DNA Binding by the ETS-domain Transcription Factor PEA3 Is Regulated by Intramolecular and Intermolecular Protein·Protein Interactions*

Amanda GreenallDagger , Nicola WillinghamDagger , Ed Cheung§, David S. Boam§, and Andrew D. SharrocksDagger §

From the Dagger  School of Biochemistry and Genetics, The Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH and the § School of Biological Sciences, University of Manchester, 2.205 Stopford Bldg., Oxford Road, Manchester M13 9PT, United Kingdom

Received for publication, December 21, 2000, and in revised form, January 16, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3·USF-1·DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eukaryotic transcription factors are grouped into families based on the type of DNA-binding domains that they possess. Due to high sequence conservation within these DNA-binding domains, members of particular families bind to similar DNA sequences. Thus, mechanisms must be in place to prevent promiscuous binding to inappropriate sites and provide specificity to ensure binding to the correct sites. One such mechanism is autoinhibition, whereby proteins block their own DNA binding by employing cis-acting inhibitory modules (reviewed in Ref. 1). These modules also often act to block other activities of transcription factors such as transcriptional activation or nuclear localization. Conversely, mechanisms exist to reverse these inhibitory interactions and allow recruitment of proteins to the correct promoter/enhancer sites at appropriate times. Activating triggers can include post-translational modifications such as phosphorylation or the binding to other coregulatory proteins or transcription factors.

The transcription factor Ets-1 represents a paradigm for this type of regulatory mechanism. Ets-1 is part of the larger ETS-domain family of proteins that bind to sequences containing the central core trinucleotide GGA (2, 3). Sequences flanking this motif permit subtle variations in binding specificities for individual family members. Due to this relatively low stringency of binding, many ETS-domain transcription factors possess autoinhibitory domains, including Elk-1 (4), PU.1 (5), and members of the PEA3 subfamily (6, 7, 8). DNA binding by Ets-1 is tightly controlled by an inhibitory module composed of alpha -helices lying N- and C-terminal to the ETS-domain (9, 10). Disruption of one of the N-terminal alpha -helices results in relief of autoinhibition and permits high affinity DNA binding (11, 12). Ets-1 has been shown to bind to DNA cooperatively with several transcription factors including USF-1 (13) and AML-1/CBFalpha 2 (14-16). In the case of the Ets-1·AML-1 complex, this cooperativity appears to result from interaction of the two proteins and subsequent mutual disruption of inhibitory modules in each protein, thereby leading to a loss of autoinhibition (13, 17, 18). Alternative routes for relief of autoinhibition have not yet been demonstrated in Ets-1, but in a different ETS-domain transcription factor Elk-1, phosphorylation has been shown to act as a trigger to relieve autoinhibition and thereby stimulate DNA binding (14, 19). Furthermore, in the case of Elk-1, DNA binding is also subject to further control and is negatively regulated in trans, by members of the Id subfamily of helix-loop-helix (HLH)1 proteins (20).

In this study we have investigated how DNA binding is regulated in a member of a different subfamily of ETS-domain transcription factors, PEA3. The PEA3 group of ETS-domain transcription factors contains three different proteins, PEA3/E1AF, ERM, and ER81 (reviewed in Ref. 21). Members of this group of proteins are conserved at both the sequence and functional levels in vertebrates as diverse as humans and zebrafish (6, 22). Biological roles for PEA3 family members include promoting muscle cell differentiation (23) and the definition of connecting muscle motor and sensory neurones (24). Embryonic expression patterns and aberrant expression in metastatic tumors, suggest a role for PEA3 proteins in regulating cell motility during organogenesis/development and during tumor progression (reviewed in Ref. 21).

We recently demonstrated that in common with the other ETS-domain transcription factors, murine PEA3 and human ERM (7, 8), DNA binding by zebrafish PEA3 is subject to stringent regulation by autoinhibitory mechanisms (6). Here we investigate the molecular basis for this inhibitory process in zebrafish PEA3 and demonstrate one route by which this autoinhibition can be relieved. Our results point to an intriguing model in which the regions that autoregulate DNA binding by PEA3 also participate in cooperative DNA binding with the partner protein USF-1, thereby coordinately regulating the loss of autoinhibition, with the stimulation of nucleoprotein complex formation.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Plasmid Constructs-- The following plasmids were used for making proteins by in vitro transcription/translation. pcDNA3Id3 (encoding full-length Id3) (25), pAS477 (encoding full-length zebrafish PEA3; amino acids 1-494), pAS283 (encoding PEA3 amino acids 341-432), pAS330 (encoding PEA3 amino acids 1-432), and pAS331 (encoding PEA3 amino acids 341-494) have been described previously (6). pAS530 (encoding PEA3 amino acids 321-494), pAS531 (encoding PEA3 amino acids 277-494), pAS532 (encoding PEA3 amino acids 258-494), pAS533 (encoding PEA3 amino acids 258-452), pAS604 (encoding PEA3 amino acids 321-452), pAS605 (encoding PEA3 amino acids 229-494), pAS774 (encoding PEA3 amino acids 321-474), and pAS1153 (encoding PEA3 amino acids 321-432) are deletion derivatives of pAS477 and were subcloned as NcoI-SacI-cleaved PCR products into the same sites in pAS37 (26). The following plasmids encode the indicated mutant derivatives of PEA3. pAS619 (PEA3-(258-452)-R339P) and pAS1165 (PEA3-(258-452)-V330P) were constructed from the template pAS533. pAS1155 (PEA3-(321-474)-E460P), pAS1156 (PEA3-(321-474)-L468P), pAS1163 (PEA3-(321-474)-K450P), pAS1164 (PEA3-(321-474)-F453P), and pAS1166 (PEA3-(321-474)-I437P) were constructed from the template pAS774. Site-directed mutations were introduced by a two-step PCR protocol using a mutagenic primer and two flanking primers as described previously (27). Details of mutagenic primers can be supplied upon request.

The following plasmids were used for expressing PEA3 derivatives as C-terminal hexahistidine/FLAG-tagged proteins in Escherichia coli. pAS609, pAS610, pAS1161, and pAS1162 (encoding PEA3 derivatives; PEA3-(321-452), PEA3-(341-432), PEA3-(341-452), and PEA3-(321-432)) were constructed by inserting the NcoI-XhoI-digested PCR-derived fragments into the same sites in pET-Hnef-PFH (28). C-terminal hexahistidine-tagged USF-1 derivatives were expressed in E. coli from the plasmids pET3his-USFII (encoding full-length USF-1), pEC12 (encoding USF-1-(144-310)) and pEC14 (encoding USF-1-(197-310)) (29). The following plasmids were used for expressing GST fusion proteins in E. coli: pGEX-Id2 (encoding full-length Id2) (30) and pAS462 (encoding PEA3-(341-432)) (6). pAS992, pAS998, and pAS999, encode GST fusions to PEA3-(1-494), PEA3-(1-337), and PEA3-(430-494), respectively, and were constructed by inserting either an NcoI/XhoI fragment from pAS501 or BamHI/XbaI- cleaved PCR products (primer pairs ADS730/753 and ADS754/755, respectively) into the same sites in pGEX-KG (31).

The following plasmids were constructed and used for use in mammalian cell transfections. pCH110 encodes a SV40 promoter-driven beta -galactosidase gene (Amersham Pharmacia Biotech). pAS501 (encoding full-length zebrafish PEA3) (6), pSG5-USF-1 (pAS849; encoding full-length human USF-1), and pSG5-USF-2 (pAS850; encoding full-length human USF-2) (32) have been described previously. pAS849 and pAS850 were also used for in vitro transcription-translation purposes. pAS1123 encodes a luciferase gene driven by two copies of the E-box/ets motif derived from the HIV-1 LTR and was constructed by inserting the annealed oligonucleotides ADS671/ADS672 into the SalI site of pAS819 (pT81, kindly provided by E. Oettgen). Both copies are inserted in the inverted orientation in comparison to the natural HIV-1 LTR.

The following plasmids were constructed for use in yeast one-hybrid assays. pAS468 encoding PEA3-(341-494) was constructed by ligating a BamHI/SpeI-cleaved PCR product (primer pairs ADS288/262 on template pAS333) into the same sites in pMSe4 (33). pAS497 and pAS613, encoding PEA3-(1-494) and PEA3-(258-494), respectively, were constructed by ligating SpeI/NcoI fragments from pAS360 and pAS343 into the same sites in pAS468.

The sequences of all plasmids constructed from PCR-derived products were verified by automated dideoxy sequencing.

Protein Expression and Purification-- In vitro transcription and translations were carried out as described previously (34). Hexahistidine-tagged and GST fusion proteins were expressed in the E. coli strains BL21(DE3) and JM101, respectively, and purified as described previously (29, 35, 36).

Gel Retardation Assays-- Gel retardation assays were performed in 12-µl reaction volumes as described previously (37). The binding sites used were the E74 site (38), the E-box/ets site found in the HIV-1 LTR (oligonucleotide pair ADS671/ADS672; top strand, 5'-TCGATCATCACGTGGCCCGAGAGCTGCATCCGGAGTAC-3') and the mutated version of the E-box/ets site (oligonucleotide pair ADS881/ADS882; top strand, 5'-TCGATCATCACGTGGCCCGAGAGCTGCATGGGGAGTAC-3'). The ets-motif and E-box are underlined and italicized, respectively. The mutated bases are indicated in boldface. In competition assays with Id3, the Id3 and PEA3 proteins were allowed to bind for 15 min at room temperature before the addition of 32P-labeled DNA probe. Antibody supershifts were carried out by adding 1-2.5 µl of anti-FLAG M2 antibody (Sigma) together with PEA3 derivatives prior to incubation with the DNA. Protein·DNA complexes were resolved on nondenaturing polyacrylamide gels cast in either 0.5× or 1× TBE (Tris-borate (90 mM), EDTA (2 mM)).

In Vitro Protein·Protein Interaction Assays-- Interactions between PEA3 and Id or USF proteins were investigated using GST pull-down assays as previously described (38).

Cell Culture, Transfection, and Reporter Gene Assays-- 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Duplicate transfection experiments were carried out in six-well plates using Superfect reagent according to the manufacturer's recommendations (Qiagen). Following transfection, cells were incubated in 0.5% fetal calf serum (Life Technologies, Inc.) for 24 h prior to cell extract preparation and luciferase assays as described previously (36). Transfection efficiencies were standardized by normalizing for the beta -galactosidase activity from cotransfected pCH110 as described previously (36).

Yeast One-hybrid Screening-- Yeast one-hybrid screening was carried out by an adaptation of the protocol described previously (33). The yeast reporter strain Ets-Lacrep was constructed by integrating the pMSv1 reporter (33) into the his3 locus of the Saccharomyces cerevisiae strain W3031A (MATalpha , ho, his3-11,15; trp1-1; ade2-1; leu2-3,112; ura3; can1-100). Untransformed Ets-Lacrep was grown in YEPD medium (2% Bactopeptone, 1% yeast extract, 2% glucose) at 30 °C with shaking. Full-length PEA3 (pAS497) was used as a bait in a yeast one-hybrid screen, using the reporter strain Ets-Lacrep and a mouse 11-day embryonic library cloned in pGAD10 (CLONTECH). The PEA3 expression vector pAS497 contains a galactose-inducible promoter. However, because induction with galactose resulted in a block in cell growth, presumably due to the toxicity of the PEA3 protein, no induction was used in the screen and we relied upon leaky expression of the bait protein. Transformations were carried out essentially as described previously (39) and potential positives were identified by filter assays for beta -galactosidase activity. Filter and liquid assays for beta -galactosidase activity were carried out essentially as described previously (33). Positive clones were verified by retransforming the Ets-Lacrep reporter strain with DNA recovered from potential "positives" in the presence and absence of the bait protein. Positive interacting clones that activated the reporter only in the presence of the bait protein were sequenced, and the identity of sequences elucidated using NCBI BLAST searches. Two screens (total of 157,000 colonies screened) resulted in the identification of 13 clones that scored positive in the above tests. One of these, pAS956, encodes the C-terminal part of mouse USF-1.

Figure Generation and Data Quantification-- All figures were generated electronically from scanned images of autoradiographic images using Picture Publisher (Micrografix) or Adobe PhotoDeluxe and PowerPoint version 7.0 (Microsoft) software. Final images are representative of the original autoradiographic images. Phosphorimaging data were quantified using Tina software (version 2.08e).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mapping and Characterization of the Autoinhibitory Cis-acting Modules in PEA3-- DNA binding by members of the PEA3 subfamily of ETS-domain transcription factors has previously been shown to be regulated by autoinhibitory mechanisms (6-8). In the case of zebrafish PEA3, three regions have been shown to be involved in regulating DNA binding (Fig. 1A) (6). Deletion of region I led to a small increase in DNA binding, whereas more dramatic increases in DNA binding were observed upon deletion of regions II and III. Combined deletion of regions II and III, to leave just the ETS DNA-binding domain, led to further enhancement of DNA binding by PEA3.


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Fig. 1.   Mapping the DNA binding inhibitory domains in PEA3. A, schematic representation of a series of truncated PEA3 constructs. The ETS DNA-binding domain is indicated by a white box and the N-terminal acidic domain by a gray box. The regions previously determined to inhibit DNA binding by PEA3 (6) are indicated (I-III). B, gel retardation analysis of N-terminally and C-terminally truncated PEA3 proteins. Equal molar quantities of the indicated in vitro translated PEA3 derivatives were bound to the E74 site.

To further define the extents of the inhibitory domains II and III, a series of finer N- and C-terminal deleted proteins were constructed (Fig. 1A). Sequential deletion of N-terminal sequences up to amino acid 321 did not result in significant increases in DNA binding (Fig. 1B, lanes 1-5). However, a large increase in the efficiency of DNA binding was observed upon deletion to amino acid 341 (corresponding to the start of the ETS-domain), which was further enhanced upon simultaneous deletion of the sequences located C-terminally to the ETS-domain (Fig. 1B; lanes 6 and 7). Thus, the N-terminal extent of inhibitory domain II is between amino acids 321 and 341. C-terminally truncated proteins were tested in the context of the protein PEA3-(321-494) that contains the intact inhibitory domain II. In this case, enhanced DNA binding was observed upon deletion to amino acid 452 (Fig. 1B, lane 9). Because PEA3-(321-474) was still inhibited (Fig. 1B, lane 10), the C-terminal extent of inhibitory domain III is located between amino acids 452 and 474. Deletion to amino acid 432 did not lead to any further increases in DNA binding, although simultaneous deletion of inhibitory domain II led to a further enhancement of DNA-binding activity (Fig. 1B, lanes 7 and 8). Thus, the inhibitory domains II and III that lie adjacent to the ETS-domain have been mapped to short 20-amino acid motifs. Deletion of either one of these inhibitory motifs results in a decrease of autoinhibition, whereas their simultaneous deletion leads to a loss of autoinhibition.

Work on the autoinhibitory mechanism used by a different ETS-domain transcription factor, Ets-1, has implicated the involvement of an inhibitory module composed of alpha -helices lying N- and C-terminal to the ETS-domain (9, 10). Disruption of one of the N-terminal alpha -helices results in relief of autoinhibition (11, 12). Little sequence conservation exists between Ets-1 and PEA3 in the regions immediately outside the ETS-domain. However, to test whether a similar mechanism involving interacting alpha -helices might operate in PEA3, proline-scanning mutagenesis was used to probe for the presence of functionally important alpha -helices in inhibitory motifs II and III. Because proline residues are incompatible with regular alpha -helices, the insertion of proline residues is expected to disrupt these secondary structure elements. Several conserved proline residues exist in the regions flanking the ETS-domain (Fig. 2A). Further proline residues were introduced in the sequences between these residues (Fig. 2A), and their effect on DNA binding by PEA3 was monitored (Fig. 2, B and C). The position of proline insertions was chosen so that the spacing between any two proline residues was less than a full helical turn. First, the effect of inserting proline residues into inhibitory motif II in the context of PEA3-(258-452) was investigated. This protein has lost the C-terminal inhibitory domain III. However, neither of the two mutations tested caused an increase in DNA binding by PEA3 (Fig. 2B). Similarly, the insertion of proline residues at several positions into inhibitory motif III in PEA3-(321-474) (containing inhibitory domains II and III), caused little stimulation of DNA binding activity. These data therefore demonstrate that it is unlikely that the formation of alpha -helices within the inhibitory motifs is a functionally important requirement for autoinhibition of DNA binding by PEA3.


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Fig. 2.   Proline scanning of the autoinhibitory motifs. A, schematic representation of full-length PEA3, the truncated protein PEA3-(258-452), and the minimal "autoinhibited" form, PEA3-(321-474). Annotation is as in Fig. 1. The sequence of regions flanking the ETS DNA-binding domain of zebrafish (z) PEA3-(321-474), its human (h) and murine (m) homologues, and the related family members ERM and ER81 is shown below. The numbers above the sequences refer to zebrafish PEA3, whereas the numbers at the ends refer to the N- and C-terminal amino acids in each sequence. Identical (shaded) and similar (asterisks) amino acids conserved among these proteins are indicated. The locations of proline residues that are either conserved (white arrows) or mutated (black arrows) are also indicated. B and C, gel retardation analysis of the indicated wild-type and proline mutants of PEA3-(258-452) (B) and PEA3-(321-474) (C) relative to PEA3-(321-452). Equal molar quantities of each in vitro translated PEA3 derivative were bound to the E74 site.

Id Proteins Act in Trans to Inhibit DNA Binding by PEA3-- It was recently demonstrated that the members of the Id family of helix-loop-helix proteins can bind to members of the TCF subfamily of ETS-domain transcription factors and subsequently inhibit their DNA binding (20). Because interaction with Id proteins is via the ETS DNA-binding domain of the TCFs, we tested whether Id proteins could also inhibit DNA binding by PEA3 (see Figs. 3A and 5A for PEA3 derivatives tested). First, we used GST pull-down assays to test whether Id2 can bind to a series of GST-PEA3 fusion proteins. Id2 could bind to both full-length PEA3 (PEA3-(1-494)) and the isolated PEA3 ETS-domain (PEA3-(341-432)) (Fig. 3B, lanes 3 and 4) but not to PEA3 derivatives that lacked the ETS-domain (Fig. 3B, lanes 5 and 6). Reciprocal experiments were carried out in which GST-Id2 fusion proteins were used to pull-down truncated PEA3 proteins. Consistent with the above results, full-length PEA3, PEA3-(1-432), and the isolated PEA3 ETS-domain (PEA3-(341-432)) could bind to Id2 (Fig. 3C). Collectively, these data demonstrate that Ids interact with PEA3 and that the ETS-domain of PEA3 is their point of interaction.


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Fig. 3.   Id proteins bind to and inhibit DNA binding by PEA3. A, schematic representation of a series of truncated PEA3 constructs. Annotation is as in Fig. 1. B and C, GST pull-down analysis of (B) 35S-labeled Id2 bound to the indicated GST-PEA3 derivatives and (C) the indicated 35S-labeled PEA3 proteins bound to GST-Id2. D, gel retardation analysis of the indicated PEA3 derivatives in the absence (lanes 1, 5, and 9) or presence of increasing molar equivalents (1-fold, lanes 2, 6, and 10; 4-fold, lanes 3, 7, and 11; 9-fold, lanes 4, 8, and 12) of Id3. Both Id3 and the PEA3 derivatives were synthesized by in vitro translation, and bound to the E74 site. The amount of binding in the presence of each concentration of Id3 relative to each PEA3 derivative in the absence of Id3 was quantified and shown graphically below each panel.

To study the functional consequences of this interaction, the effect of Id proteins on DNA binding by PEA3 was investigated. Several PEA3 derivatives (Fig. 3A) were tested that lack one of the inhibitory motifs and, therefore, exhibit detectable DNA binding. DNA binding by all the PEA3 derivatives tested was inhibited upon titration of increasing concentrations of Id3 (Fig. 3D). Moreover, the degree of inhibition was similar in all cases and because the minimal DNA-binding region tested is the ETS-domain (in PEA3-(341-432)), this part of the protein therefore represents the target for inhibition. Thus, binding of Id proteins to the ETS-domain of PEA3 results in inhibition of DNA binding in trans, in an analogous manner to the cis-acting inhibition mediated by the autoinhibitory module.

Isolation of USF-1 as an Interaction Partner for PEA3-- Because DNA binding by PEA3 is tightly inhibited by intramolecular autoinhibitory motifs, this inhibition must be relieved to permit its recruitment to DNA. One such mechanism might involve interaction with a second transcription factor or coregulatory protein (reviewed in Ref. 1). We therefore employed a yeast one-hybrid assay to identify proteins that might act to potentiate the activity of PEA3, either via providing linkages to the basal machinery, recruitment to the DNA or a combination of both. The system we used was adopted from a previous study (33) and is depicted in Fig. 4A. A stable yeast strain was constructed that contains an integrated LacZ reporter construct driven by 5 ets binding sites, Ets-Lacrep. Subsequently, expression vectors for PEA3 were introduced into the Ets-Lacrep strain, in combination with a cDNA library encoding GAL4 activation domain fusion proteins derived from day-11 mouse embryos. This stage was selected because mouse PEA3 is strongly expressed at this time point (40) and zebrafish PEA3 is strongly expressed at an equivalent developmental stage (6). Several positive clones were isolated; one of which contains the C-terminal half of the basic helix-loop-helix-leucine zipper (bHLHZip) transcription factor USF-1 (Fig. 4B). Upon retransformation, USF-1 was only able to efficiently activate the LacZ reporter in the presence of a cotransformed vector encoding PEA3 (Fig. 4C). Liquid beta -galactosidase assays were subsequently performed to demonstrate the requirement of both proteins and to map the interaction determinants in PEA3 needed for maximal activation of the LacZ reporter gene. In the presence of the empty expression vectors, little reporter gene activity was observed (Fig. 4D), whereas in the presence of either vectors encoding USF-1 alone or one of a series of truncated PEA3 proteins, modest activation of the reporter was observed (black and white bars; Fig. 4D). However, in the presence of USF-1 and full-length PEA3 (PEA3-(1-494)), a large increase in reporter gene activity was observed. Similarly, large increases in reporter gene activity were observed in the presence of USF-1 and either of the truncated PEA3 proteins PEA3-(258-494) and PEA3-(341-494) (blue bars; Fig. 4D). These data therefore demonstrate that USF-1 represents a true interaction partner for PEA3 and that a region(s) between amino acids 341-494 is sufficient for binding to USF-1 in this assay.


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Fig. 4.   Identification of USF-1 as a PEA3 interaction partner by yeast one-hybrid screening. A, schematic representation of the beta -galactosidase reporter gene used in the yeast one-hybrid screen. The reporter contains a basal cycP promoter driven by 5× ets DNA-binding sites (33). PEA3 binds to these sites in vivo and recruits proteins such as USF-1 from the library to activate transcription of the LacZ gene. The GAL4 activation domain in the USF-1 fusion protein is indicated by a red box. B, schematic representation of the domain structure of full-length USF-1 and the USF-1 fusion protein isolated from the yeast two-hybrid screen. The bHLHZip DNA-binding domain and GAL4 activation domain in the fusion protein are indicated by white and red boxes, respectively. C, beta -galactosidase filter assay of yeast strain Ets-Lacrep, containing the USF-1 expression vector and either the empty expression vector pMSe4 or expression vector containing full-length PEA3. D, quantitative liquid beta -galactosidase assays of yeast strain Ets-Lacrep, containing the indicated combinations of the empty expression vectors pGAD10 and pMSe4 or the same vectors containing the indicated PEA3 derivatives (in pMSe4) or USF-1-(160-310)* (in pGAD10). Data are presented (average of duplicate samples) relative to PEA3 expression vector alone (taken as 1).

PEA3 and USF-1 Interact in Vitro and Synergistically Activate Transcription in Vivo-- GST pull-down assays were used to verify the interactions between PEA3 and USF-1. A series of truncated PEA3 proteins fused to GST were constructed (Fig. 5A) and tested for their ability to interact with in vitro translated full-length USF-1 (Fig. 5B). Of the constructs tested, full-length PEA3 and the isolated ETS DNA-binding domain (PEA3-(341-432)) were able to bind to USF-1 (Fig. 5B, lanes 2 and 3). Neither the region N-terminal (PEA3-(1-337)) nor the region C-terminal (PEA3-(430-494)) to the ETS-domain could bind to USF-1 (Fig. 5B, lanes 4 and 5). Deletion of the bHLHZIP domain of USF-1 resulted in a loss of binding to PEA3, indicating that the N-terminal region of USF-1 is insufficient for binding in solution. Therefore, the DNA-binding domain of USF-1 is required for the formation of such complexes (data not shown).


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Fig. 5.   PEA3 interacts with USF-1 in vitro and synergistically activates transcription with USF-1 in vivo. A, schematic representation of a series of truncated PEA3 constructs fused to GST. Annotation is as in Fig. 1. B, GST pull-down analysis of 35S-labeled full-length USF-1 bound to the indicated GST-PEA3 derivatives. C, schematic representation of the luciferase reporter gene used in transfection experiments. The reporter contains a basal TK promoter driven by 2× E-box-ets DNA-binding sites from the HIV-1 LTR (Fig. 6A) (13). PEA3 binds to the ets motif, whereas USF binds to the E-box to activate transcription of the Luc gene. D, the indicated combinations of USF-1/USF-2 and PEA3 expression vectors (500 and 200 ng, respectively) were transfected into 293 cells in the presence of the ets/E-box-Luc reporter (100 ng). Data are normalized for the activity of a cotransfected beta -galactosidase expression vector and presented (average of duplicate samples) relative to the ets/E-box-Luc reporter alone (taken as 1) and are representative of three independent experiments.

To verify that PEA3 and USF-1 can functionally interact on a physiologically relevant site in vivo, we analyzed their ability to regulate the activity of a luciferase reporter gene in mammalian cells driven by a composite element from the HIV-1 long terminal repeat (LTR). This element is composed of a relatively high affinity E-box and a weak ets (PEA3 binding) motif and has previously been shown to be synergistically regulated by USF-1 and a different ETS-domain transcription factor, Ets-1 (13). When PEA3 or USF-1/USF-2 alone were transfected into 293 cells, moderate levels of reporter gene activity were observed (2.1- and 4.5-fold, respectively). However, in the presence of both PEA3 and USF-1/USF-2, synergistic activation was observed (11.7-fold).

Collectively, these data therefore demonstrate that the interactions between PEA3 and USF-1 can be verified in vitro and that these proteins work together to activate transcription in vivo from a physiologically relevant site.

PEA3 and USF-1 Positively Regulate Each Other's DNA Binding Activity-- USF-1 and Ets-1 have previously been shown to be able to cooperatively bind to the E-box-ets motif in the HIV-1 LTR (Fig. 6A) (13). Because interactions between PEA3 and USF-1 have been demonstrated, we tested whether similar cooperativity could be observed between these two proteins on this DNA binding site. Two different protein derivatives were tested in each case, consisting of either the minimal DNA-binding domains of each protein (PEA3-(341-432) and USF-1-(197-310)), the full-length USF-1 protein (USF-1- (1)), or an extended PEA3 protein encompassing additional motifs outside the ETS-domain (PEA3-(341-452)) (Fig. 6B). These proteins were purified to near homogeneity from overexpressing bacterial strains.


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Fig. 6.   PEA3 stimulates the DNA binding activity of USF-1 and cooperatively binds DNA with USF-1. A, sequence of the composite E-box/ets element from the HIV-1 LTR. The cores of the E-box and ets binding sites are boxed. B, schematic representation of a series of truncated PEA3 and USF-1 constructs. Annotation is as in Figs. 1 and 4. C-E, gel retardation analysis using the indicated combinations of PEA3 and USF-1 proteins and the E-box/ets binding site. All proteins were expressed and purified from bacteria. C, DNA binding by USF-1 derivatives (USF-(1-310), 0.5 pmol; USF-(197-310), 1.5 pmol) in the absence (lanes 1 and 5) or presence of increasing amounts (6 pmol, lanes 2 and 6; 25 pmol, lanes 3 and 7; 50 pmol, lanes 4 and 8) of PEA3-(341-432). D, DNA binding by USF-(1-310) (0.5 pmol) in the absence (lanes 1 and 5) or the presence of increasing amounts (6 pmol, lanes 2 and 6; 25 pmol, lanes 3 and 7; 60 pmol, lanes 4 and 8) of the indicated PEA3 derivatives. The DNA·protein complexes alone are shown, as extended electrophoresis was used to allow maximum resolution and the free binding site was run off the end of the gels. E, antibody supershift experiments in the presence of PEA3 (25 pmol) derivatives and USF-(1-310) (0.5 pmol) to detect the presence of the indicated FLAG-tagged PEA3 derivatives in USF-1 complexes. Complexes containing USF-1 and PEA3 are indicated by black and gray arrows, respectively. White arrows refer to a higher order USF-1·PEA3 complex (D) and antibody induced supershifted complexes (E). The asterisk refers to a band that is not reproducibly observed.

Because the PEA3 ETS-domain was sufficient for interaction with USF-1, we first tested whether this domain could stimulate DNA binding by USF-1 (Fig. 6C). PEA3-(341-432) was able to stimulate DNA binding by both full-length USF-1 (USF-1-(1-310)) and the bHLHZip DNA-binding domain of USF-1 (USF-1-(197-310)). However, at the concentrations used here, PEA3 binding to the E-box-ets motif was not visible at the exposure shown, indicating that, in comparison to USF-1, it has a low affinity for this site (see also Fig. 6E, lane 2). Next we compared the ability of PEA3-(341-432) and the longer PEA3 protein PEA3-(321-452), which contains additional regions that flank the ETS-domain (Fig. 6B), to stimulate DNA binding by full-length USF-1 (Fig. 6D). Both these PEA3 derivatives were able to stimulate the formation of a USF-1 complex. However, extended electrophoresis permitted resolution of a novel lower mobility complex that formed only in the presence of PEA3-(321-452) (Fig. 6D, lanes 5-8). This complex most likely contains PEA3, in addition to USF-1. To test this hypothesis, antibody supershift experiments were carried out using an antibody directed against the FLAG tag in the PEA3 derivatives. Addition of anti-FLAG antibody gave a small supershift in the presence of PEA3-(341-432), but efficiently shifted a significant proportion of the complex formed with PEA3-(321-452) (Fig. 6E, compare lanes 3 and 6). Higher concentrations of antibody had little further effect on PEA3-(341-432) but resulted in loss of the supershifted complex formed with PEA3-(321-452) (data not shown; Fig. 7C).


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Fig. 7.   Identification of PEA3 and USF-1 domains required for ternary complex formation. A-C, gel retardation analysis using the indicated combinations of PEA3 and USF-1 proteins and the E-box-ets binding site. Schematic representations of the truncated PEA3 and USF-1 constructs used in each experiment are shown above or to the left of each set of lanes, respectively. Annotation is as in Figs. 1 and 4. The DNA·protein complexes alone are shown as extended electrophoresis was used to allow maximum resolution and the free binding site was run off the end of the gels. The USF-1 (5 pmol) and PEA3 (3 pmol) proteins used were expressed in and purified from bacteria. Anti-FLAG antibodies were added to detect the presence of FLAG-tagged PEA3 derivatives in protein·DNA complexes. In this experiment a high concentration of antibody was used that results in abolition of ternary complex formation rather than a supershift as in Fig. 6. Complexes containing USF-1 are indicated by black arrows, whereas white arrows refer to ternary USF-1·PEA3 complexes. D, the location of the USF binding and autoinhibitory motifs on the linear PEA3 sequence. The dotted line indicates that this part of the C-terminal inhibitory motif, although not sufficient, might be required for inhibiting DNA binding by PEA3. E, model showing a proposed mechanism for the relief of autoinhibition by interaction with USF-1. The inhibitory motifs are depicted as black elipses. Direct interactions between the ETS-domain of PEA3 and USF-1 and putative interactions with the autoinhibitory motifs of PEA3 are indicated by two-way arrows. An alternative route for relief of autoinhibition other than USF-1 binding is indicated by a question mark. This might involve interactions with other proteins or post-translational modification.

Collectively, these data therefore indicate that, although both PEA3-(341-432) and PEA3-(321-452) stimulate DNA binding by USF-1, only the latter forms a stable ternary DNA-bound complex with USF-1. Therefore, although the ETS DNA-binding domain of PEA3 is sufficient to stimulate DNA binding by both full-length USF-1 and the USF-1 bHLHZip DNA-binding domain, additional PEA3 sequences are required for the formation of stable ternary complexes.

Mapping the Regions of PEA3 Required for Ternary Complex Formation with USF-1-- To identify the regions in USF-1 and to further define the regions in PEA3 that are required to permit ternary complex formation, a series of truncated PEA3 and USF-1 derivatives were tested for their ability to form ternary DNA-bound complexes (Fig. 7, A-C). In this series of experiments, the largest PEA3 protein tested was PEA3-(321-452), which was shown previously to be sufficient to form ternary complexes with full-length USF-1 (Fig. 6D). Two additional PEA3 proteins that lack either the N- (PEA3-(341-452)) or C-terminal (PEA3-(321-432)) extensions to the ETS-domain were tested, to determine whether either of these regions were sufficient to permit ternary complex formation. A concentration of USF-1 proteins that gave >50% probe binding was used in these experiments, and the gels were run for extended times to permit complex resolution. Of the proteins tested, only PEA3-(321-452) was able to form a ternary complex with full-length USF-1 (USF-1-(1-310)) (Fig. 7A, lane 15). This complex was abolished upon addition of anti-FLAG antibody (Fig. 7A, lane 16). In contrast, the FLAG antibody had no effect on complexes formed in the presence of the truncated PEA3 derivatives (Fig. 7A, lanes 4, 8, and 12), indicating that PEA3 was not part of these complexes. Thus both the N- and C-terminal extensions to the PEA3 ETS-domain are required in combination to form ternary complexes with USF-1. Interestingly, these same regions participate in intramolecular autoinhibition of DNA binding by PEA3, indicating an intriguing link between stimulation of DNA binding by PEA3 and relief of autoinhibition (Figs. 1 and 7D; see "Discussion")

To map the ternary complex determinants in USF-1, two truncated USF-1 derivatives were tested. Although USF-1-(144-310) could form a complex with PEA3-(321-452) (Fig. 7B, lane 7), the isolated bHLHZip domain in USF-1-(197-310) was unable to form a ternary complex with PEA3-(321-452) (Fig. 7C, lane 3). None of the other truncated PEA3 derivatives tested could form complexes with either of these truncated USF-1 proteins (Fig. 7B, lane 3; data not shown). Therefore, the region immediately preceding the bHLHZip domain (amino acids 144-197) is required for ternary complex formation with PEA3.

Thus in the case of both PEA3 (amino acids 321-341 and 433-452) and USF-1 (amino acids 144-197), regions flanking their DNA-binding domains are required for the formation of stable DNA-bound ternary complexes.

DNA Binding Sites for PEA3 and USF-1 Are Required for Ternary Complex Formation-- Results from the yeast one-hybrid system indicated that PEA3 and USF-1 can interact on DNA in the absence of USF-1 binding sites (Fig. 4). To test whether the formation of such complexes could be detected in vitro and whether reciprocal complexes on a USF-1 site could be detected, we analyzed the binding of PEA3 and USF-1 to sites that contained only an ets or an E-box binding site (Fig. 8).


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Fig. 8.   Both ets and E-box binding sites are required for ternary complex formation. Gel retardation analysis using the indicated combinations of PEA3 and USF-1 proteins and the indicated binding sites. The ets binding site is the high affinity E74 site, whereas the E-box site is identical to the E-box-ets site used in Figs. 6 and 7 with two point mutations in the ets binding motif. The USF-1 and PEA3 proteins were as described in Fig. 7. Complexes containing USF-1 are indicated by black arrows, whereas white arrows refer to ternary USF-1·PEA3 complexes.

Binding of PEA3-(321-452) to the ets site was barely detectable due to the presence of the autoinhibitory domains (Fig. 8, lane 3). The addition of USF-1 did not lead to an enhancement of binding or the formation of a ternary complex (Fig. 8, lane 2). In the presence of both E-box and ets sites, ternary complex formation could be readily detected (Fig. 8, lane 5; see Figs. 6 and 7). However, upon mutation of the ets site, although USF-1 binding to the E-box was unaffected, ternary complex formation was abolished (Fig. 8, lanes 7 and 8).

Collectively, these results therefore demonstrate that in vitro, specific protein·DNA contacts by both PEA3 and USF-1 are required for the formation of ternary complexes.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Autoinhibition is emerging as a novel mechanism for regulating the activities of eukaryotic transcription factors. There are numerous examples where autoinhibition results in a reduction of the DNA binding activity of the transcription factor (reviewed in Ref. 1). Subsequently, this autoinhibition is relieved, either in response to activation of signal transduction pathways or the presence of a coregulatory partner protein. Indeed, the ETS-domain family provides multiple additional examples of combinatorial interactions that have been investigated in detail, where DNA binding by at least one component of a complex is enhanced (reviewed in Refs. 2 and 3). Here, we have located the cis-acting autoinhibitory motifs in the ETS-domain transcription factor PEA3 and identified two proteins that act in trans to modify DNA binding by PEA3. The Id proteins act to inhibit DNA binding, whereas USF-1 acts to promote DNA binding by PEA3. Thus a complex network of intra- and intermolecular protein·protein interactions serve to modify the DNA binding potential of PEA3.

Mechanisms for Negatively Regulating DNA Binding by PEA3-- DNA binding by PEA3 subfamily members is regulated by cis-acting autoinhibitory domains (6-8). Regions in the C- and N-terminal part of PEA3 are involved in regulating its DNA binding potential. Here we have further localized the C-terminal inhibitory domains in PEA3 to two short regions that lie on either side of the ETS DNA-binding domain (summarized in Fig. 8D). These regions are highly conserved among all members of the PEA3 subfamily (45% identical; 76% similar) and between species (79 and 87% identical between human and zebrafish PEA3) (Fig. 2A). These similarities are particularly striking considering that the overall identity among all PEA3 subfamily members is only 20% outside the ETS-domains (6). This sequence conservation suggests that the autoinhibitory mechanism is evolutionarily conserved among PEA3 subfamily members and hence further emphasizes the likely importance of this regulatory mechanism.

The two inhibitory motifs that we have identified act in concert to inhibit DNA binding by PEA3 as deletion of either one leads to a substantial relief of autoinhibition. Each domain on its own can only partially inhibit DNA binding by PEA3 (Fig. 1). These observations indicate that the two motifs might form a single autoinhibitory module as observed in Ets-1 (9, 10). However, unlike Ets-1, where this module is composed of three alpha -helices, our results indicate that the formation of alpha -helices does not appear to be required for the function of this module in PEA3. Furthermore, there is essentially no sequence conservation between the autoinhibitory modules of Ets-1 and PEA3. Thus, although both these modules serve to inhibit DNA binding, their molecular mechanisms of action are likely to differ. It is currently unclear how DNA binding is inhibited, however, this module might either mask the DNA binding surface or, alternatively, inhibit DNA binding by allosterically affecting the DNA binding surface. Indeed, a similar model has been proposed for autoinhibition of Ets-1 (11, 12).

In addition to the cis-acting autoinhibitory motifs, we also demonstrate that trans-acting proteins (the Id proteins) can inhibit DNA binding by PEA3. The Id proteins were previously demonstrated to bind to the ETS-domain and inhibit DNA binding by members of the TCF subfamily (20). Similarly, the Id proteins bind to the ETS DNA-binding domain in PEA3 (Fig. 3). However, one key difference is that the Id proteins inhibit PEA3 binding to the high affinity E74 site, whereas the TCFs are only affected on lower affinity sites. This might reflect intrinsic differences between these different proteins in the precise mode of DNA recognition and/or Id protein binding. The mechanism used by Id proteins to inhibit DNA binding by ETS-domain transcription factors is unknown, although they may act by analogous mechanisms proposed for the cis-acting autoinhibitory motifs discussed above.

Interaction with USF-1 Enhances DNA Binding by PEA3-- In this study, we have identified USF-1 as an interaction partner for PEA3 and demonstrate that USF-1 recruits PEA3 into DNA-bound complexes. This recruitment might merely reflect the role of protein·protein interactions in enhancing the binding of PEA3. However, USF-1 might also lead to relief of autoinhibition. This latter hypothesis is supported by the observation that the two motifs in PEA3 that are required for ternary complex formation with USF-1 overlap with the two motifs that constitute the autoinhibitory module (Fig. 8D). Again, these two motifs are required in combination to permit ternary complex formation as deletion of either motif abolishes cooperative binding with PEA3 (Fig. 8C). Thus an important regulatory module has been identified that is involved in stimulating the formation of nucleoprotein complexes and inhibiting DNA binding. USF-1 appears to act as a switch that changes the activity of this module.

It is noteworthy that the ability of USF-1 to form ternary complexes with PEA-3 is dependent on a region between amino acids 144-196, which is conserved between USF family members and contains a USF-specific region (USR) (41, 42 and references therein). The USR plays an important role in many of the functions of USF, including transcriptional activation, nuclear localization (41, 42), and regulatory phosphorylation (29) and also been proposed to recruit either cell-specific coactivators or repressors. Thus in both PEA3 and USF-1, important regulatory domains play additional roles in promoting complex formation.

USF-1 was also isolated as an interaction partner for a different ETS-domain transcription factor, Ets-1, by using the same one-hybrid system (13). Cooperative DNA binding between USF-1 and Ets-1 and synergistic transcriptional activation was also observed, although the mechanisms of cooperativity appear to differ. For example, in the case of USF-1 and Ets-1, the respective DNA-binding domains of each protein were sufficient for interaction in solution and to cooperatively form ternary complexes. However, in the case of USF-1 and PEA3, such reciprocal cooperativity was not observed unless additional regions flanking each of the DNA-binding domains were present. Although the ETS DNA-binding domain of PEA3 was sufficient to stimulate DNA binding by the bHLHZip DNA-binding domain of USF-1, no stable ternary complexes were formed. This is consistent with our observation that the ETS DNA-binding domain was sufficient for interaction with USF-1 in GST pull-down assays (Fig. 5) and that a minimal PEA3 construct that lacks the N-terminal region flanking the ETS-domain was sufficient to interact with USF-1 in the yeast one-hybrid assay (Fig. 4). Indeed, the cooperativity we see (DNA binding by PEA3 is barely detectable in the absence of USF-1), appears significantly more than that observed between Ets-1 and USF-1. In this regard, a key difference between our own and the previous study is, therefore, the quantities of proteins used (we used 30-fold less ETS-domain protein), and it is possible that, at higher concentrations, we may also have detected residual cooperativity between the isolated DNA-binding domains. Thus, with PEA3 and USF-1, although it is clear that it is possible to promote complex formation in the absence of autoinhibitory modules and E-box binding sites in vivo by protein overexpression (as observed with Ets-1/USF-1), other factors likely contribute to DNA·protein complex formation. Indeed, two DNA binding sites and the autoinhibitory regions of each protein are absolutely required for the formation of protein·DNA complexes in vitro. We believe that this is more indicative of the true physiological situation.

Thus one model for how these proteins interact and cooperate, is that interactions occur between the ETS- and bHLHZip DNA-binding domains, which can promote DNA binding but, in the case of PEA3 and USF-1, are insufficient to permit the formation of ternary complexes. The regions flanking these domains are required for stable complex formation (Fig. 7E). In light of our results and the study of Ets-1 interactions with AML-1 (13, 15), it will be interesting to re-evaluate the possible role of additional motifs in Ets-1 in cooperative DNA binding with USF-1.

It is currently unclear how cooperativity in the USF-1·PEA3 complex is achieved. It is likely that protein·protein interactions between USF-1 and PEA3 serve to increase the affinity of PEA3 for the binding site. In addition, disruption or reorientation of the autoinhibitory module might transmit signals in an allosteric manner to the ETS DNA-binding domain of PEA3. Further experimentation is required to probe these possibilities further.

    ACKNOWLEDGEMENTS

We thank Margaret Bell and Linda Shore for excellent technical assistance; Bob Liddell for DNA sequencing; Paul Shore, Shen-Hsi Yang, and members of our laboratory for comments on the manuscript and stimulating discussions; Michael Sieweke, Thomas Graf, John Norton, Michelle Sawadogo, and Elke Oettgen for reagents.

    FOOTNOTES

* This work was supported by grants from the Biotechnology and Biological Sciences and Research Council (GR/H09676 to D. B.) and from the Cancer Research Campaign (CRC) and a Lister Institute of Preventative Medicine Research Fellowship (to A.D.S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 44-161-275-5979; Fax: 44-161-275-5082; E-mail: a.d.sharrocks@man.ac.uk.

Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011582200

    ABBREVIATIONS

The abbreviations used are: HLH, helix-loop helix; bHLHZip, basic helix-loop-helix-leucine zipper; PCR, polymerase chain reaction; GST, glutathione S-transferase; HIV-1, human immunodeficiency virus type 1; USR, USF-specific region.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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