(Received for publication, April 7, 1995; and in revised form, June 22, 1995)
From the
Transcriptional activation of target genes by the human
progesterone receptor is thought to involve direct or indirect
protein-protein interactions between the progesterone receptor and
general transcription factors. A key role in transcription plays the
general transcription factor TFIID, a multiprotein complex consisting
of the TATA-binding protein and several tightly associated factors
(TAFs). TAFs have been shown to be required for activated transcription
and are, thus, potential targets of activator proteins. Using in
vitro interaction assays, we could identify specific interactions
between the progesterone receptor and the TATA-binding
protein-associated factor dTAF110. The
dTAF
110 domain responsible for the interaction is distinct
from that reported to suffice for binding to Sp1. Somewhat
surprisingly, deletion analysis indicated that the previously
identified activation functions 1 and 2 of the progesterone receptor
are not required for this interaction but pointed to an important role
of the DNA binding domain. In cotransfection experiments and an in
vitro transcription assay, the DNA binding domain of the
progesterone receptor displayed significant activation potential. These
findings, taken together, suggest that an interaction between the
progesterone receptor and TAF
110 may represent an
important step in the mechanism of activation.
In eukaryotes, at least seven basal transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ) are required for basal levels of transcription at promoters by RNA polymerase II in vitro (reviewed in (1) ). In mammalian systems, these factors assemble into functional initiation complexes in a highly ordered, stepwise fashion beginning with the binding of TFIID to the TATA box (1, 2, 3) , while in yeast a number of factors seem to be brought in as a preassembled RNA polymerase II holoenzyme complex(4) . Sequence-specific activators that bind to specific regulatory elements within distal promoter regions or enhancers are capable of stimulating the rate of transcription initiation. Although the precise mechanism is presently unknown, there is increasing support for models proposing that protein-protein interactions between activators and basal transcription factors play a decisive role in this process(5) . Such interactions might help to recruit these basal transcription factors or the preassembled RNA polymerase II holoenzyme complex to the TATA box, stabilize intermediates in the assembly of the initiation complex, or activate certain components by inducing conformational changes.
Biochemical
and molecular characterization of TFIID has shown that it is a
multi-subunit complex consisting of the TATA-binding protein (TBP) ()and a number of TBP-associated factors
(TAFs)(6, 7, 8) , ranging from 20 to 250 kDa
in size. In cell-free transcription systems reconstituted from
partially purified factors, recombinant TBP is able to support basal
transcription of TATA-only promoters(9, 10) . However,
unlike TFIID, TBP fails to mediate transcriptional stimulation by
various activators, indicating that TAFs are required for this
process(8, 10) . Consequently, a model has been
proposed, suggesting that different activators might interact with
different TAFs in the TFIID complex(3, 7) . Indeed,
interactions with subunits of Drosophila(11, 12, 13, 14, 15, 16) and
human (15, 17, 18) TFIID complexes have been
demonstrated for a growing number of transcription activators.
The progesterone receptor (PR) is a ligand-inducible transactivator that belongs to the superfamily of nuclear receptors(19, 20, 21) . Activated PR facilitates the formation of initiation complexes through binding to progesterone response elements (PREs) located in the promoter region of target genes(22) . Molecular analysis has shown that PR, like other transcription activators, consists of separable domains responsible for DNA binding (DBD) and transcriptional activation(23) . The amino-terminal activation function (AF-1) is constitutively active, whereas the activation function located within the carboxyl-terminal part (AF-2) requires hormone for its activity(24, 25) .
In the present study, possible
interactions of hPR with a subunit of the TFIID complex were examined
by protein-protein interaction assays. Our results show that the
110-kDa subunit of Drosophila TFIID (dTAF110) is
specifically bound by hPR in vitro and that this interaction
is mediated by specific domains of both proteins. Transfection studies
and in vitro transcription experiments revealed that the part
of hPR which mediates the interaction contains a previously
unidentified activation function.
Similarly, a cDNA encoding a His-tagged hTBP was constructed, starting from the expression vector pTM-hTFIID(9) . In the final pBlueBac derivative, the first ATG codon of the hTBP cDNA was preceded by a DNA sequence encoding MSHHHHHHTSGSPSLI.
Transfer vectors were cotransfected with linearized baculovirus DNA (Pharmingen) into Sf21 cells. Baculoviruses expressing the desired proteins were identified by SDS-PAGE, Western blotting with appropriate antibodies, or electrophoretic mobility shift assay with extracts derived from cells infected with plaque-purified recombinant viruses.
For protein expression, a 250-ml culture of E. coli TG1 containing pQE-hPR(DBD) was grown at 37 °C in
TB medium containing 100 µg/ml ampicillin to an A of 0.6. Expression was then induced by addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM. Cells were harvested 4 h later.
Bacterial pellets were resuspended in 10 ml of NaP buffer (300 mM NaCl, 50 mM NaH
PO
, 2 mM
-mercaptoethanol). Cells were lysed by sonication, and insoluble
material was removed by centrifugation for 30 min and 13.000 rpm in a
Beckmann JA20 rotor. To purify the MH
-hPR(DBD) protein, 100
µl of a 50% slurry of Ni-NTA resin (Qiagen) equilibrated in NaP
buffer was added following incubation at 4 °C for 1 h. Subsequent
washing steps included washing with NaP buffer, buffer D100 (20%
glycerol, 100 mM KCl, 25 mM HEPES, pH 7.9, 10 mM
-mercaptoethanol), buffer D100 plus 5 mM imidazole,
and buffer D100 plus 20 mM imidazole. MH
-hPR(DBD)
was eluted with buffer D100 plus 100 mM imidazole and 40
nM EGTA, dialyzed against 20 mM HEPES, pH 7.6, 50
mM KCl, 10% glycerol, 0.1 mM EGTA, 1.5 mM MgCl
, 1 µM ZnSO
, 2 mM dithiothreitol and stored at -80 °C. All buffers
contained protease inhibitors. Preparation of an E. coli mock
extract was performed following the same protocol, except that
untransformed E. coli TG1 cells were grown in media lacking
ampicillin.
Construction of expression vectors
encoding amino-terminally deleted dTAF110 mutants was
performed by PCR-based approaches using pT
110 as template and
appropriate primers generating 5`-NdeI and 3`-XbaI
overhangs. The PCR fragments were reinserted into NdeI/XbaI-cut pT
110, creating the vectors
TAF128-921, TAF373-921, and TAF666-921. In
TAF128-921 and TAF666-921, naturally occurring ATGs
encoding the methionine residues 128 and 666, respectively, served as
initiation codons within the NdeI site, while in
TAF373-921 a methionine codon was introduced upstream of amino
acid 373. TAF373-787 was constructed by cleavage of
TAF373-921 with XhoI and XbaI, Klenow
treatment, and religation. The construct TAF
138-307 was
created by cleavage of pT
110 with ClaI and SalI,
Klenow treatment, and religation of the vector fragment.
Numbers
included in the vector names indicate the amino acids of the
dTAF110 open reading frame present in the construct,
except for TAF
138-307, where the deleted residues are
indicated.
For the expression of human TFIIB a pSG5-based vector containing the complete open reading frame of human TFIIB was kindly provided by D. Reinberg (Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ). Luciferase was expressed from the control template provided with the TNT kit (Promega).
Interaction assays using oligonucleotide-bound hPR
were performed basically as described by Hoey et
al.(12) . 5`-Biotinylated DNA fragments containing two
PREs and a TATA box were synthesized by PCR using the vector
PRETATA-CAT (see below) as template and the
oligonucleotides Biotin-TGTAAAACGACGGCCAG and CGAATTCCTGCAGCCC as
primers. Binding of the 171-base pair fragments to streptavidin-agarose
beads (Pierce) was performed by overnight incubation at 4 °C in
buffer T (25 mM HEPES, pH 7.6, 1 mM EDTA, 0.01
mM ZnSO
, 2 mM dithiothreitol, 10%
glycerol, 0.01% Nonidet P-40) containing 0.1 M NaCl (buffer T
0.1 M) with subsequent removal of unbound DNA by three washing
steps. 15 µl of beads (50% suspension) lacking or containing 1
µg of DNA were incubated in the absence or presence of 5 µg of
partially purified MH
-hPR0 in buffer T 0.1 M and
0.1 µg/µl bovine serum albumin. After one wash with buffer T
0.1 M and two washes with buffer T 0.05 M, 4-8
µl of TNT lysate containing
[
S]methionine-labeled protein were added in a
total volume of 0.5 ml of buffer T 0.05 M and incubated for 2
h at 4 °C with constant agitation. Subsequently, the beads were
washed four times with 1 ml of buffer T 0.05 M. Bound proteins
were eluted with 40 µl of buffer T 1.0 M, and half of the
eluates were separated by SDS-PAGE. After fixing and treatment with
Enlightning (DuPont NEN), [
S]methionine-labeled
proteins were visualized by autoradiography.
For interaction assays
using proteins directly immobilized to a matrix, the His-tagged
proteins were bound to Ni-NTA-agarose beads (Qiagen, 10 µl of a 50%
suspension) by incubation in buffer T 0.1 M for 1 h at 4
°C. Washing of the beads, incubation with
[S]methionine-labeled proteins, and analysis
were done as described above.
To construct the eukaryotic expression vector
pSGN-MH-hPR0, the His-tagged cDNA was excised by NheI restriction from the corresponding pBlueBac transfer
vector (see above) and inserted into the NheI site of pSGN.
pSGN had been derived from pSG5 (30) by inserting an NheI 8-mer linker into the refilled BglII site.
pSGN-hPR(C) encoded a methionine residue followed by aa 556-639
of hPR0, and ARG and was made by a PCR-based approach. Correct reading
frames and the absence of mutations in PCR-amplified fragments were
verified by automated sequencing (ALF DNA-Sequencer, Pharmacia Biotech
Inc.) with appropriate primers.
Cos7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum and antibiotics. One day prior to transfection,
10 cells were seeded onto 6-cm dishes. Transfections were
performed using the Lipofectin method according to the
manufacturer's (Life Technologies, Inc.) recommendation. Briefly,
reporter DNA (2 µg) and expression vector (100 ng) were diluted
into 100 µl of OPTIMEM (Life Technologies, Inc.) and made up to a
total of 2.5 µg of DNA by the addition of empty expression vector.
This solution was mixed with an equal volume of OPTIMEM containing 6
µg of Lipofectin. 15 min after mixing, 1.8 ml of OPTIMEM was added,
and the liposome/DNA mixture was poured onto the cells, which had been
washed with phosphate-buffered saline and serum-free OPTIMEM. After 4
h, the solution was removed, and 3 ml of phenol red-free
Dulbecco's modified Eagle's medium containing 10% fetal
calf serum was added. Progesterone treatment was started by adding 3
µl of a 0.1 mM stock in ethanol; controls received 3
µl of ethanol. Cells were harvested 40 h after transfection.
Preparation of cell extracts and CAT assays were done as
described(31) .
Figure 1:
DNA-bound
hPR interacts specifically with dTAF110. A, in vitro protein-protein interaction assays with DNA-bound hPR
and radiolabeled dTAF
110. Streptavidin-agarose beads
lacking (-DNA) or containing (+DNA) 1 µg of biotinylated
PRE
TATA-DNA were preincubated in the absence(-) or
presence (+) of 5 µg of partially purified
MH
-hPR0. Each sample was tested for binding of
dTAF
110 by incubation with 4 µl of reticulocyte lysate
containing [
S]methionine-labeled
dTAF
110 as described under ``Materials and
Methods.'' Lane 1 contains 1 µl (25%) of the input
lysate. For a lower amount of input material, compare Fig. 5A. The molecular weights of radiolabeled marker
proteins separated in lane 2 are indicated. The position of
bound dTAF
110 is marked on the rightside of the autoradiogram. B, in vitro interaction
assay performed as in A with
[
S]methionine-labeled luciferase (luc). C, same as A, except that 8 µl of reticulocyte
lysate containing radiolabeled TFIIB were
added.
Figure 5:
Mapping of the dTAF110 domain
required for interaction with MH
-hPR0 and MH
-C. A, domain structure of wild type TAF
110
(TAF1-921(wt)) and various deletion constructs. The structural
motifs of dTAF
110 (hatched, S/T-rich; filled, Q-rich; stippled, charged) are shown as
described by Hoey et al.(12) . Restriction sites used
for creating carboxyl-terminal deletion constructs are indicated at the top. At the rightside of the panel, the potential of the different constructs for
interaction with MH
-hPR0 and MH
-C is
summarized. The regions of dTAF
110 shown to be sufficient
for interaction with Sp1A and Sp1B (12) and hPR and
dTAF
30
(48) are indicated at the bottom. B and C, interaction assays were
performed with Ni-NTA-agarose beads lacking(-) or containing
(+) 0.1 nmol of MH
-hPR0 (B) or 0.6 nmol of
MH
-C (C) and 4 µl of reticulocyte lysate
containing the radiolabeled dTAF
110 construct indicated at
the top of each lane.
The sensitivity of the assay
involving DNA-bound hPR was relatively low, probably due to gradual
dissociation of hPR-dTAF110 complexes from the DNA during
the washing steps. We thus analyzed whether the interaction would also
occur with MH
-hPR0 immobilized directly to Ni-NTA-agarose
beads through the amino-terminal His-tag. As shown in Fig. 2A, dTAF
110 was indeed bound on beads
loaded with MH
-hPR0 (lane4) but not on
control beads without MH
-hPR0 (lane3).
Furthermore, a His-tagged fusion protein representing XDCoH (kindly
provided by E. Pogge v. Strandmann and G.U. Ryffel), the Xenopus homologue of DCoH(36) , did not bind dTAF
110 (Fig. 2B). These results confirm that specific
interactions between hPR and dTAF
110 can also occur with
hPR immobilized to Ni-NTA-agarose beads.
Figure 2:
hPR
interacts with dTAF110 immobilized to a Ni-NTA matrix. A, partially purified MH
-hPR0 (10 µg) was
bound to Ni-NTA-agarose beads (lane 4) and tested for binding
of [
S]methionine-labeled dTAF
110 as
described under ``Materials and Methods.'' In lane
3, control beads lacking bound protein were used. Lane 1 contains 2% of the input of dTAF
110. Where indicated (lanes 5-8), EtBr was included in the binding reaction.
The position of bound dTAF
110 is indicated on the rightside of the autoradiograph. B, same as A, except that 5 µg of His-tagged XDCoH were immobilized
on the beads. Lane 1 contains 25% of the dTAF
110
input.
Even though the interaction
could be observed with hPR immobilized to Ni-NTA-agarose, an
involvement of DNA could not be excluded as in vitro transcription/translation lysate programmed with a significant
amount of dTAF110 expression plasmid was used in the
experiment. Therefore, the interaction assay was performed in the
presence of increasing amounts of the DNA intercalator ethidium bromide
(EtBr), which inhibits DNA binding and thus allows to discriminate
between DNA-dependent and DNA-independent protein
interactions(37) . As shown in Fig. 2A, EtBr up
to 100 µg/ml (lanes 5 and 6), a concentration
shown to inhibit DNA-dependent protein associations(37) , had
little effect on the interaction, while at relatively high
concentrations of 200 and 400 µg/ml (lanes 7 and 8) significant inhibition was obtained. However, even at the
highest EtBr concentration used, the association of hPR and
dTAF
110 could not be completely inhibited. The partial
sensitivity to EtBr suggests that there is a DNA-independent component
reflecting bona fide protein-protein interactions, which are possibly
further stabilized through DNA.
Figure 3:
Expression and purification of hPR and
deletion mutants as His-tagged fusion proteins in insect cells. A, at the top a schematic representation of the
domain structure of the hPR (formB) is given.
Homology regions A/B to E are indicated according to Meyer et
al.(38) . Below, the structures of the His-tagged
fusion proteins are shown. The hatchedbox represents
the core activating region of AF-1(38) . The His-tag at the
amino-terminal end of the proteins is depicted as a filledbox. At the rightside of the panel, the dTAF110-binding potential of the
different constructs is shown. B, SDS-PAGE analysis of the
recombinant PR proteins. Proteins were prepared from
baculovirus-infected cells as described under ``Materials and
Methods.'' 5 µg of each protein preparation as determined by
the Bradford assay were separated by SDS-PAGE and visualized by silver
staining. Molecular weights of marker proteins (lane 1) are
given.
To
analyze the interaction potential of the various hPR mutants, we
employed the assay in which the receptor proteins were immobilized to
Ni-NTA-agarose because of the advantage that one can also examine
mutants, which do not contain a DBD (MH-AB
core and
MH
-B
core). Using this set of mutants, we could
demonstrate that progressive deletion of AF-1 and AF-2 sequences up to
a construct containing only amino acids 538-555 of AF-1 and amino
acids 556-639 of the DBD (MH
-C) did not eliminate
binding of dTAF
110 (Fig. 4A). In contrast,
two different deletion mutants lacking domain C but containing amino
acids 538-555 of AF-1 (MH
-AB
core and
MH
-B
core) showed negligible binding of
dTAF
110, even when 6-fold higher amounts of the receptor
proteins were tested (Fig. 4B). Thus, our experiments
show that homology region C containing the DNA binding domain plays an
important role in the binding of dTAF
110, while AF-2 and
the core and modulatory regions of AF-1 are clearly not required. Minor
variations in the level of dTAF
110 binding observed with
the different receptor proteins are likely to be due to different
degrees of purity or, in the case of MH
-C, due to an
overestimation of the protein content by the protein determination
method (see Fig. 3B).
Figure 4:
Amino
acids 538-639 of hPR including the DBD are sufficient for binding
of dTAF110. A, Ni-NTA-agarose beads lacking (lane 3) or containing (lanes 4-10) 100 nmol of
the various hPR constructs indicated at the top of each lane were analyzed for binding of
[
S]methionine-labeled dTAF
110. 2.5%
of the dTAF
110 input is shown in lane 1. B,
interaction assays were performed as in A, except that beads
containing two different amounts of MH
-C (lanes 4 and 5), MH
-AB
core (lanes 6 and 7), or MH
-B
core (lanes 8 and 9) were used.
Next, we analyzed a number of amino-terminal deletion
constructs for their binding to MH-hPR0 (Fig. 5B, lowerpanel).
dTAF
110 constructs lacking amino acids 1-127
(TAF128-921) or 1-372 (TAF373-921) associated
efficiently with MH
-hPR0 (lanes 16 and 18). Further truncation up to amino acid 665 created a protein
(TAF666-921) that showed increased binding to the Ni-NTA matrix (lane 19) but no specific association with
MH
-hPR0-loaded beads (compare lanes 19 and 20). Consistently, an internal deletion mutant lacking amino
acids 138-307 of dTAF
110 (TAF
138-307)
retained its ability to interact with MH
-hPR0 (lanes 21 and 22). Thus, the amino-terminal border of the hPR
interaction domain maps between amino acids 373 and 666 of
dTAF
110. As summarized in Fig. 5A, using
MH
-C instead of MH
-hPR0 to determine the
carboxyl- and amino-terminal borders of the hPR interaction domain of
dTAF
110 gave identical results (Fig. 5C).
In addition, a deletion construct lacking both amino- and
carboxyl-terminal sequences (TAF373-787) was tested for its
interaction with MH-hPR0 and various hPR deletion
constructs. As shown in Fig. 6, TAF373-787 bound
efficiently to MH
-hPR0 (lane 4) and all of the hPR
deletion constructs shown to be capable of binding TAF1-921,
including MH
-C (lane 10). Therefore,
carboxyl-terminal sequences spanning amino acids 373-787 of
dTAF
110 are sufficient for specific interaction with amino
acids 538-639 of hPR.
Figure 6:
Amino acids 373-787 of
dTAF110 are sufficient for interaction with hPR and
various deletion mutants. Protein-protein interaction assays with
Ni-NTA-agarose beads lacking (lane 3) or containing 100 nmol
of the indicated PR constructs (lanes 4-10) were
performed as described in Fig. 5A with 4 µl of
radiolabeled TAF373-787-containing reticulocyte lysate. 2.5% of
the input is shown in lane 1.
Figure 7:
The DNA binding domain of hPR contains a
transactivation function. A, Cos7 cells were cotransfected
with expression vectors encoding the indicated PR proteins and a CAT
reporter construct with two PREs in front of a minimal promoter
(PRETATA-CAT). Control transfections were performed using
the pSGN expression vector lacking a cDNA insert. CAT activities in
untreated (openbars) and progesterone-treated cells (filledbars) were normalized to the values obtained
with the empty expression vector. Similar results were obtained in CV1
cells. B, nuclear extracts from rat liver were incubated
without recombinant protein or the indicated amounts of E. coli mock extract, MH
-hPR(DBD) and MH
-hPR0. In vitro transcription reactions were performed as described
under ``Materials and Methods.'' Appropriate autoradiographs
were scanned with a laser densitometer, and signals representing
correctly initiated PRE
TATA transcripts were normalized to
the internal control (TATA-G400).
For analysis of
the transcriptional activation potential of the DBD of the hPR in
vitro, we used a cell-free transcription system, which uses rat
liver nuclear extract as a source for general transcription
factors(29, 34, 35) . Two different test
genes containing a TATA box or two PREs and a TATA box in front of a
G-free cassette (33) were transcribed simultaneously in all
reactions. To determine fold activations, correctly initiated
transcripts from PRETATA were normalized to the amount of
transcripts obtained from the template containing the TATA box only. As
the DNA binding activity in vitro of the E.
coli-expressed His-tagged amino acids 556-664 of hPR
(MH
-hPR(DBD)) purified by Ni-NTA chromatography was higher
than of the Sf21-expressed amino acids 538-639
(MH
-C), we decided to use MH
-hPR(DBD) in our in vitro transcription experiments. Amino acids 556-664
of hPR are homologous to the minimal deletion mutant of the human
estrogen receptor, which is sufficient for stable binding to DNA in
vitro(40) . As shown in Fig. 7B, addition
of recombinant MH
-hPR0 activated transcription
approximately 15-fold, whereas addition of an E. coli mock
extract had no effect. Saturating amounts of MH
-hPR(DBD)
activated transcription to about 13% of the activity observed with
MH
-hPR0, which agrees well with the value obtained in the
transfection experiments. Together, these data prove that the DBD of
hPR contains significant transactivation potential.
To detect the interactions between hPR and
dTAF110 in vitro and to define the domains
involved, we used partially purified hPR proteins bound to a Ni-NTA
matrix and in vitro synthesized dTAF
110. In this
assay, EtBr caused a clear inhibition (Fig. 2), indicating that
DNA is somehow involved. However, since dTAF
110 alone does
not stably bind to DNA (Fig. 1A, lane 5), we
exclude the possibility that the association of dTAF
110
with hPR loaded beads is simply due to capturing of DNA molecules
containing bound dTAF
110 through nonspecific interactions
with the DNA binding domain of hPR. Furthermore, we are convinced that
the binding of dTAF
110 to the Ni-NTA matrix is due to an
interaction with the hPR and not mediated by a natural poly-histidine
containing protein, for example TFIIA(41, 42) , either
present in the reticulocyte lysate or copurified with the PR proteins
from infected Sf21 cells for the following reasons. First, we
were able to detect hPR-dependent dTAF
110 binding also in
a different assay, in which the hPR was bound to biotinylated DNA
fragments immobilized to streptavidin beads (Fig. 1A).
Second, the PR dependence of dTAF
110 binding to
Ni-NTA-agarose beads (Fig. 2A, 4, 5, and 6) rules out
the possibility that dTAF
110 is directly bound by a
natural poly-histidine containing protein present in the reticulocyte
lysate used to synthesize the labeled dTAF
110 proteins.
Third, the negative results obtained with two different hPR mutants
(MH
-AB
core, MH
-B
core, Fig. 4B), purified from infected Sf21 cells by
our standard procedure, strongly argue against copurified
poly-histidine containing Spodoptora proteins as being
responsible for dTAF
110 binding. Furthermore, in a
chromatographic analysis of a MH
-C preparation on a
Superose 12 gel filtration column, only one peak of binding activity
was observed, which perfectly coincided with the single
MH
-C polypeptide peak at 13 kDa (data not shown). However,
since the dTAF
110 proteins used in this study were assayed
without further purification, we cannot exclude a model in which the
hPR-dTAF
110 interaction is mediated or stabilized by an
additional protein(s) present in the translation lysate. Highly
purified recombinant dTAF
110 will be required to
investigate this issue.
Prior to this study, two activators have
been shown to interact with dTAF110, namely Sp1 and
CREB(11, 12, 43) . Although the interaction
between CREB and dTAF
110 has not been mapped to a
particular region of dTAF
110(11) , sequence
similarities of CREB and Sp1 activation domains suggest that these
activators may target the same domain of
dTAF
110(43) . As indicated in Fig. 5A, work of Tjian's group (12) has
shown that the amino-terminal 308 amino acids of dTAF
110
suffice for interaction with Sp1. In contrast, our experiments
establish that the interaction with hPR does not involve the
amino-terminal region but depends on a more carboxyl-terminal
nonoverlapping domain of dTAF
110. Together with the
results of the Sp1-dTAF
110 interaction, this study
identifies dTAF
110 as the first TAF containing at least
two distinct interaction surfaces mediating contacts with activators.
Due to the multiplicity of activators and the limited number of TAFs,
we expect that multiple interaction domains within TAF polypeptides
will turn out as a more general feature of these important molecules.
Work of the groups of Tjian and Roeder (44, 45, 46, 47, 48) has
indicated that dTAF110 makes additional contacts with
several other TAFs of the TFIID complex. While most TAF-TAF
interactions have not been mapped to particular domains of
dTAF
110, Yokimori et al.(48) demonstrated that dTAF
30
binds to
sequences in the carboxyl-terminal part of dTAF
110 (aa
572-921). In contrast to hPR, dTAF
30
binding
was absolutely dependent on the very carboxyl-terminal end of
dTAF
110. Therefore, the sequence requirements for binding
of hPR and dTAF
30
to dTAF
110 are clearly
different.
Perhaps the most surprising result of this study is that
an hPR construct (MH-C) containing only 18 amino acids of
AF-1 (aa 538-555) and the DBD (aa 556-639) is sufficient
for interaction with dTAF
110. Since two overlapping
mutants (MH
-AB
core, MH
-B
core)
containing these 18 amino acids of AF-1 are not sufficient for binding
to dTAF
110, the interaction motif is at least partly
contained within the DBD of hPR. Another example for an interaction
between a DBD and a TAF was recently described by Chiang and Roeder (18) , who demonstrated that the DBD of Sp1 binds to a 55-kDa
subunit of human TFIID in vitro. Our results thus support the
concept that TAFs can interact with multiple distinct domains of
transactivators(18) .
By cotransfection and in vitro transcription experiments, we were able to demonstrate that the
DBD of hPR, which has not been tested for transactivation potential in
the studies defining AF-1 and AF-2 of
hPR(24, 25, 38) , is able to mediate
transcriptional activation through PREs located in front of a minimal
promoter, albeit with reduced efficiency compared to wild type hPR (Fig. 7). The presence of a previously unidentified activation
function within the hPR domain mediating the interaction with
dTAF110 in vitro clearly supports our notion that
this interaction reflects an important step in the mechanism of
transactivation by hPR.
Interestingly, mutational analysis of the rat and human glucocorticoid receptors (49, 50) and the Xenopus estrogen receptor (51) has also revealed the presence of a transactivation function within the respective DBDs. While activation by the glucocorticoid receptor DBDs pointed to a role of a basic region immediately following the second zinc finger(49, 50) , activation by the DBD of the estrogen receptor may be mediated by a short acidic tract at the carboxyl terminus of the DBD(51) . Thus, the presence of an activation function located in the DBD might be a more general feature of steroid receptors.
Analogous to the studies that identified interactions of
Sp1 and CREB with dTAF110(11, 12) , we
have investigated interactions between a mammalian activator and a Drosophila TAF, i.e. between proteins from
evolutionary widely separated organisms. Since the cDNA encoding the
human homologue of dTAF
110 is not available yet, we have
not been able to prove that it also interacts with hPR. However, the
part of dTAF
110 sufficient for binding to hPR includes
regions with extensive sequence similarity to the human homologue
hTAF
130.
Our results thus raise the
possibility that this part of TAF
110 contains a conserved
motif mediating interactions with the highly conserved DBDs of members
of the steroid receptor superfamily.
Ing et al.(52) have reported that hPR and a truncated chicken PR
synthesized in reticulocyte lysate and E. coli, respectively,
interact specifically with the basal transcription factor TFIIB. In
contrast, using baculovirus-expressed hPR that is transcriptionally
active (Fig. 7B), we have been unable to detect
association of hPR and TFIIB (Fig. 1B). It remains to
be seen whether the different sources of the receptors or experimental
differences may account for the discrepancy between the two sets of
data. Interestingly, the region required for interaction with TFIIB has
been mapped to a 168-amino acid fragment of chicken PR, which includes
the highly conserved DBD(52) . Since we have shown that the
corresponding region of hPR mediates interaction with
dTAF110, it is feasible that the DBDs of PRs are not only
involved in the formation of receptor dimers(21, 25) ,
in the recognition of the PREs(21) , and in interactions with a
TAF, but also in protein-protein interactions with TFIIB(52) .
Since the DBD of hPR accounts only for a small part of the overall
transactivation potential of hPR (Fig. 7), it is likely that hPR
contacts additional, yet unidentified components of the initiation
complex, possibly through AF-1 and AF-2. Multiple interactions with
members of the transcription machinery have been proposed for a growing
number of transcriptional activators, including VP16 and the
glucocorticoid receptor(13, 53) . Such a multiplicity
of protein-protein contacts may enable the hPR to affect subsequent
rate-limiting steps in the process of preinitiation complex formation
and contribute to the highly synergistic activation observed with
multiple PRs bound to closely adjacent PREs.