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Molecular Biology and Genetics |
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Similarly, other chromosomal translocations lead to the fusion of the NH2-terminal activation domain of EWS to parts, or to the whole, of various DNA-binding transcription factors. The t(12;22)(q13;q12) translocation found in malignant melanoma of soft parts (soft tissue clear cell sarcomas) is characterized by the fusion of EWS to the ATF1 transcription factor that is endowed with a basic leucine-zipper DNA-binding domain (16) . The resulting EWS-ATF1 fusion protein is a constitutively active transcription factor deregulating ATF1 target genes and may thereby cause cell transformation (17 , 18) . In desmoplastic small round-cell tumors, the t(11;22)(p13;q12) translocation leads to the fusion of EWS to the WT1 gene (19 , 20) . In contrast to the transcriptional repressor WT1, the EWS-WT1 protein up-regulates the expression of the platelet-derived growth factor-A, a potent mitogen and chemoattractant that may contribute to the generation of desmoplastic small round-cell tumors (21) . Furthermore, EWS is fused to CHOP/GADD153, a member of the CCAAT/enhancer-binding protein family, in myxoid liposarcomas characterized by a t(12;22)(q13;q12) translocation (22) , and a t(9;22)(q22;q12) translocation in myxoid chondrosarcomas results in the fusion of EWS to CHN/TEC, a nuclear orphan receptor (23 , 24) .
Whereas the EWS fusion proteins are DNA-binding transcription factors capable of cell transformation, little is known about the EWS proto-oncoprotein itself. In its COOH-terminal portion, EWS possesses a potential RNA-binding domain and consistently is able to bind to RNA in vitro (3 , 25) . Furthermore, the homologous TLS/FUS protein binds to RNA in vivo and displays characteristics of a heterogeneous ribonuclear protein (26) . Other evidence pointing to a function of EWS in RNA-processing stems from its interaction with the ZFM1/SF1 splicing factor (27) . On the other hand, the transcriptional potency of the NH2-terminal domain of EWS observed in its various tumorigenic fusion proteins suggested that EWS may function as a transcription factor. In support of such a notion, EWS as well as its close homologues, TLS/FUS and hTAFII68, bind to various subunits of the basal transcription factor TFIID and to RNA polymerase II (28) .
In this report, we studied the potential involvement of EWS in gene transcription. The nuclear EWS protein is shown to act as a transcriptional activator in a cell-type- and promoter-specific manner. In addition, we demonstrate that EWS physically and functionally interacts with two homologous coactivators, CBP and p300 (29 , 30) . Altogether, our results identify for the first time a physiological role for the EWS proto-oncoprotein as a modulator of gene activity by collaborating with CBP and p300.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-36 expression vectors (33)
were as reported. The Gal4 reporter plasmid (Gal42-tk80-luc) contains two Gal4 DNA-binding sites upstream of a thymidine kinase minimal promoter fused to the firefly luciferase gene (34)
; the c-fos luciferase reporter plasmid (fl711) contains the human c-fos promoter from nucleotides -711 to +39. Bacterial expression vectors for GST-CBP fusion proteins have been described before (35)
.
Immunostaining.
Mink lung Mv1Lu cells were seeded on coverslips and transiently transfected with Myc-tagged EWS expression vectors. Cells were fixed with formaldehyde and stained with monoclonal 
-Myc antibodies (9E10) followed by staining with secondary antibodies coupled to FITC essentially as described (36)
.
Reporter Gene Assays.
Rabbit kidney epithelial-like RK13, Mv1Lu, human embryonal kidney 293T, or mouse embryonic fibroblast AKR cells were grown to 25% confluence on 6-cm dishes and then transiently transfected by the calcium phosphate coprecipitation method. One µg of a luciferase reporter plasmid, 0.2 µg of a ß-galactosidase expression vector (pEQ176), and the indicated amounts of protein expression vectors were cotransfected. Thirty-six h after transfection, cells were lysed, and luciferase activity was measured in a luminometer. Determination of ß-galactosidase activity served to control for transfection efficiency (37)
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Coimmunoprecipitations.
Transiently transfected 293T cells, which had been plated on 6-cm dishes, were lysed in 650 µl of lysis buffer [2.5 mM Tris (pH 7.1), 7.5 mM Na4P2O7, 12.5 mM NaCl, 0.25% Triton X-100, 12.5 mM NaF, 0.5 mM Na3VO4, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 0.5 mM phenylmethylsulfonyl fluoride, and 0.2 mM DTT) for 5 min at 4°C. After scraping of the dishes, the suspension was transferred to a 1.5-ml tube, briefly vortexed, and tumbled for 45 min; then debris was removed by centrifugation (20,800 x g) for 10 min. The supernatant was precleared with 20 µl of protein A beads (Repligen) for 1 h, centrifuged (6 min; 20,800 x g), and transferred to a new 1.5-ml tube. After addition of 1 µl of mouse monoclonal antibodies (either -HA 12CA5 or -Myc 9E10), the mixture was tumbled for 2 h and then another 90 min with 20 µl of protein A beads. Beads were pelleted (1 min; 960 x g), washed three times with 500 µl lysis buffer and again pelleted, and, after a final wash, centrifuged for 1 min at 20,800 x g. The pellets were taken up in 25 µl Laemmli sample buffer, boiled, and then loaded onto SDS polyacrylamide gels. Proteins were revealed by chemiluminescence (enhanced chemiluminescence detection kit, Amersham/Pharmacia) after Western blotting.
Coimmunoprecipitation of endogenous p300 and EWS was performed in a similar way. Four µl of polyclonal -p300 (C-20; Santa Cruz Biotechnologies) antibodies were used, and the coimmunoprecipitated EWS protein was detected by Western blotting using a 1:1000 dilution of -EWS (C-19; Santa Cruz Biotechnologies) antibodies.
GST Pull-down Assays.
GST-CBP fusion proteins were bound to glutathione agarose beads as described before (35)
. A protein extract of 293T cells transiently transfected with HA-tagged EWS was incubated with the loaded glutathione beads, the beads were washed, and bound EWS protein was revealed by -HA Western blotting (32)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Myc antibodies. As shown in Fig. 1
, the full-length EWS protein (EWS2–656) is exclusively localized to the cell nucleus. No staining of nontransfected cells was observed, demonstrating the specificity of our staining procedure (data not shown). Identical nuclear localization of Myc-tagged EWS was detected in HeLa cells or upon utilization of an HA-tagged EWS protein (data not shown), indicating that the nuclear localization of EWS is not affected by the nature of the tag used nor by the cell line studied.
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. However, we additionally observed some cytoplasmic staining with these truncations, suggesting that the COOH terminus is required for the absolute nuclear localization observed with the full-length EWS protein. Furthermore, deletion of 245 or 373 NH2-terminal amino acids resulted in molecules that were present throughout the cytoplasm and nucleoplasm (Fig. 1
, bottom panels), suggesting that both NH2- and COOH-terminal amino acids are responsible for the nuclear localization of EWS.
The COOH Terminus of EWS Represses the NH2-Terminal Activation Domain.
Previously, it has been shown that the NH2 terminus of EWS encodes a potent transactivation domain (see Fig. 2
for a sketch of the EWS protein) when fused to various DNA-binding domains (2
, 38) . However, up to now it has not been determined whether the full-length EWS protein is a transcriptional activator. Because EWS is not a DNA-binding protein capable of interacting with specific gene promoter elements, we fused EWS to the DNA-binding domain of the yeast transcription factor Gal4. This allowed us to measure the transcriptional activity of the Gal4-EWS fusion protein with a luciferase reporter gene driven by two Gal4 DNA-binding sites. Surprisingly, Gal4-EWS did not activate transcription and was even slightly less active than the Gal4 DNA-binding domain alone (Fig. 2)
. Furthermore, deletion of the NH2-terminal transactivation domain in Gal4-EWS246–656 and Gal4-EWS374–656 resulted in significant repression of luciferase activity to only 
30% of the control. These data suggest that the COOH-terminal half of EWS may function as a transcriptional repressor.
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; Gal4-EWS2–502). Additional deletion of COOH-terminal amino acids eliminating most of the potential RNA-binding domain (Fig. 2
; see Gal4-EWS2–374) enhanced transcriptional activation, whereas additional deletion of the first RGG domain was without significant effect (Fig. 2
; see Gal4-EWS2–246). Altogether, COOH-terminal amino acids of EWS can efficiently repress its NH2-terminal activation domain.
The EWS Protein Is a Transcriptional Regulator.
Despite our inability to observe any transcriptional activity of the Gal4-EWS fusion protein, we speculated that the genuine EWS protein nevertheless might be able to regulate transcription. Thus, we analyzed the impact of EWS overexpression on various luciferase reporter genes in RK13 cells. Whereas the promoters of the human c-fos proto-oncogene (39)
, of the Xenopus homeobox Xvent-2 gene (40)
, and of the human ErbB2 (HER2/Neu) gene (41)
were stimulated by EWS more than 4-fold in a dose-dependent manner (Fig. 3a)
, the Smad protein-dependent 3TP-lux reporter (42)
was only 1.6-fold activated, and the viral CMV reporter not at all. Thus, EWS acts as a promoter-specific transcriptional coactivator in RK13 cells.
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. In AKR cells, these three promoters were also activated by EWS, albeit to a lesser degree than in RK13 cells. Similar as in RK13 cells, the 3TP-lux and CMV reporter plasmids were not activated in AKR cells (Fig. 3b)
. Surprisingly, overexpression of EWS in Mv1Lu cells led to only a marginal activation (<1.6-fold) of the c-fos and Xvent-2 promoters, and no activation at all was observed in 293T cells. Furthermore, the ErbB2 promoter was repressed by 40% in Mv1Lu cells but slightly (1.3-fold) activated in 293T cells. In line with the experiments in RK13 cells, the 3TP-lux and CMV reporters were basically unaffected by EWS overexpression in both Mv1Lu and 293T cells (Fig. 3b)
. Thus, EWS functions as a transcriptional activator in a cell-type-dependent manner.
Interaction of EWS with CBP and p300.
To investigate whether EWS may specifically collaborate with certain transcription factors, we analyzed three different Gal4 fusion proteins containing activation domains of the transcription factor Elk-1 (34)
, the cofactor CBP (35)
, or the viral transcription factor VP16 (43)
. Whereas EWS expression did not affect the vector control or the Gal4-Elk fusion protein, the COOH-terminal activation domain of CBP was 5.7-fold more active in the presence of than in the absence of EWS (Fig. 4a)
. In contrast, the Gal4-VP16 protein was slightly repressed by EWS. These results demonstrate that EWS functions as a cofactor for selected transactivation domains, further corroborating that EWS is involved in gene transcription.
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-Myc immunoprecipitations were performed, and the presence of p300 was analyzed by -HA immunoblotting. As shown in Fig. 4b
, p300 did coimmunoprecipitate with EWS, whereas no p300 was detectable in the immunoprecipitate in the absence of coexpressed Myc-tagged EWS. Similarly, EWS interacted specifically with p300 in a reverse order coimmunoprecipitation experiment (-HA immunoprecipitation with subsequent -Myc immunoblotting; Fig. 4c
). Finally, we analyzed whether endogenous EWS and p300 form a complex in vivo. To that end, 293T cell extracts were subjected to immunoprecipitation with -p300 antibodies. As shown in Fig. 4d
(Lane 2), EWS coimmunoprecipitated with p300. We conclude that EWS and p300 interact with each other in vivo.
Mapping of Interaction Domains in EWS and CBP/p300.
We then determined which regions of EWS are important for the interaction with p300. Deletion of the last 154 amino acids in EWS2–502 abolished binding to p300 in our coimmunoprecipitation assay (Fig. 5a)
, and EWS2–374 and EWS2–246 were also consistently incapable of binding to p300. Conversely, deletion of the first 245 or 373 amino acids did not abolish binding of EWS to p300 (see EWS246–656 and EWS374–656). However, EWS246–656 interacted less efficiently with p300 than EWS374–656, suggesting that amino acids 246–373 may exert a negative effect on p300-binding. Similarly, we analyzed the interaction of EWS with CBP. As observed with p300, full-length EWS2–656 interacted with CBP, whereas EWS2–502 did not (Fig. 5b)
. Altogether, the COOH-terminal half of EWS interacts with the homologous proteins CBP and p300.
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), and we wanted to test which of these could bind to EWS. To that end, various GST-CBP fusion proteins were purified, bound to glutathione agarose and incubated with a cell extract containing HA-tagged EWS. Bound EWS was revealed by -HA immunoblotting. As expected from the activation of Gal4-CBP1891–2441 by EWS (see Fig. 4a
), GST-CBP1891–2441 bound EWS (Fig. 5d)
. Surprisingly, another domain in CBP, amino acids 451–721, also interacted with EWS, demonstrating that two independent EWS interaction domains reside within CBP.
CBP/p300 Are Required for EWS Function.
To determine whether CBP/p300 are required for EWS-mediated activation of the c-fos promoter, we studied two truncations of EWS that are incompetent in binding to CBP/p300, EWS2–502, and EWS2–246. In contrast to full-length EWS2–656, both of these COOH-terminal truncations did not activate the c-fos promoter (Fig. 6a)
. Conversely, EWS246–656 that is able to interact with CBP/p300 is capable of activating c-fos (Fig. 6a)
. These data would be in line with the hypothesis that EWS requires CBP/p300 for its function as a transcriptional activator.
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, E1A on its own did not influence c-fos expression, but it abolished EWS-mediated c-fos activation. Furthermore, an E1A mutant that is defective in CBP/p300-binding, E1A-2-36 (33)
, did not significantly suppress c-fos activation by EWS. These results indicate that EWS is indeed dependent on CBP/p300 to function as a transcriptional coactivator. |
DISCUSSION |
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The exclusively nuclear localization of EWS was dependent on the presence of both its NH2-terminal activation domain and COOH-terminal amino acids. Interestingly, the NH2-terminal activation domain on its own (EWS2–246) was predominantly localized to the cell nucleus. Because the EWS NH2-terminal activation domain can be fused to various DNA-binding domains of heterologous transcription factors because of chromosomal translocations in tumor cells (1 , 2) , its presence may ensure that the resulting chimeric transcription factors are localized to the cell nucleus, wherein they constitutively up-regulate target genes resulting in cell transformation.
In contradiction to our data, Felsch et al. (46) reported that the EWS protein is primarily cytoplasmic and associated with ribosome containing fractions. However, these data were obtained solely by subcellular fractionation experiments that are prone to the leaking of nuclear proteins into the cytoplasm. In contrast, another research laboratory (47) also performed subcellular fractionation and revealed an Mr 85,000 protein by means of an antibody directed against IQ domains. This Mr 85,000 protein was present in the nuclear extract but not in the cytoplasmic or membrane fractions. Subsequent purification and protein sequencing identified the Mr 85,000 protein as the IQ domain containing EWS protein (47) , thus corroborating our finding of nuclear localization of EWS.
Overexpression of EWS in RK13 and AKR cells led to the activation of the c-fos, Xvent-2, and ErbB2 promoters, indicating that EWS functions as a transcriptional cofactor. However, EWS does not appear to be a general coactivator, because the 3TP-lux or the CMV promoter were unaffected by EWS expression in RK13 and AKR cells. Furthermore, EWS only negligibly, if at all, affected the c-fos, Xvent-2, and ErbB2 promoters in Mv1Lu and 293T cells, suggesting that EWS functions in a cell-type-specific manner. Because EWS does not contain any obvious DNA-binding domain, nor has it been reported to bind to double-stranded DNA, EWS may be recruited to promoters by protein-protein interactions. This could involve specific DNA-binding transcription factors, or components of the basal transcription machinery. Given that EWS interacts with the basal transcription factor TFIID as well as with the RNA polymerase II holoenzyme (28) , the latter possibility appears to be highly likely. Yet, EWS may also be recruited to gene promoters through interaction with CBP/p300, which themselves interact with specific transcription factors such as the c-fos regulating proteins CREB and Elk-1 (30 , 39) .
The COOH-terminal amino acids 246–656 of EWS were sufficient to activate the c-fos promoter, and the degree of activation was comparable with that of full-length EWS. Thus, NH2-terminal amino acids of EWS are not necessary for the functional activation of c-fos by EWS. This is in contrast to EWS fusion proteins, which require EWS NH2-terminal amino acids for both gene activation and cell transformation.
Whereas EWS246–656 activates c-fos, this COOH-terminal portion of EWS exhibits repressing activity on its own as well as on the NH2-terminal activation domain when fused to the DNA binding domain of the yeast protein Gal4. These results suggest that COOH-terminal EWS amino acids must be excluded, and indeed are excluded, from the fusions to the DNA-binding domains of ETS transcription factors, ATF1, WT1, CHOP/GADD153, or CHN/TEC, to elicit the tumorigenic potential of these respective fusion proteins in various types of cancer (1 , 2) . Indeed, while this manuscript was under preparation, Li and Lee (48) demonstrated that a fusion of full-length EWS to ATF1 was transcriptionally repressed, and that this repression was collaboratively mediated by the three arginine-glycine-glycine motif-rich regions of EWS. Again, this suggests that EWS domains perform different functions in the genuine EWS protein versus the EWS fusion proteins found in cancer cells.
The physical association of EWS with CBP/p300 appears to be required for the ability of EWS to enhance gene expression, because deletion of the COOH terminus that mediates binding to CBP/p300 abolished c-fos activation by EWS. Also, EWS-mediated c-fos activation was suppressed by the adenoviral E1A protein that has been shown to antagonize CBP/p300 function (44 , 45) . Importantly, an E1A mutant deficient in CBP/p300-binding, and thereby unable to obstruct CBP/p300 function, did not suppress EWS-mediated c-fos activation, strongly corroborating that EWS activates gene activity by collaborating with the cofactors CBP and p300.
Interestingly, CBP encompasses two independent interaction domains for EWS, amino acids 451–721 and 1891–2441. Amino acids 451–721 have been found to interact with transcription factors such as CREB, c-Myb, and the viral Tax protein, whereas the COOH terminus of CBP accommodates binding to receptor-activated Smad proteins and cofactors of nuclear hormone receptors (29 , 30 , 49) . As such, the widely expressed EWS protein (50) may compete with many different transcription factors for the limiting amounts of CBP/p300 within the cell, thereby potentially influencing transcription factor activity by squelching (29 , 51) .
CBP/p300 are essential coactivators (52 , 53) and have long been suspected to be tumor suppressors (30) . In support of this notion, it has recently been shown that loss of heterozygosity at the CBP locus is associated with hematological malignancies (54) , and that p300 mutations in epithelial cancers may be pathogenic (55) . Thus, the EWS protein may be an important accessory factor of the tumor suppressors CBP/p300 in the control of cell proliferation. In addition, CBP/p300 have been implicated in many developmental processes (56) , which may require collaboration with the interaction partner EWS. In line with a potential role of EWS in development are our findings that EWS can regulate c-fos expression, which is required for proper bone development and hematopoiesis (57) , Xvent-2 gene activity, which controls pattern formation in the mesoderm (40) , as well as activity of ErbB2, which is involved in neural and cardiac development (41) .
The EWS transactivation domain is phosphorylated by v-Src and thereby stimulated (58) . Other tyrosine kinases, probably via their SH3 domains, may also interact with the proline-rich NH2 terminus of EWS (59) . Tyrosine phosphorylation mediated by these protein kinases could give rise to the binding of other signaling molecules containing SH2 domains (58) , whereby EWS could be regulated in its transcriptional properties through a variety of mitogenic signaling pathways. In addition, EWS may be linked to Ca2+ signal transduction pathways because its IQ domain interacts with calmodulin, whose binding to EWS may be inhibited by PKC phosphorylation within the IQ domain (47) . Thus, a plethora of intracellular signaling pathways could use EWS to modulate gene transcription, both in normal and in cancer cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Mayo Foundation, the Mayo Clinic Cancer Center and a scholarship (to R. J.) from the Sidney Kimmel Foundation for Cancer Research. 
2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: (507) 266-4393; Fax: (507) 284-1767; E-mail: janknecht.ralf{at}mayo.edu
3 The abbreviations used are: EWS, Ewing’s sarcoma; CBP, CREB-binding protein; GST, glutathione S-transferase; HA, hemagglutinin; WT1, Wilms’ tumor.
Received 8/ 8/00. Accepted 1/17/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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