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.

MATERIALS AND METHODS

Baculovirus Expression

()

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.

Preparation of Sf21 Cell Extracts and Affinity Purification of Recombinant Proteins

Expression in Escherichia coli and Protein Purification

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 NaHPO, 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.

Expression Vectors for in Vitro Translation

Construction of expression vectors encoding amino-terminally deleted dTAF110 mutants was performed by PCR-based approaches using pT110 as template and appropriate primers generating 5`-NdeI and 3`-XbaI overhangs. The PCR fragments were reinserted into NdeI/XbaI-cut pT110, 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 TAF138-307 was created by cleavage of pT110 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 TAF138-307, 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).

In Vitro Protein-Protein Interaction Assays

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.

SDS-PAGE and Silver Staining

Transient Transfection and CAT Assay

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) .

In Vitro Transcription

RESULTS

hPR Interacts Specifically with dTAF110 in Vitro

Figure 1: DNA-bound hPR interacts specifically with dTAF110. A, in vitro protein-protein interaction assays with DNA-bound hPR and radiolabeled dTAF110. Streptavidin-agarose beads lacking (-DNA) or containing (+DNA) 1 µg of biotinylated PRETATA-DNA were preincubated in the absence(-) or presence (+) of 5 µg of partially purified MH-hPR0. Each sample was tested for binding of dTAF110 by incubation with 4 µl of reticulocyte lysate containing [S]methionine-labeled dTAF110 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 dTAF110 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 TAF110 (TAF1-921(wt)) and various deletion constructs. The structural motifs of dTAF110 (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 dTAF110 shown to be sufficient for interaction with Sp1A and Sp1B (12) and hPR and dTAF30 (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 dTAF110 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, dTAF110 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 dTAF110 (Fig. 2B). These results confirm that specific interactions between hPR and dTAF110 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 dTAF110 as described under ``Materials and Methods.'' In lane 3, control beads lacking bound protein were used. Lane 1 contains 2% of the input of dTAF110. Where indicated (lanes 5-8), EtBr was included in the binding reaction. The position of bound dTAF110 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 dTAF110 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 dTAF110 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.

Amino acids 538-639 of hPR Are Sufficient for Interaction with dTAF110

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-ABcore and MH-Bcore). 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 dTAF110 (Fig. 4A). In contrast, two different deletion mutants lacking domain C but containing amino acids 538-555 of AF-1 (MH-ABcore and MH-Bcore) showed negligible binding of dTAF110, 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 dTAF110, while AF-2 and the core and modulatory regions of AF-1 are clearly not required. Minor variations in the level of dTAF110 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 dTAF110. 2.5% of the dTAF110 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-ABcore (lanes 6 and 7), or MH-Bcore (lanes 8 and 9) were used.

Amino Acids 373-787 of dTAF110 Are Sufficient for Interaction with hPR

Next, we analyzed a number of amino-terminal deletion constructs for their binding to MH-hPR0 (Fig. 5B, lowerpanel). dTAF110 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 dTAF110 (TAF138-307) 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 dTAF110. 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 dTAF110 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 dTAF110 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.

Sequences Comprising the dTAF110 Interaction Domain of hPR Mediate Transactivation in Vivo and in Vitro

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 PRETATA 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.

DISCUSSION

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 dTAF110. In this assay, EtBr caused a clear inhibition (Fig. 2), indicating that DNA is somehow involved. However, since dTAF110 alone does not stably bind to DNA (Fig. 1A, lane 5), we exclude the possibility that the association of dTAF110 with hPR loaded beads is simply due to capturing of DNA molecules containing bound dTAF110 through nonspecific interactions with the DNA binding domain of hPR. Furthermore, we are convinced that the binding of dTAF110 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 dTAF110 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 dTAF110 binding to Ni-NTA-agarose beads (Fig. 2A, 4, 5, and 6) rules out the possibility that dTAF110 is directly bound by a natural poly-histidine containing protein present in the reticulocyte lysate used to synthesize the labeled dTAF110 proteins. Third, the negative results obtained with two different hPR mutants (MH-ABcore, MH-Bcore, Fig. 4B), purified from infected Sf21 cells by our standard procedure, strongly argue against copurified poly-histidine containing Spodoptora proteins as being responsible for dTAF110 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 dTAF110 proteins used in this study were assayed without further purification, we cannot exclude a model in which the hPR-dTAF110 interaction is mediated or stabilized by an additional protein(s) present in the translation lysate. Highly purified recombinant dTAF110 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 dTAF110 has not been mapped to a particular region of dTAF110(11) , sequence similarities of CREB and Sp1 activation domains suggest that these activators may target the same domain of dTAF110(43) . As indicated in Fig. 5A, work of Tjian's group (12) has shown that the amino-terminal 308 amino acids of dTAF110 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 dTAF110. Together with the results of the Sp1-dTAF110 interaction, this study identifies dTAF110 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 dTAF110, Yokimori et al.(48) demonstrated that dTAF30 binds to sequences in the carboxyl-terminal part of dTAF110 (aa 572-921). In contrast to hPR, dTAF30 binding was absolutely dependent on the very carboxyl-terminal end of dTAF110. Therefore, the sequence requirements for binding of hPR and dTAF30 to dTAF110 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 dTAF110. Since two overlapping mutants (MH-ABcore, MH-Bcore) containing these 18 amino acids of AF-1 are not sufficient for binding to dTAF110, 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 dTAF110 is not available yet, we have not been able to prove that it also interacts with hPR. However, the part of dTAF110 sufficient for binding to hPR includes regions with extensive sequence similarity to the human homologue hTAF130.Our results thus raise the possibility that this part of TAF110 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.

FOOTNOTES

*

This work was supported by Deutsche Forschungsgemeinschaft Grant SFB354 (to L. K.-H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Max Delbrück Centrum für Molekulare Medizin, Robert-Rössler-Str. 10, D-13122 Berlin, Germany.

¶

To whom correspondence should be addressed. Tel.: 49-201-723-3178; Fax: 49-201-723-5904.

()

The abbreviations used are: TBP, TATA-binding protein; TAF, TBP-associated factor; PR, progesterone receptor; PRE, progesterone response elements; DBD, DNA binding domain; hPR, human progesterone receptor; CAT, chloramphenicol acetyltransferase; aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

()

L. Klein-Hitpass, unpublished data.

()

R. Tjian and N. Tanese, personal communication.

ACKNOWLEDGEMENTS

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