INTRODUCTION

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Chromosomal translocations, which are hallmarks of both hematopoietic and solid malignancies (28), frequently involve the fusion of two independent genes to encode a novel chimeric protein with aberrant functions. Alveolar rhabdomyosarcoma, characterized by the t(2;13)(q35;q14) translocation (14, 31), is a malignant tumor of skeletal muscle and is one of the most common soft tissue sarcomas of childhood, accounting for more than half of such cases in pediatric patients (27). This translocation creates the Pax3-FKHR fusion gene. This gene, which encodes a protein of 837 amino acids, comprises the 5' sequences of the Pax3 gene, a member of the paired class homeodomain family of transcription factors (32, 33), and the 3' sequences of the FKHR gene, which encodes a member of the forkhead family of transcription factors (6). The fusion protein maintains the integrity of the Pax3 DNA binding motifs (paired domain and homeodomain), but the transactivation domain of Pax3 is replaced by the bisected FKHR DNA binding domain and transcriptional activation domain. As a result, the Pax3-FKHR fusion protein exhibits higher transcriptional activity than does Pax3 alone (13), suggesting that the oncogenic potential of Pax3-FKHR results from this gain-of-function mutation.

Many chromosomal translocations result in fusion proteins that involve transcription factors. A DNA binding-independent model has been described to explain the oncogenicity of the fusion protein E2a-Pbx1. In acute lymphoblastic leukemia, the t(1;19)(q23;p13) translocation fuses the N-terminal transcriptional activation domains of E2a to the C-terminal region of the homeodomain protein Pbx1 (21, 26). Transformation by the E2a-Pbx1 fusion protein is dependent on protein-protein interactions via the homeodomain cooperativity motif of Pbx1 (9) and is independent of DNA binding by the homeodomain of Pbx1 (23).

Pax3-FKHR has been demonstrated to transform chicken embryo fibroblasts in culture (30), but the structural requirements for its oncogenicity have not yet been defined. In the present study, we have analyzed the structure-function relationship of the Pax3-FKHR domains necessary for transcriptional activation and transformation. Our results show that paired domain-mediated DNA binding is not necessary for transformation. However, the integrity of the third -helix of the Pax3 homeodomain, in conjunction with a region of the FKHR transactivation domain, is essential for Pax3-FKHR-mediated oncogenicity. These results suggest a model in which homeodomain-mediated DNA binding, with an additional role of homeodomain-mediated protein-protein interactions, is important for the oncogenic potential of Pax3-FKHR.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Expression constructs and mutagenesis. All expression plasmids were constructed in the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, Calif.). The murine Pax3 cDNA, pBH3.2, kindly provided by Peter Gruss (Max Planck Institute, Göttingen, Germany), encodes a protein that has over 98% homology with human Pax3 and is completely identical in its DNA binding regions (15, 20). The isolation of the Pax3-FKHR fusion protein cDNA was previously reported (14, 31). The Pax3-FKHR point mutants Un-1, BU35, V265A, S268A, N269A, and S268N269A were created by standard PCR site-directed mutagenesis (18). The internal deletion mutants PD-NH2, HD-C, and HD were constructed by overlap extension PCR (19), and the C-terminal deletion mutants 772, 728, and 672 were generated by PCR amplification and standard cloning techniques. All mutants were confirmed by sequence analysis. Further C-terminal truncation proteins (503 and 606) were constructed by using restriction enzyme sites ScaI and NdeI at nucleotide positions 1497 and 1805, respectively (Fig. 1). The chloramphenicol acetyl transferase (CAT) reporter construct (pTK-CAT) containing six direct repeats of both the paired domain and homeodomain consensus binding sequences (PRS-9) [(PRS-9) PTKCAT] was a gift from Martyn Goulding (The Salk Institute, San Diego, Calif.).

Electrophoretic mobility shift assay. Protein-DNA binding reaction mixtures (20 µl) included 2 µl of in vitro-translated protein (Promega) and 3 µg of poly(dI-dC) in buffer containing 20 mM HEPES (pH 7.6), 50 mM NaCl, 5 mM MgCl₂, 0.5 mM dithiothreitol, and 10% glycerol (13). Protein expression was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with detection by autoradiography of [³⁵S]methionine-labeled protein. Unlabeled protein was incubated at room temperature for 20 min with 10⁵ dpm of the oligonucleotide probe PRS-9, which contains both the paired domain and the homeodomain recognition sequences (7); this probe was labeled with [-³²P]dCTP (6,000 Ci/mmol; Amersham, Arlington, Ill.). Incubation of protein with competitor (1,500-fold molar excess of unlabeled PRS-9 probe) or with 1 µl of Pax3 rabbit antiserum was performed at room temperature for 10 min prior to the addition of labeled probe. DNA-protein complexes were resolved on a 6% native polyacrylamide gel, containing 2.5% glycerol in 25 mM Tris-HCl (pH 8.3) and 190 mM glycine, at 35 mA for 1.5 h. Gels were fixed in 10% acetic acid and dried, and labeled oligonucleotides were visualized by autoradiography.

Retroviral stocks and transformation assays. The various forms of Pax3-FKHR cDNA were cloned into the EcoRI site of the vector pSR(HindIII)-tk-Neo (29), kindly provided by Charles Sawyers (UCLA Medical School, Los Angeles, Calif.). Retroviral stocks were generated by calcium phosphate cotransfection (16) of 293T cells, kindly provided by David Baltimore (California Institute of Technology, Pasadena, Calif.), with various constructs and an ecotropic packaging plasmid (2), as described previously (24). Culture supernatants containing virus were collected between 36 and 72 h posttransfection, filtered, and subsequently used to infect NIH 3T3 cells, which were demonstrated to be transformed by EWS-FLI, kindly provided by Christopher Denny (University of California at Los Angeles). Soft agar assays were performed by resuspending 2 × 10⁴ cells in 1 ml of 1× Iscove's modified Dulbecco's medium containing 15% fetal bovine serum and 0.3% Noble agar (Difco). The cell suspension was then layered over 2 ml of 0.6% bottom agar in 35-mm-diameter dishes. Colonies were scored 2 and 3 weeks after seeding.

Antibody production and immunoprecipitation. The Pax3 antiserum was produced by immunization of rabbits with a bacterially expressed, His₆ epitope-tagged Pax3 protein (a six-histidine-tagged Pax3 protein containing only the paired domain and the homeodomain). To confirm overexpression of the Pax3-FKHR constructs, NIH 3T3 cells infected with high-titer retroviruses encoding either Pax3-FKHR or the Pax3-FKHR mutant derivatives were metabolically labeled at 48 h after infection with [³⁵S]methionine (0.5 mCi/ml; New England Nuclear, Beverly, Mass.) for 2 h, lysed in dissociation buffer (0.5% SDS, 1 mM dithiothreitol, 50 mM Tris-HCl [pH 7.4], 1 mM EDTA), and boiled for 5 min. Cell lysates were diluted fourfold with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholic acid, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). The lysate supernatants were then centrifuged for 30 min at 4°C at 10,000 × g and incubated with the Pax3 rabbit polyclonal antiserum for 2 h, at 4°C, on a rotary shaker. Immune complexes were collected with protein A-Sepharose (Pharmacia, Piscataway, N.J.) and washed four times with radioimmunoprecipitation assay buffer containing 0.1% SDS. Pellets were resuspended in 30 µl of 2× Laemmli SDS-PAGE buffer, boiled for 5 min, and centrifuged to remove debris. Proteins were resolved on an SDS-10% PAGE gel. The gel was incubated in ENHANCE (New England Nuclear), dried, and autoradiographed at 80°C overnight.

Immunofluorescence. NIH 3T3 cells were infected with retroviruses encoding either Pax3-FKHR or the Pax3-FKHR mutant derivatives. At 24 h postinfection, 2 × 10⁴ cells were replated onto coverslips in 35-mm-diameter dishes and cultured for an additional 24 h. Cells were fixed with 2% paraformaldehyde for 15 min at room temperature, rinsed twice with phosphate-buffered saline (PBS), and permeabilized for 10 min in 2% paraformaldehyde containing 1% Triton X-100. The fixed cells were incubated for 1 h with a 1:200 dilution of the Pax3 rabbit polyclonal antiserum. After being washed with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (1:1,000; Sigma Chemical Co., St. Louis, Mo.) for 30 min, washed with PBS, and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.). Slides were examined with an Olympus BH2 fluorescent microscope.

Transient transfections and transactivation assays. NIH 3T3 cells (3 × 10⁵) were plated in 60-mm-diameter dishes and transfected the following day, at approximately 70% confluency, by the Lipofectamine method (Gibco/BRL, Grand Island, N.Y.), in accordance with the manufacturer's specifications. The Lipofectamine-DNA precipitate was formed in a total of 600 µl of serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine. The precipitate consisted of 1 µg of the (PRS-9)pTK-CAT reporter plasmid described above (7), 250 ng of expression plasmid (containing Pax3, Pax3-FKHR, or the mutant derivatives), and 1 µg of the secreted alkaline phosphatase (SEAP) control plasmid under control of the MAP1 promoter (5). The Lipofectamine-DNA precipitate was incubated on the cell monolayer for 5 h in 3.0 ml of DMEM supplemented with only 2 mM glutamine, after which the cell monolayer was fed with 5 ml of DMEM supplemented with 10% fetal calf serum and 2 mM glutamine. After 48 h, the medium was assayed for SEAP activity, as previously described (5), and the cells were harvested and assayed for CAT activity, as previously described (1). The percentage of [¹⁴C]chloramphenicol acetylation was quantitated from thin-layer chromatography plates by using a PhosphorImager (Molecular Dynamics). The transfection efficiency was normalized relative to the SEAP activity. Each experiment was repeated three times in duplicate plates.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Pax3-FKHR mutant constructs and DNA binding activity. To define the Pax3-FKHR domains required for transformation, we generated a series of Pax3-FKHR mutants (Fig. 1). We made three mutants that disrupt DNA binding through the Pax3 paired domain: Un-1 contains a glycine-to-serine mutation at codon 48 (G48S), which is analogous to the mutation in the Pax1 paired domain of the undulated mouse mutant (2); BU35 contains an arginine-to-leucine mutation at codon 56 (R56L), which has been reported in a family with Waardenburg syndrome type 1 (20); and PD-NH2 has a deletion in the N-terminal -helices of the paired domain that are involved in making DNA contacts (35). We also constructed six mutants involving the homeodomain: S268A contains a serine-to-alanine mutation within the third -helix of the homeodomain of Pax3-FKHR, which reduces Pax3 homodimerization upon binding to DNA (12); V265A, N269A, and S268N269A contain point mutations of amino acids within the homeodomain recognition helix that were demonstrated to be involved in making direct DNA contacts (35). HD-C lacks the entire third -helix from glutamic acid 260 to lysine 276 (E²⁶⁰-K²⁷⁶) of the homeodomain. This helix, called the recognition helix, is believed to mediate homeodomain-DNA contacts, homeodomain-mediated protein-protein interactions, and Pax3 homodimerization on DNA (12, 34). HD represents an in-frame deletion of the entire homeodomain from glutamine 219 to glycine 279 (Q²¹⁹-G²⁷⁹). Finally, we made progressive C-terminal deletions in the FKHR transactivation domain (772 to 503 as shown in Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the Pax3, Pax3-FKHR, and Pax3-FKHR mutant proteins. Two mutants containing a single amino acid change in the paired domain were constructed: Un-1 (G48S) and BU35 (R56L), which recapitulate naturally occurring mutations in Pax1 and Pax3, respectively (2, 20). PD-NH2 contains an in-frame deletion of the paired-domain helices that are involved in DNA contacts (Asn53 to Thr93). S268A contains a single amino acid change in the homeodomain, which mediates Pax3 dimerization (12). V265A, N269A, and S268N269A contain single or double amino acid changes in the homeodomain recognition helix that have been shown to make direct DNA contacts (35). HD-C and HD contain in-frame deletions of the homeodomain DNA recognition helix (Glu269 to Lys276) and of the entire homeodomain (Gln219 to Gly279), respectively. 772, 728, 672, 606, and 503 contain progressive C-terminal deletions in the FKHR transactivation domain (see Materials and Methods. bi., bisected.

View larger version (117K): [in this window] [in a new window]   FIG. 2.   Gel retardation analysis with the paired recognition sequence (PRS-9) oligonucleotide probe, which contains both the paired domain and the homeodomain recognition sequences. (A) Only the Pax3-FKHR mutants containing an intact paired domain and homeodomain are able to efficiently bind to the probe. In vitro-translated Pax3, Pax3-FKHR, or Pax3-FKHR mutant proteins (2 µl) were each incubated with end-labeled PRS-9 oligonucleotide by using the binding conditions described in Materials and Methods. Protein-DNA complexes were separated on a 6% nondenaturing gel. The mobility of the Pax3 protein is indicated by an open arrowhead, and the mobility of the Pax3-FKHR, S268A, 772, 728 and 672 proteins is indicated by a closed arrowhead. 606 and 503 show a slightly lower mobility than that of Pax3-FKHR, as expected. The Pax3-FKHR and Pax3 protein-DNA complexes could be competed with a molar excess of unlabeled PRS-9 oligonucleotide probe. (B) Pilot in vitro translation reactions were performed with [35S]methionine-labeled proteins to confirm that comparable amounts of Pax3, Pax3-FKHR, and the Pax3-FKHR mutants were synthesized. The labeled proteins were separated by SDS-10% PAGE, and their molecular sizes were estimated by comparison with the rainbow protein molecular size markers (Amersham), whose positions are indicated to the left of the gel. Molecular sizes of the proteins are as follows: Pax3, 56 kDa; Pax3-FKHR, Un-1, BU35, V265A, S268A, N269A, and S268N269A, 97 kDa; PD-NH2, 90 kDa; HD-C, 94 kDa; HD, 90 kDa; 503, 56 kDa; 606, 67 kDa; 672, 75 kDa; 728, 81 kDa; and 772, 86 kDa.

Oncogenic potential of Pax3-FKHR and its mutant derivatives. To examine the transforming properties of the Pax3-FKHR protein, wild-type Pax3-FKHR and its mutant derivatives were individually cloned into the retroviral vector pSR(HindIII)-tk-Neo (29). NIH 3T3 fibroblasts were infected with replication-deficient retroviral stocks and subsequently assayed for their ability to exhibit anchorage-independent growth in soft agar. Cells infected with the Pax3-FKHR virus displayed macroscopically visible colonies in soft agar (Fig. 3B and 4E) and possessed a highly refractile, spindle-shaped morphology under cell culture conditions (Fig. 4F). In contrast, cells infected with either the vector (Fig. 3A and 4A) or with Pax3 alone (Fig. 4C) showed no visible colony formation and displayed a flat morphology when grown on plastic (Fig. 4B and D, respectively). Immunoprecipitation with the Pax3 antiserum of Pax3-FKHR and the Pax3-FKHR mutant derivatives from metabolically labeled cell lysates confirmed that all cells expressed similar amounts of mutant proteins and that none of the mutations interfered with the expression of the Pax3-FKHR fusion (Fig. 5).

View larger version (96K): [in this window] [in a new window]   FIG. 3.   Soft agar assays to assess the tumorigenicity of Pax3-FKHR and the Pax3-FKHR mutants. NIH 3T3 fibroblasts were infected with high-titer retroviral stocks containing pSR(HindIII)-tk-Neo (A), Pax3-FKHR (B), Un-1 (C), PD-NH2 (D), HD-C (E), HD (F), 728 (G), or 772 (H) and cloned in 0.3% soft agar. Colonies were visualized and counted 2 to 3 weeks after plating.

View larger version (133K): [in this window] [in a new window]   FIG. 4.   Cell morphology of NIH 3T3 cells infected with high-titer retroviral stocks containing pSR(HindIII)-tk-Neo (A and B), Pax3 (C and D), or Pax3-FKHR (E and F). Cells were grown on plastic, and colonies were visually scored 2 and 3 weeks after plating. Colonies shown are representative of the total population.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   Overexpression of Pax3, Pax3-FKHR, and Pax3-FKHR deletion mutants in retrovirally infected NIH 3T3 fibroblasts. Retrovirally infected NIH 3T3 cells were metabolically labeled with [35S]methionine and lysed as described in Materials and Methods. Cell lysates were immunoprecipitated with Pax3 antiserum, separated on an SDS-10% PAGE gel, and visualized by autoradiography. Molecular sizes were estimated by comparison with protein molecular size markers (see legend to Fig. 2), which are indicated, in kilodaltons, to the left of the gel.

View larger version (63K): [in this window] [in a new window]   FIG. 6.   Determination of the nuclear localization of Pax3, Pax3-FKHR, and HD by immunofluorescence. NIH 3T3 cells (2 × 104) infected with either pSR(HindIII)-to-Neo (A), Pax3 (B), HD (C), or Pax3-FKHR (D) were plated on glass coverslips. After 48 h, the cells were fixed, permeabilized, immunostained with Pax3 antiserum, and incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody, as described in Materials and Methods.

View larger version (38K): [in this window] [in a new window]   FIG. 7.   Transformation and transcriptional activity of Pax3, Pax3-FKHR, and Pax3-FKHR mutants. Pax3, Pax3-FKHR, and Pax3-FKHR mutant proteins (as described in the legend to Fig. 1) are represented schematically (left column). To determine transformation (right column), NIH 3T3 fibroblasts were infected with high-titer retroviral stocks containing Pax3, Pax3-FKHR, or Pax3-FKHR mutant proteins and grown on 0.3% soft agar. Cell colonies were counted 2 to 3 weeks after plating. The values represent the number of colonies present on a 35-mm-diameter dish and an average of two independent determinations. To determine transactivation (Transact.) activity (right column), NIH 3T3 cells were transfected with 1 µg of the (PRS-9) pTK-CAT reporter construct, 1 µg of MAP1-SEAP, and 250 ng of an expression plasmid encoding either Pax3, Pax3-FKHR, or a Pax3-FKHR mutant protein. Quantification of the CAT activity was performed as described in Materials and Methods. Transfection efficiency was normalized relative to SEAP activity as described previously (5). The CAT activity in the presence of Pax3-FKHR was assigned a value of 100%, and all other activities are reported relative to this value. The error bars represent the standard deviation from the average value of three individual experiments performed in duplicate.

Transcriptional activity of Pax3-FKHR mutants. To relate transformation to transcriptional activity, wild-type Pax3-FKHR and its mutant derivatives were tested in transient-transfection assays using a Pax3-responsive reporter plasmid (see Materials and Methods). Pax3-FKHR demonstrated approximately ninefold greater transcriptional activity than Pax3 did (Fig. 7), consistent with previous reports (3, 13). Proteins mutated in the paired domain and deletion mutants of the homeodomain of Pax3 (Un-1, BU35, PD-NH2, HD, and HD-C), which had no detectable DNA binding ability, were all transcriptionally inactive in NIH 3T3 cells (Fig. 7). Given that the S268A mutant retains its DNA binding potential (Fig. 2A), these results are consistent with the requirement of DNA binding for transcriptional activity. Surprisingly, proteins with point mutations in the homeodomain (V265A, N269A, and S268N269A), which retained only residual DNA binding capability, maintained nearly wild-type transcriptional activity. This finding suggests that in addition to affecting the DNA binding abilities of Pax3-FKHR, these point mutants may also interfere with the reported inhibitory action of the homeodomain (4) (see below).

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

The oncogenic potential of Pax3-FKHR is independent of its transcriptional activity. By creating a series of mutations targeting key structural regions of Pax3-FKHR, we have identified the domains necessary for transformation and transactivation by the fusion protein. We found that both the integrity of the third -helix of the Pax3 homeodomain and a small region of the FKHR transactivation domain are absolutely required for effective transformation of NIH 3T3 mouse fibroblasts. However, the paired domain-mediated DNA binding is dispensable.

Pax3 homeodomain-mediated DNA binding is essential for transformation. The two point mutants BU35 and Un-1 recapitulate naturally occurring mutations in the paired domains of Pax3 and Pax1, respectively (2, 20). These point mutations occur in the region of the paired domain that is involved in making DNA contacts (35) and prevent Pax3 and Pax1 from binding DNA (22). Introduction of these point mutations into the chimeric protein, as well as deletion of the region of the paired domain that is involved in making DNA contacts (PD-NH2), destroyed the ability of Pax3-FKHR to bind DNA. Surprisingly, these mutants transformed cells as efficiently as did wild-type Pax3-FKHR. From this we conclude that Pax3 paired domain-mediated DNA binding is not required for the transformation by the fusion protein.

Transformation requires the proximity of a small region of the FKHR transactivation domain. Mutant proteins containing an intact FKHR transactivation domain and a mutated homeodomain (N269A, S268N269A, HD-C, and HD) or an intact homeodomain and a mutated FKHR transactivation domain (728, 672, 606, and 503) were unable to transform NIH 3T3 cells, indicating that both the third -helix of the Pax3 homeodomain and an element that lies between amino acids 728 and 772 of the FKHR transactivation domain must be present for efficient transformation by Pax3-FKHR. This gain-of-function mutation, in which transformation activity is critically dependent on the presence of two independent, nontransforming domains being brought into proximity in the fusion protein, is similar to the mechanism by which another homeodomain-containing fusion protein, E2a-Pbx1, induces transformation (26). E2a-Pbx1-mediated transformation is dependent on the homeodomain cooperativity motif, a small region proximal to the homeodomain in Pbx1 (9), and on two transcriptional activation domains in E2a being brought close to one another in the fusion protein (23).

The additional role of homeodomain-mediated protein-protein interactions. In addition to making homeodomain-mediated DNA contacts, the Pax3 recognition helix also mediates protein-protein interactions (34). Several pieces of evidence support an additional role of protein-protein interactions in the oncogenic potential of Pax3-FKHR. First, Pax3-FKHR mutants that are unable to bind DNA (UN-1, BU35, and PD-NH2) exhibit a wild-type oncogenic potential. These mutants contain the homeodomain structural components necessary for protein-protein interactions that would enable them to transform NIH 3T3 fibroblasts in the absence of DNA binding. Second, the reported inhibitory action of the homeodomain is no longer present in the nontransforming homeodomain point mutants N269A and S268N269A. This indicates that these point mutants disrupt homeodomain-mediated protein-protein interactions that are necessary for the inhibitory action of the homeodomain and which may be necessary for Pax3-FKHR's oncogenic potential. Finally, protein-protein interactions have been implicated in the regulation of the biological activity of several homeodomain-containing proteins. For example, the homeodomain protein Msx-2 represses transcription by interacting with the basal transcriptional machinery in a DNA binding-independent manner (25). The Drosophila homeodomain protein fushi tarazu, through specific protein-protein interactions with the orphan nuclear receptor Ftz-F1, mediates the regulation of the genes engrailed and wingless (17). Finally, protein interactions between the homeodomain protein Pbx and several Hox proteins modulate the DNA binding specificity and transcriptional regulation of the Hox proteins (8). Therefore, protein-protein interactions, mediated through the homeodomain, may assist in the transforming ability of Pax3-FKHR.