mutants to support activation by unrelated
activators G4-VP16 and G4-AP2, whose activities are not enhanced by
coexpression of hTAFII28 (reference 22
and data not shown), was also tested. Comparable activities were seen
with wild-type TBP spm3, L270A-E271A, and L275R, whereas the activity
of mutant C was severely reduced (Fig. 6A and B). These results show
that mutant L270A-E271A conserves its intrinsic ability to mediate activation but is specifically unable to functionally cooperate with
hTAFII28, while mutant C is generally defective in its
ability to mediate activation.
| |
DISCUSSION |
The 2-helix of the hTAFII28 histone fold plays a
critical role in functional interactions with TBP.
We have
identified specific amino acids in the hTAFII28 2-helix
which are required for synergy with TBP in mammalian cells. Simultaneous mutation of amino acids E164 and E168, which are close
together on the solvent-exposed surface of the 2-helix (Fig.
7), abolishes functional cooperation with
TBP. This same mutation also results in a loss of interaction with TBP.
Therefore, the hydrophilic face of the 2-helix, which is not
involved in intermolecular interactions with hTAFII18,
mediates interaction with other proteins, one being TBP.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 7.
(A) A ribbon representation of the hTAFII28
histone fold. The locations of the N-, 1-, and 2-helices are
shown along with the LN and L1 loops. For the sake of clarity, the
3-helix has been deleted. The 2-helix has been aligned so that
the hydrophobic interface with hTAFII18 is oriented to the
rear, while the exposed residues are oriented toward the viewer. The
side chains of several pertinent amino acids are depicted. (B) Surface
contour map of the hTAFII28/hTAFII18
heterodimer. The locations of the hTAFII28 2-helix and
amino acids I152, E164, and E168 are indicated. M154 was also colored
but is invisible on the surface, showing that it is completely buried
in the interface with hTAFII18.
|
|
Functional synergy and interaction with TBP are also abolished by
mutation of V151 and I152. Of these two amino acids, only I152 is on
the exposed surface (Fig. 7). As none of the other mutations on the
hydrophobic face of the 2-helix abolish interaction with TBP, it is
probable that it is mutation of I152 which is responsible for the loss
of interaction with TBP. While I152 is not adjacent to E164 and E168,
it is nevertheless part of an epitope on the exposed face of the
2-helix involved in interactions with TBP. Furthermore, by analogy
with the results obtained with E164 and E168, it is probable that it is
the mutation of I152 which also causes the loss of synergy with TBP.
The m19 and m5 mutants discussed above, which have lost the ability to
synergize with TBP, have changes in amino acids on the exposed surface
of the 2-helix. However, synergy is also lost upon mutation of M154
(m20) on the hydrophobic surface. Since M154 is not on the exposed
surface of the 2-helix (Fig. 7A), it is unlikely that it contributes
to physical interactions with TBP, and indeed, coprecipitation of
mutant m20 with TBP is comparable to that of the wild type.
As M154 is on the hydrophobic interface with TAFII18, this
rather suggests that heterodimerization with TAFII18 is
involved in coactivator activity. However, m20 does not abolish
heterodimerization with hTAFII18. Indeed, mutation of
several other residues (I157 and V162), which must surely disrupt the
hydrophobic interactions between the hTAFII28 and
hTAFII18 2-helices, does not abolish heterodimer
formation and has no effect on synergy. Therefore, the tight
hTAFII28-hTAFII18 interaction, involving not
only the two 2-helices but also the strong interface formed by the
hTAFII28 N- and hTAFII18 1-helices
(4), cannot be totally disrupted by mutation of only one or
two amino acids in the 2-helix. Such mutations are nevertheless
likely to significantly affect heterodimer stability or conformation.
Our results show that the perturbations introduced by mutation of I157
and V162 do not affect synergy, whereas the changes in structure and/or
stability induced by mutation of M154 are not compatible with function.
Therefore, although it is not clear why M154 is particularly sensitive
to mutation, the result obtained with this mutant indicates that
interaction with hTAFII18 is required for coactivator activity.
Mutation of exposed residues of the N- and 1-helices of the
histone fold had no effect on synergy with TBP (18a),
further highlighting the unique and critical role played by the
2-helix in this process. All together, our results would suggest
that this reflects the ability of this helix to interact with factors essential for NR AF-2 activity via the exposed face and a requirement for heterodimerization with TAFII18.
Possible molecular mechanisms underlying the coactivator activity
of hTAFII28.
Our results show that specific residues
in the H1' -helix of TBP are required for interactions with
hTAFII28. Interaction with hTAFII28 was
severely reduced by mutation of G272 and L275. Mutation E271R had no
effect on interaction, while mutation L270A-E271A and deletion of the
C-terminal domain of TBP led to increased interaction with
hTAFII28. The effect of these mutations was specific to hTAFII28, since they did not affect, either
positively or negatively, interactions with coexpressed
hTAFII18 or hTAFII20 (18a).
While mutations in hTAFII28 which affect interaction with
TBP also affect synergy, reciprocal TBP mutations which clearly diminish interactions with hTAFII28 do not abolish the
synergy between these two proteins. This synergy is, however, abolished by the L270A-E271A mutation in TBP which increases interaction with
hTAFII28. This does not reflect a general loss of TBP
function, since this mutant supports activation by VP16 and AP2. This
mutant therefore exhibits a selective defect in its ability to
synergize with hTAFII28, which correlates with the
increased interaction.
How do TAFII28 and TBP synergize to enhance activation by
NRs? Our results would be consistent with the incorporation of
hTAFII28 and TBP spm3 into a TFIID complex comprising
endogenous TAFIIs. Indeed, we and others have previously
shown that a fraction of transfected TBP spm3 and hTAFII28
(and indeed other TAFIIs) do associate with the endogenous
cellular TFIID (12, 22, 32). Transfected
hTAFII28 may be assembled into TFIID complexes via TAF-TAF
rather than TAF-TBP interactions, explaining why the mutations in TBP
do not abolish functional cooperation. Heterodimerization with
TAFII18 is an obvious alternative interaction allowing
incorporation of TAFII28 into a TFIID complex.
In our experiments, incorporation of hTAFII28 and TBP spm3
into endogenous TFIID complexes does not necessarily arise by the modification of existing TFIID complexes. In the course of the 48 h of the experiment, several cell divisions take place and de novo
TFIID complexes are assembled. These new complexes are assembled under
conditions where the intracellular concentration of TAFII28
and/or TBP spm3 is much higher than normal. Such conditions would of
course favor the incorporation of these proteins during assembly of
TFIID complexes. The net result would be to increase the concentration
of TFIID complexes containing TAFII28 and TBP spm3.
Further evidence that association of hTAFII28 with
endogenous TFIID is required for activation comes from the observation that expression of hTAFII28 leads to enhanced activation by
the NR AF-2s even in the absence of TBP spm3 (we have previously made similar observations with TAFII135; for discussion, see
reference 24). This is in keeping with our
observation that expression of hTAFII28 alone suffices to
increase activation with a reporter with a TATA element
(22). The lower increase seen here with the TGTA-containing
reporter reflects the low affinity of the endogenous TBP for this
element, which is only partially compensated by the expression of
TAFII28. These results show that hTAFII28 expression facilitates NR AF-2-dependent activation via the
endogenous TFIID, an observation most readily explained by the
incorporation of hTAFII28 into endogenous TFIID.
Taken altogether, our results would suggest that the synergistic
activation observed here requires the formation of TFIID complexes with TBP spm3, which allows efficient recognition of the TGTA
element, and hTAFII28, which facilitates the efficient use
of the complexes containing TBP spm3 by the NR AF-2s.
The TFIID complexes comprising hTAFII28 and/or TBP spm3 may
efficiently mediate NR AF-2 activity due to interactions between hTAFII28 and factors required for NR AF-2 activity. Further
evidence for this comes from consideration of the effect of mutations
in hTAFII28. Since the hTAFII28 mutations
abrogate coactivator activity and this is not simply a consequence of
loss of interaction with TBP, it is probable that these mutations also
affect interactions with another protein(s) which is indispensable for
transcriptional activation.
The phenotype of the TBP mutant L270A-E271A is particularly interesting
in this respect. As discussed above, the hTAFII28 2-helix may interact with a protein other than TBP required for transcriptional activity. It is therefore possible that the increased interaction with this TBP mutant impairs these other
hTAFII28 interactions, resulting in a loss of coactivator
activity. While we cannot exclude more complex scenarios, altogether
our observations would be consistent with a model in which
hTAFII28 coactivator activity involves dynamic
hTAFII28-TBP interactions which have to be dissociated to
allow hTAFII28 to subsequently interact with other proteins
required for transcriptional enhancement.
One surprising observation was that deletion of the C-terminal residues
of the TBP H2' helix led to a loss of function. This TBP mutant not
only was defective in its ability to synergize with
hTAFII28 but was generally defective, since it did not
efficiently mediate activation by the VP16 activation domain. These
C-terminal residues are not known to be required for interaction with
other components of the preinitiation complex (6, 37), and
the mutations of Y329 and K337 had only mild effects on activated transcription in mammalian cells (6). Nevertheless, two
mutations in the equivalent region of yTBP (E236P and F237D) led to
defects in activated transcription but did not affect interaction with the TATA element (34). The F237D mutation led to a general
loss of TBP interactions with other basal transcription factors,
possibly due to an altered conformation. While the molecular basis for the loss of function seen with C-terminal deletion in hTBP is not
clear, our results show that this region is necessary for TBP function
in mammalian cells.
We thank A. Berk for expression vectors and reporter plasmids;
P. Chambon for support; L. Perletti and M. Vigneron for critical comments; S. Vicaire and D. Stephane for DNA sequencing; Y. Lutz and
the MAb facility; the staff of cell culture and oligonucleotide facilities; B. Boulay, J. M. Lafontaine, R. Buchert, and C. Werlé for illustrations; and Roussel-Uclaf for providing
1,25(OH)2D3.
G.M. and A.-C.L. were supported by fellowships from the Ligue Nationale
contre le Cancer and the Association pour la Recherche contre le
Cancer. This work was supported by grants from the CNRS, the INSERM,
the Hôpital Universitaire de Strasbourg, the Ministère de
la Recherche et de la Technologie, the Association pour la Recherche
contre le Cancer, the Ligue Nationale contre le Cancer, and the Human
Frontier Science Programme.
| 1.
|
Ali, S.,
Y. Lutz,
J. P. Bellocq,
M. P. Chenard-Neu,
N. Rouyer, and D. Metzger.
1993.
Production and characterization of monoclonal antibodies recognising defined regions of the human oestrogen receptor.
Hybridoma
12:391-405[Medline].
|
| 2.
|
Apone, L. M.,
C. A. Virbasius,
F. C. Holstege,
J. Wang,
R. A. Young, and M. R. Green.
1998.
Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA.
Mol. Cell
2:653-661[Medline].
|
| 3.
|
Bell, B., and L. Tora.
1999.
Regulation of gene expression by multiple forms of TFIID and other novel TAFII-containing complexes.
Exp. Cell Res.
246:11-19[Medline].
|
| 4.
|
Birck, C.,
O. Poch,
C. Romier,
M. Ruff,
G. Mengus,
A. C. Lavigne,
I. Davidson, and D. Moras.
1998.
Human TAFII28 and TAFII18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family.
Cell
94:239-249[Medline].
|
| 5.
|
Brou, C.,
S. Chaudhary,
I. Davidson,
Y. Lutz,
J. Wu,
J. M. Egly,
L. Tora, and P. Chambon.
1993.
Distinct TFIID complexes mediate the effect of different transcriptional activators.
EMBO J.
12:489-499[Medline].
|
| 6.
|
Bryant, G. O.,
L. S. Martel,
S. K. Burley, and A. J. Berk.
1996.
Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo.
Genes Dev.
10:2491-2504[Abstract/Free Full Text].
|
| 7.
|
Burley, S. K., and R. G. Roeder.
1996.
Biochemistry and structural biology of transcription factor IID (TFIID).
Annu. Rev. Biochem.
65:769-799[Medline].
|
| 8.
|
Caron, C.,
G. Mengus,
V. Dubrowskaya,
A. Roisin,
I. Davidson, and P. Jalinot.
1997.
Human TAFII28 interacts with the human T cell leukemia virus type I Tax transactivator and promotes its transcriptional activity.
Proc. Natl. Acad. Sci. USA
94:3662-3667[Abstract/Free Full Text].
|
| 9.
|
Collart, M. A.
1996.
The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters.
Mol. Cell. Biol.
16:6668-6676[Abstract].
|
| 10.
|
Dubrovskaya, V.,
A. C. Lavigne,
I. Davidson,
J. Acker,
A. Staub, and L. Tora.
1996.
Distinct domains of hTAFII100 are required for functional interaction with transcription factor TFIIF beta (RAP30) and incorporation into the TFIID complex.
EMBO J.
15:3702-3712[Medline].
|
| 11.
|
Eisenmann, D. M.,
K. M. Arndt,
S. L. Ricupero,
J. W. Rooney, and F. Winston.
1992.
SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae.
Genes Dev.
6:131-131.
|
| 12.
|
Farmer, G.,
J. Colgan,
Y. Nakatani,
J. L. Manley, and C. Prives.
1996.
Functional interaction between p53, the TATA-binding protein (TBP), and TBP-associated factors in vivo.
Mol. Cell. Biol.
16:4295-4304[Abstract].
|
| 13.
|
Grant, P. A.,
D. Schieltz,
M. G. Pray-Grant,
D. J. Steger,
J. C. Reese,
J. R. Yates, and J. L. Workman.
1998.
A subset of TAFs are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation.
Cell
94:45-53[Medline].
|
| 14.
|
Grant, P. A.,
D. E. Sterner,
L. J. Duggan,
J. L. Workman, and S. L. Berger.
1998.
The SAGA unfolds: convergence of transcription regulators in chromatin-modifying complexes.
Trends Cell Biol.
8:193-197.
[Medline] |
| 15.
|
Holstege, F. C.,
E. G. Jennings,
J. J. Wyrick,
T. I. Lee,
C. J. Hengartner,
M. R. Green,
T. R. Golub,
E. S. Lander, and R. A. Young.
1998.
Dissecting the regulatory circuitry of a eukaryotic genome.
Cell
95:717-728[Medline].
|
| 16.
|
Jacq, X.,
C. Brou,
Y. Lutz,
I. Davidson,
P. Chambon, and L. Tora.
1994.
Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor.
Cell
79:107-117[Medline].
|
| 17.
|
Kim, J. L., and S. K. Burley.
1994.
1.9 A resolution refined structure of TBP recognizing the minor groove of TATAAAAG.
Nat. Struct. Biol.
1:638-653[Medline].
|
| 18.
|
Lavigne, A. C.,
G. Mengus,
M. May,
V. Dubrovskaya,
L. Tora,
P. Chambon, and I. Davidson.
1996.
Multiple interactions between hTAFII55 and other TFIID subunits. Requirements for the formation of stable ternary complexes between hTAFII55 and the TATA-binding protein.
J. Biol. Chem.
271:19774-19780[Abstract/Free Full Text].
|
| 18a.
| Lavigne, A.-C., Y.-G. Gangloff, L. Carré, G. Mengus, C. Birck, O. Poch, C. Romier, D. Moras, and I. Davidson. Unpublished data.
|
| 19.
|
Lescure, A.,
Y. Lutz,
D. Eberhard,
X. Jacq,
A. Krol,
I. Grummt,
I. Davidson,
P. Chambon, and L. Tora.
1994.
The N-terminal domain of the human TATA-binding protein plays a role in transcription from TATA-containing RNA polymerase II and III promoters.
EMBO J.
13:1166-1175[Medline].
|
| 20.
|
Madison, J. M., and F. Winston.
1997.
Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae.
Mol. Cell. Biol.
17:287-295[Abstract].
|
| 21.
|
Martinez, E.,
T. K. Kundu,
J. Fu, and R. G. Roeder.
1998.
A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID.
J. Biol. Chem.
273:23781-23785[Abstract/Free Full Text].
|
| 22.
|
May, M.,
G. Mengus,
A. C. Lavigne,
P. Chambon, and I. Davidson.
1996.
Human TAFII28 promotes transcriptional stimulation by activation function 2 of the retinoid X receptors.
EMBO J.
15:3093-3104[Medline].
|
| 23.
|
Mazzarelli, J. M.,
G. Mengus,
I. Davidson, and R. P. Ricciardi.
1997.
The transactivation domain of adenovirus E1A interacts with the C terminus of human TAFII135.
J. Virol.
71:7978-7983[Abstract].
|
| 24.
|
Mengus, G.,
M. May,
L. Carre,
P. Chambon, and I. Davidson.
1997.
Human TAFII135 potentiates transcriptional activation by the AF-2s of the retinoic acid, vitamin D3, and thyroid hormone receptors in mammalian cells.
Genes Dev.
11:1381-1395[Abstract/Free Full Text].
|
| 25.
|
Mengus, G.,
M. May,
X. Jacq,
A. Staub,
L. Tora,
P. Chambon, and I. Davidson.
1995.
Cloning and characterization of hTAFII18, hTAFII20 and hTAFII28: three subunits of the human transcription factor TFIID.
EMBO J.
14:1520-1531[Medline].
|
| 26.
|
Michel, B.,
P. Komarnitsky, and S. Buratowski.
1998.
Histone-like TAFs are essential for transcription in vivo.
Mol. Cell
2:663-673[Medline].
|
| 27.
|
Moqtaderi, Z.,
M. Keaveney, and K. Struhl.
1998.
The histone H3-like TAF is broadly required for transcription in yeast.
Mol. Cell
2:675-682[Medline].
|
| 28.
|
Nakajima, T.,
C. Uchida,
S. F. Anderson,
J. D. Parvin, and M. Montminy.
1997.
Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors.
Genes Dev.
11:738-747[Abstract/Free Full Text].
|
| 29.
|
Natarajan, K.,
B. M. Jackson,
E. Rhee, and A. G. Hinnebusch.
1998.
yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex.
Mol. Cell
2:683-692[Medline].
|
| 30.
|
Nikolov, D. B., and S. K. Burley.
1994.
2.1 A resolution refined structure of a TATA box-binding protein (TBP).
Nat. Struct. Biol.
1:621-637[Medline].
|
| 31.
|
Ogryzko, V. V.,
T. Kotani,
X. Zhang,
R. L. Schlitz,
T. Howard,
X. J. Yang,
B. H. Howard,
J. Qin, and Y. Nakatani.
1998.
Histone-like TAFs within the PCAF histone acetylase complex.
Cell
94:35-44[Medline].
|
| 32.
|
Sadovsky, Y.,
P. Webb,
G. Lopez,
J. D. Baxter,
P. M. Fitzpatrick,
E. Gizang-Ginsberg,
V. Cavailles,
M. G. Parker, and P. J. Kushner.
1995.
Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein.
Mol. Cell. Biol.
15:1554-1563[Abstract].
|
| 33.
|
Saluja, D.,
M. F. Vassallo, and N. Tanese.
1998.
Distinct subdomains of human TAFII30 are required for interactions with glutamine-rich transcriptional activators.
Mol. Cell. Biol.
18:5734-5743[Abstract/Free Full Text].
|
| 34.
|
Stargell, L. A., and K. Struhl.
1996.
A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo.
Mol. Cell. Biol.
16:4456-4464[Abstract].
|
| 35.
|
Strubin, M., and K. Struhl.
1992.
Yeast and human TFIID with altered DNA-binding specificity for TATA elements.
Cell
68:721-730[Medline].
|
| 36.
|
Tanese, N.,
D. Saluja,
M. F. Vassallo,
J. L. Chen, and A. Admon.
1996.
Molecular cloning and analysis of two subunits of the human TFIID complex: hTAFII130 and hTAFII100.
Proc. Natl. Acad. Sci. USA
93:13611-13616[Abstract/Free Full Text].
|
| 37.
|
Tang, H.,
X. Sun,
D. Reinberg, and R. H. Ebright.
1996.
Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex.
Proc. Natl. Acad. Sci. USA
93:1119-1124[Abstract/Free Full Text].
|
| 38.
|
Verrijzer, C. P., and R. Tjian.
1996.
TAFs mediate transcriptional activation and promoter selectivity.
Trends Biochem. Sci.
21:338-342[Medline].
|
| 39.
|
Walker, S. S.,
W. C. Shen,
J. C. Reese,
L. M. Apone, and M. R. Green.
1997.
Yeast TAFII145 required for transcription of G1/S cyclin genes and regulated by the cellular growth state.
Cell
90:607-614[Medline].
|
| 40.
|
Wieczorek, E.,
M. Brand,
X. Jacq, and L. Tora.
1998.
Function of TAFII-containing complex without TBP in transcription by RNA polymerase II.
Nature
393:187-191[Medline].
|
| 41.
|
Xiao, J. H.,
I. Davidson,
H. Matthes,
J. M. Garnier, and P. Chambon.
1991.
Cloning, expression, and transcriptional properties of the human enhancer factor TEF-1.
Cell
65:551-568[Medline].
|
| 42.
|
Yamit-Hezi, A., and R. Dikstein.
1998.
TAFII105 mediates activation of anti-apoptotic genes by NF-kappaB.
EMBO J.
17:5161-5169[Medline].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
Present address: European Molecular Biology Laboratory, 69012 Heidelberg, Germany.
Present address: Institut de Pharmacologie et de Biologie
Structurale, CNRS, UPR 9062, 31077 Toulouse, France.
