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• W2120015511 abstract "Article1 July 1999free access The Pax3–FKHR oncoprotein is unresponsive to the Pax3-associated repressor hDaxx Andrew D. Hollenbach Andrew D. Hollenbach Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Jack E. Sublett Jack E. Sublett Department of Developmental Neurobiology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Craig J. McPherson Craig J. McPherson Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Gerard Grosveld Corresponding Author Gerard Grosveld Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Andrew D. Hollenbach Andrew D. Hollenbach Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Jack E. Sublett Jack E. Sublett Department of Developmental Neurobiology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Craig J. McPherson Craig J. McPherson Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Gerard Grosveld Corresponding Author Gerard Grosveld Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA Search for more papers by this author Author Information Andrew D. Hollenbach1, Jack E. Sublett2, Craig J. McPherson1 and Gerard Grosveld 1 1Department of Genetics, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA 2Department of Developmental Neurobiology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN, 38105 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:3702-3711https://doi.org/10.1093/emboj/18.13.3702 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Pax3–FKHR fusion protein is present in alveolar rhabdomyosarcoma and results from the t(2;13) (q35;q14) chromosomal translocation. Its oncogenic activity is dependent on a combination of protein–DNA and protein–protein interactions mediated by the Pax3 homeodomain recognition helix. In this report we demonstrate that human Daxx (hDaxx) interacts with Pax3 in vivo and with DNA-bound Pax3 in vitro. This interaction is mediated primarily through the homeodomain recognition helix with the additional involvement of the octapeptide domain and its N-terminal flanking amino acids. Through this interaction hDaxx represses the transcriptional activity of Pax3 by ∼80%. The Pax3–FKHR fusion is unresponsive to this repressive effect despite an observed endogenous interaction with hDaxx in a rhabdomyosarcoma tumor cell line. Therefore, these data support the model that fusion of FKHR to Pax3 not only adds a strong transactivation domain, but also deregulates transcriptional control of Pax3 by overriding the natural repressive effect of hDaxx. Introduction Alveolar rhabdomyosarcoma, a malignant tumor of skeletal muscle, is characterized by the t(2;13) (q35;q14) translocation (Shapiro et al., 1993; Galili et al., 1995) that results in the fusion of Pax3, a member of the paired class homeodomain family of transcription factors, to FKHR, a member of the forkhead family of transcription factors. The Pax3–FKHR fusion, which has been demonstrated to be a more potent transcriptional activator than Pax3 (Fredericks et al., 1995; Bennicelli et al., 1996; Lam et al., 1999), maintains the integrity of the Pax3 DNA-binding motifs (paired domain and homeodomain). However, the Pax3 transactivation domain is replaced by the bisected, nonfunctional FKHR DNA-binding domain (DBD) and the FKHR transactivation domain. A recent structure–function analysis of Pax3–FKHR demonstrated that, in conjunction with a small region of the FKHR activation domain, the integrity of the recognition helix of the Pax3 homeodomain (the third α-helix) is necessary for the oncogenic activity of Pax3–FKHR in NIH 3T3 fibroblasts (Lam et al., 1999). In this report it is demonstrated that point mutants in the third α-helix of the homeodomain diminish homeodomain-mediated Pax3–FKHR DNA binding with a correlative reduction in the oncogenic activity of the fusion protein. However, point mutants in the paired domain that completely disrupt Pax3–FKHR DNA binding ability while retaining an intact homeodomain maintain a wild-type oncogenic activity. These results suggest that the oncogenicity of Pax3–FKHR is independent of paired domain-mediated DNA binding but instead requires both protein–DNA and protein–protein interactions mediated through the Pax3 homeodomain recognition helix (Lam et al., 1999). Two recent reports have identified proteins that interact with the homeodomain of Pax3. The retinoblastoma protein (Rb) was demonstrated to repress Pax3 transcriptional activity 2- to 3-fold by a direct interaction with the first two α-helices of the homeodomain (Wiggan et al., 1998). HIRA, a mammalian homologue of a Saccharomyces cerevisiae transcriptional co-repressor, was demonstrated to interact with the Pax3 homeodomain (Magnaghi et al., 1998). However, it remains to be determined whether Pax3 is regulated by HIRA and whether the Pax3–FKHR fusion protein is affected by the regulatory actions of these proteins. The Pax3–FKHR fusion protein may alter the regulation of transcription of Pax3 target genes in one of two ways. First, after the Pax3 homeodomain mediates the binding of Pax3–FKHR to the promoter regions of Pax3 target genes the presence of the more potent FKHR transactivation domain may overcome the natural regulatory actions of proteins that interact with the Pax3 homeodomain recognition helix. Alternatively, the FKHR portion of the fusion may create steric hindrance and prevent these regulatory proteins from interacting with Pax3. In either case it is important to identify additional regulatory proteins that may be involved. The murine homologue of hDaxx was previously identified as a putative Fas receptor binding protein, implicated in the enhancement of Fas-mediated apoptosis (Yang et al., 1997; Chang et al., 1998, 1999). Human Daxx (hDaxx) was also isolated using a yeast one-hybrid screen to detect transcription factors that regulate a steroidogenic factor 1like element from the human steroidogenic acute regulatory protein gene promoter (Kiriakidou et al., 1997). In addition, hDaxx has been demonstrated to interact with the centromeric protein CENP-C in a cell cycle-dependent manner (Pluta et al., 1998). In this report we demonstrate that hDaxx is also involved in the regulation of Pax3 transcriptional activity. This regulation is mediated by the direct interaction of hDaxx with the Pax3 homeodomain recognition helix and the N-terminally extended octapeptide domain. We also show that hDaxx is associated endogenously with the Pax3–FKHR fusion protein. However, Pax3–FKHR does not respond to the repressive action of hDaxx. These results provide some of the first evidence for a mechanism that explains, in part, the oncogenic activity of Pax3–FKHR. Results Isolation and structural characteristics of hDaxx In order to identify proteins that interact with Pax3 and that may contribute to the oncogenicity of Pax3–FKHR, the Pax3 moiety of Pax3–FKHR was fused to the LexA DNA-binding domain and used in a yeast two-hybrid analysis (Gyuris et al., 1993). One of 11 initial clones isolated from an oligo(dT)-primed HeLa cell cDNA library was identical to the C-terminal 120 amino acids of hDaxx. hDaxx encodes a protein of 740 amino acids and contains a Ser/Pro/Thr-rich C-terminus, which we found to interact with Pax3, and a region rich in acidic amino acids (Figure 1A), both motifs commonly found in transcriptional regulators (Mitchell and Tjian, 1989). hDaxx was also demonstrated to contain a region predicted to form a coiled coil like structure (Pluta et al., 1998). In addition, we observed that hDaxx contains two regions predicted to form paired amphipathic helices (PAHs) (Figure 1B) that share conserved amino acids with the four PAHs present in the yeast transcriptional co-repressor Sin3 and its mammalian homologue Sin3a (Figure 1C) (Wang et al., 1990; Ayer et al., 1995). Figure 1.Schematic representation of hDaxx structural characteristics. (A) The domains of hDaxx are presented as follows: the Ser/Pro/Thr-rich domain (S/P/T) is depicted by the shaded box, the acidic rich domain (D/E) by the horizontal lines, the region of coiled-coil (C-C) by the cross-hatched box, and the two paired amphipathic helices (PAHs) by the diagonal stripes. The vertical lines above the structure indicate predicted phosphorylation sites and the region of hDaxx required for the interaction with Pax3 is indicated. (B) Amino acid sequences of helices A and B of the two PAHs represented as Edmundson helical wheels. The amino acid sequence of PAH1 is closest to the wheel and hydrophobic residues are indicated. (C) Sequence similarity of hDaxx and yeast Sin3 (Wang et al., 1990) and mammalian Sin3a (Ayer et al., 1995) PAHs. Amino acids are represented by the single letter code. Download figure Download PowerPoint hDaxx is expressed in all tissue types (data not shown; Kiriakidou et al., 1997), suggesting that hDaxx may have an alternative function in cells not expressing the Fas receptor. The reported interaction between Daxx and the Fas receptor would indicate that hDaxx should localize to the cytoplasm of cells. However, overexpressed six-histidine epitope-tagged hDaxx localized exclusively to the nucleus of transfected COS1 cells, consistent with the presence of two nuclear localization signals in the hDaxx sequence (Kiriakidou et al., 1997). In direct agreement with this observation, we have also noted a strict nuclear staining for hDaxx, which was identical to the cellular localization of hemagglutinin (HA)-Pax3, indicating a co-localization of hDaxx and Pax3 to the nucleus (data not shown). We have also confirmed a strict nuclear localization of both endogenous and overexpressed hDaxx in all cell lines examined (COS1, NIH 3T3 and Jurkat; data not shown). In all cell lines examined, a C-terminal antibody against hDaxx identified three distinctly migrating species with apparent molecular weights of 70, 97 and 110 kDa (Figure 2, lane 2). To rule out the possibility that the three species of hDaxx are N-terminal degradation products of the full-length protein, hDaxx was N-terminally FLAG-epitope tagged and overexpressed in NIH 3T3 fibroblasts. A Western analysis of these cell lysates, utilizing both the C-terminal hDaxx antibody (Figure 2, lane 2) and the N-terminal FLAG antibody (Figure 2, lane 1), identified the same three species of hDaxx. This confirms the presence of both the N-terminus and the C-terminus of hDaxx in all three species and that these species do not result from degradation of hDaxx. Northern analysis also demonstrated that the hDaxx mRNA exists as a single transcript of 2.4 kb (data not shown; Kiriakidou, 1997) indicating that the three species of hDaxx are not the result of alternatively spliced transcripts. Instead, the three forms of hDaxx may arise from different post-translational modifications of the protein. Consistent with this, a PROSITE analysis of hDaxx (Bairoch et al., 1997) predicted 34 phosphorylation sites located near key structural regions of the protein (Figure 1A). Incubation of NIH 3T3 fibroblasts with [32P]orthophosphate specifically labeled the apparent 110 kDa species, but not the 97 or 70 kDa species (Figure 2, lane 3). A Western analysis of the immunoprecipitated protein demonstrated the ability of the FLAG antibody to precipitate all forms of hDaxx (Figure 2, lane 4). In addition, incubation of hDaxx containing cell lysates in the absence of phosphatase inhibitors resulted in the presence of only the 70 kDa form of hDaxx (Figure 2, lane 5). These results directly demonstrate that the different species of hDaxx result, in part, from a post-translational hyperphosphorylation. Figure 2.hDaxx is present in all cell lines as three distinctly migrating species. NIH 3T3 cells overexpressing an N-terminal FLAG epitope tagged hDaxx (FLAG-hDaxx) were lysed and separated by SDS–PAGE, and the presence of FLAG-hDaxx was detected with either an N-terminal anti-FLAG antibody (lane 1) or a C-terminal anti-hDaxx antibody (lane 2) as described in the Materials and methods. A schematic of the structural characteristics of hDaxx and the location of the respective epitopes is presented. To examine the phosphorylation status of hDaxx, FLAG-hDaxx was directly labeled with [32P]orthophosphate and immunoprecipitated from COS1 cell lysates (lane 3). The FLAG antibodies precipitated all forms of hDaxx as demonstrated by a direct Western blot using the anti-hDaxx antibody (lane 4). In addition, Jurkat cells overexpressing full-length hDaxx were lysed in either the presence (lane 6) or absence (lane 5) of phosphatase inhibitors, incubated at 37°C for 12 h, and the presence of hDaxx was detected using the C-terminal hDaxx antibody. Download figure Download PowerPoint Physical interaction between Pax3 and hDaxx To demonstrate a direct physical interaction between Pax3 and hDaxx in vivo, COS1 cells were co-transfected with influenza virus HA epitope tagged Pax3 (HA-Pax3) and full-length hDaxx, followed by co-immunoprecipitation. Immunoprecipitation of HA-Pax3 from cell lysates expressing similar levels of both proteins (Figure 3A, lanes 3 and 4) co-precipitated the 70 kDa species of hDaxx (Figure 3A, lane 2) consistent with an interaction between Pax3 and non-phosphorylated hDaxx. This interaction was specific for HA-Pax3, since no hDaxx was detected when the identical co-immunoprecipitation was carried out on cells overexpressing hDaxx alone (Figure 3A, lane 1). To examine the interaction between hDaxx and DNA-bound Pax3, full-length Pax3 and the interacting region of hDaxx (hDaxx635–740) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and subjected to electrophoretic mobility shift analysis. GST–Pax3 shifted the radiolabeled Pax3-specific PRS-9 oligonucleotide probe, which contains both Pax3 paired and homeodomain recognition sequences (Figure 3B, lane 3). This complex was supershifted by the addition of Pax3-specific antiserum and could be specifically competed by a molar excess of unlabeled probe (Figure 3B, lanes 4 and 5). Increasing amounts of GST–hDaxx635–740, added prior to DNA binding, resulted in a titratable supershift of the Pax3–DNA complex (Figure 3B, lanes 6–8). The addition of purified GST to the binding reaction was unable to supershift the Pax3–DNA complex (Figure 3B, lane 9), demonstrating that the observed supershift is specific for hDaxx and not a result of non-specific binding of GST to GST–Pax3. In the same manner GST–hDaxx635–740 alone was unable to shift the Pax3-specific probe (Figure 3B, lane 10). Therefore, we conclude that hDaxx binds directly to Pax3 both in vivo and to DNA-bound Pax3 in vitro. Figure 3.The 70 kDa species of hDaxx interacts with Pax3 in vivo and to DNA-bound Pax3 in vitro. (A) Pax3 interacts with the 70 kDa species of hDaxx in vivo. COS1 cells overexpressing equivalent amounts of hDaxx and HA-Pax3 (lanes 3 and 4) were lysed in the presence of protease and phosphatase inhibitors and immuno- precipitated with an anti-HA antibody followed by detection with an anti-hDaxx antibody (lane 2). As a negative control cells overexpressing hDaxx alone were subjected to an identical treatment (lane 1). The mobilities of HA-Pax3 and hDaxx are indicated by the solid and open arrows, respectively. (B) hDaxx interacts with DNA-bound Pax3 in vitro. Bacterially expressed GST–Pax3 and the indicated amounts of bacterially expressed GST–hDaxx635–740 or GST were subjected to an electrophoretic mobility shift assay (EMSA) with an end labeled PRS-9 oligonucleotide probe, which contains both the Pax3 paired and homeodomain recognition sequences. The mobilities of DNA bound Pax3 and the Pax3–hDaxx complex are indicated by the solid and open arrows, respectively. Download figure Download PowerPoint Specificity of the Pax3 and hDaxx interactions In order to identify the domains of Pax3 required for the interaction with hDaxx, deletion mutants targeting key structural domains of Pax3 (Figure 4) were used as the bait and hDaxx635–740 was used as the interacting protein in the LexA yeast two-hybrid system (Gyuris et al., 1993). The interaction was detected by a quantitative liquid β-galactosidase assay (Adams et al., 1997). The introduction of the deletion mutants did not affect the stability of the proteins as determined by immunoprecipitation of metabolically labeled protein from NIH 3T3 fibroblasts (data not shown). Deletion of the N-terminal Pax3 domain responsible for transcriptional inhibition (ΔNH2) (Chalepakis et al., 1994), the region of the paired domain involved in making DNA contacts (PDND) (Xu et al., 1995) or the entire paired domain (ΔPD) had no effect on hDaxx binding, indicated by wild-type β-galactosidase activity (Figure 4). However, deletion of an additional 20 amino acids past the paired domain (ΔPD2) or deletion of the paired and octapeptide domains (ΔPDOD2) resulted in ∼2- and 5-fold decreases in β-galactosidase activity, respectively, with no additional decrease in activity upon further deletion (ΔPDOD) (Figure 4). This is consistent with a role of the octapeptide domain and its N-terminal flanking amino acids, which will be referred to as the N-terminally extended octapeptide domain, in the interaction of Pax3 with hDaxx. In the same manner, deletion of the recognition helix of the homeodomain (HD3D) resulted in no significant β-galactosidase activity above background (Figure 4). When the HD3D mutant was used in independent experiments with PAPI, a calcium-binding protein was shown to interact primarily with the Pax3 paired domain (data not shown), wild-type β-galactosidase activity was seen providing additional evidence that the HD3D deletion mutant does not significantly affect the stability of the protein. From this we conclude that the homeodomain recognition helix in conjunction with the N-terminally extended octapeptide domain are necessary for the interaction between Pax3 and hDaxx. Suprisingly, the homeodomain deletion mutant (ΔHD) retained an activity slightly higher than full-length Pax3. This enhanced activity may result from the removal of a cis-acting inhibitory action associated with the Pax3 homeodomain (Bennicelli et al., 1996), or more simply removal of the homeodomain may make paired domain- and octapeptide domain-mediated interactions more accessible. Figure 4.The Pax3 homeodomain recognition helix and the N-terminally extended octapeptide domain are required for interaction with hDaxx. Pax3, the indicated Pax3 deletion mutants, Pax7 and Pax4 were fused in-frame to the LexA bait vector. The Pax3 and Pax7 paired domains are represented by the diagonal stripes, the octapeptide domains by the shaded box, and the homeodomains by the dark hashed box. The Pax4 paired domain is represented by the horizontal lines and the Pax4 homeodomain by the cross-hatched box. The ability of the mutants to interact with the partial hDaxx clone, hDaxx635–740, was determined by the yeast two-hybrid liquid β-galactosidase assay (see Materials and methods). The results are presented as β-galactosidase activity corrected for the background activity of the deletion mutants in the absence of hDaxx635–740. Errors represent the standard deviation from four independent determinations. Download figure Download PowerPoint Because different members of the Pax family of transcription factors show variable sequence and domain conservation (Stuart et al., 1995), Pax4 and Pax7 were chosen to determine the specificity of hDaxx interactions with other members of the Pax family of transcription factors. Pax4, which does not contain an octapeptide domain and is only 45% identical to Pax3 in the homeodomain, had no detectable binding ability (Figure 4). Conversely, Pax7, which contains an octapeptide domain and is 97% identical to Pax3 in the homeodomain (Stuart et al., 1995), resulted in a β-galactosidase activity nearly three times higher than that for Pax3 (Figure 4). This would suggest that hDaxx635–740 binds to Pax7 with a higher affinity than that of Pax3. This result is further consistent with the role of the octapeptide domain and the homeodomain recognition helix in the interaction of hDaxx with both Pax3 and Pax7. hDaxx acts as a transcriptional repressor The presence of transcriptional regulatory domains in hDaxx and the sequence similarity of the PAHs in hDaxx to the co-repressor Sin3 suggest that hDaxx may act as a transcriptional regulator. To test this hypothesis full-length hDaxx was fused to the GAL4 DBD and the effect of GAL4–hDaxx on the constitutively active reporter construct 1X-CRE-TK-CAT, which contains one GAL4 DNA-binding site, was determined. Transient co-transfection of NIH 3T3 fibroblasts with the reporter construct and increasing amounts of GAL4–hDaxx or hDaxx (pcDNA3-hDaxx) resulted in an ∼70% decrease in transcriptional activity (Figure 5A, shaded bars). This repression was dependent on the tethering of hDaxx to DNA through the GAL4 DBD since hDaxx alone was unable to repress transcription in this system (Figure 5A, open bars). This result directly demonstrates the ability of hDaxx to act as a transcriptional repressor in this experimental setting. Despite the sequence similarity between the PAHs of hDaxx and Sin3, domains responsible for the recruitment of histone deacetylase activity by Sin3 (Alland et al., 1997; Hassig et al., 1997; Heinzel et al., 1997; Kadosh and Struhl, 1997; Nagy et al., 1997; Zhang et al., 1997), we were unable to demonstrate a relief of repression when the transient transfections were repeated in the presence of the histone deacetylase inhibitor, Trichostatin A (data not shown). This suggests that the inhibitory effect of hDaxx involves mechanisms other than the recruitment of histone deacetylases. Figure 5.Transcriptional regulation by hDaxx. (A) hDaxx represses transcription when tethered to DNA through the GAL4 DBD. The indicated amounts of the GAL4–hDaxx fusion (pM2-hDaxx, shaded bars) or hDaxx alone (pcDNA3-hDaxx, open bars) were co-transfected into NIH 3T3 cells with a chloramphenicol acetyl transferase (CAT) reporter construct containing one GAL4 DNA binding site. Forty-eight hours post-transfection CAT activity was determined as described previously (Ausubel et al., 1996). All values were normalized for co-transfected secreted alkaline phosphatase activity (Bram et al., 1993) and are presented as the percentage of CAT activity in the absence of hDaxx (pM2 only). Error bars represent the standard deviation from four independent determinations. (B) hDaxx represses Pax3 but not Pax3–FKHR transcriptional activity. Pax3 (shaded bars) or Pax3–FKHR (open bars) and increasing amounts of hDaxx were co-transfected into NIH 3T3 cells with the (PRS-9)TK-CAT reporter construct, which contains both the Pax3 paired and homeodomain recognition sequences. After 48 h CAT activity was determined as described (Ausubel et al., 1996). All values were normalized for co-transfected secreted alkaline phosphatase activity (Bram et al., 1993) and are presented as the percent maximal activity relative to Pax3 or Pax3–FKHR transcriptional activity in the absence of hDaxx. Error bars represent the standard deviation from eight independent determinations. Download figure Download PowerPoint To determine the effect of hDaxx on Pax3 transcriptional activity, NIH 3T3 cells were transiently transfected with Pax3, increasing amounts of full-length hDaxx, and the reporter construct (PRS-9)TK-CAT, which contains both Pax3 paired and homeodomain recognition sequences (Chalepakis et al., 1994). Increasing amounts of hDaxx caused a titratable inhibitory effect on Pax3 transcription resulting in ∼80% repression of Pax3 transcriptional activity (Figure 5B, shaded bars). This is a level of repression similar to that seen for the action of the co-repressors Sin3a and Sin3b on Ume6 and Myc respectively (Allend et al., 1997; Kadosh and Struhl, 1997). To confirm that overexpression of hDaxx has no effect on the protein levels of Pax3, similar transcription experiments were repeated using co-infection of cells with Pax3 and hDaxx retrovirus, a system that allows a higher level of overexpression of Pax3 and hDaxx. Under these conditions it was found that overexpression of hDaxx represses Pax3 transcription just as well as in transient co-transfections but it had no detectable effect on the level of Pax3 protein expression, as determined by Western analysis (data not shown). The observed repression of reporter gene expression was also not a result of hDaxx-induced apoptosis since TUNEL assays and visual inspection of morphology of cells overexpressing both Pax3 and hDaxx, determined by immunofluorescence, confirmed the absence of any detectable apoptosis in all transcription assays. In fact we were unable to detect any hDaxx-induced apoptosis in either NIH 3T3 fibroblasts overexpressing co-transfected Fas receptor and hDaxx or in Jurkat cells overexpressing transfected hDaxx (data not shown). Therefore we conclude that under the conditions used in this study hDaxx acts as a Pax3-associated transcriptional repressor. Pax3–FKHR is unresponsive to repression by hDaxx When the Pax3–FKHR fusion protein was used in the identical transcription assays as for Pax3, we found that hDaxx exerted only a minimal repressive action on Pax3–FKHR transcriptional activity (Figure 5B, open bars). The inability of hDaxx to repress Pax3–FKHR transcriptional activity may be caused by the additional FKHR sequences preventing hDaxx from binding to Pax3. To determine whether the presence of FKHR prevents the interaction between Pax3 and hDaxx, Pax3–FKHR was used as the interacting protein in a yeast two-hybrid analysis. In this system Pax3–FKHR alone was transcriptionally active when used as the bait protein. Therefore, Pax3–FKHR was cloned into the library plasmid, pJG4-5, and the hDaxx partial clone hDaxx635–740 was cloned into the bait plasmid, pEG202. Co-expression of the hDaxx635–740 clone and Pax3–FKHR resulted in β-galactosidase activity far above background levels (Figure 6A), demonstrating an interaction between hDaxx and Pax3–FKHR in yeast. Figure 6.Pax3–FKHR interacts with hDaxx. (A) Pax3–FKHR interacts with hDaxx635–740 in yeast. hDaxx635–740 was fused in-frame to the LexA bait vector and Pax3–FKHR was fused in frame to the LexA library vector. The results are presented as β-galactosidase activity corrected for the background activity of hDaxx635–740 in the absence of Pax3–FKHR. Errors represent the standard deviation from four independent determinations. (B) In vivo interaction between Pax3–FKHR and hDaxx. NIH 3T3 cells overexpressing both hDaxx and Pax3–FKHR were lysed in the presence of protease and phosphate inhibitors and subsequently incubated with magnetic beads containing the Pax3-specific oligonucleotide, e5 (Pax3 site resin), which contains both paired and homeodomain recognition sequences, or the control beads that contained no oligonucleotide (resin). Proteins retained by the beads were separated by 10% SDS–PAGE and the presence of Pax3–FKHR (right panel) or hDaxx (left panel) were determined by Western analysis. (C) Endogenous interaction between Pax3–FKHR and hDaxx. Rh30 cells (5×106) that naturally express both Pax3–FKHR and hDaxx were lysed in the presence of protease and phosphatase inhibitors and submitted to the Pax3 site resin analysis as described for (B). Download figure Download PowerPoint To demonstrate an interaction between Pax3–FKHR and hDaxx in vivo, both proteins were overexpressed in NIH 3T3 cells and the interaction was determined by using a Pax3-specific oligonucleotide affinity resin (Figure 6B). A Western blot of the resulting proteins retained by the Pax3 site-specific resin demonstrate that in addition to binding overexpressed Pax3–FKHR, primarily the 110 kDa species of hDaxx was also retained (Figure 6B). The retention of hDaxx by the resin was directly dependent on the binding of Pax3–FKHR since neither Pax3–FKHR nor hDaxx bound to the resin alone (Figure 6B). Despite the retention of overexpressed Pax3 by the Pax3 site-specific resin we were unable to demonstrate the retention of hDaxx from cell lysates overexpressing both Pax3 and hDaxx (data not shown). This suggests that the affinity of hDaxx for Pax3 is not nearly as great as the affinity of hDaxx for Pax3–FKHR. Finally, to show an endogenous interaction between Pax3–FKHR and hDaxx the Pax3-specific oligonucleotide affinity resin was used to isolate Pax3–FKHR and associated proteins from Rh30, a rhabdomyosarcoma cell line that endogenously expresses both Pax3–FKHR and hDaxx (data not shown). Once again a Western blot analysis of the endogenous proteins retained by the Pax3 site-specific resin demonstrated the retention of Pax3–FKHR and primarily the 110 kDa species of hDaxx, and to a minor extent the 97 and 70 kDa species of hDaxx (Figure 6C). The retention of hDaxx by the resin was directly dependent on the binding of Pax3–FKHR to the Pax3-specifi" @default.
• W2120015511 created "2016-06-24" @default.
• W2120015511 creator A5010642130 @default.
• W2120015511 date "1999-07-01" @default.
• W2120015511 modified "2023-10-18" @default.
• W2120015511 title "The Pax3-FKHR oncoprotein is unresponsive to the Pax3-associated repressor hDaxx" @default.
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• W2120015511 doi "https://doi.org/10.1093/emboj/18.13.3702" @default.
• W2120015511 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1171447" @default.
• W2120015511 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10393185" @default.
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