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• W2000639640 abstract "Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation. Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation. IntroductionThe Ewing family tumors (EFTs) 2The abbreviations used are: EFTEwing family tumorPol IIpolymerase IIUppuridine phosphorylaseTsp2thrombospondin 2IGFBP3insulin-like growth factor binding protein 3qRT-PCRquantitative RT-PCRU6 818U6 polymerase III promoterqPCRquantitative PCRUntra6mouse chromosome 6. are a family of malignant diseases that have traditionally been labeled Ewing's sarcoma, primitive neuroectodermal tumors, and Askin tumors (1Bernstein M. Kovar H. Paulussen M. Randall R.L. Schuck A. Teot L.A. Juergens H. Oncologist. 2006; 11: 503-519Crossref PubMed Scopus (338) Google Scholar). The genetic hallmark of EFTs is a reciprocal translocation between the N terminus of the EWS gene and the C terminus of one of five ETS transcription factor family members: FLI1, ERG, ETV-1, ETV4, or FEV (2Turc-Carel C. Philip I. Berger M.P. Philip T. Lenoir G. C. R. Seances Acad. Sci. III. 1983; 296: 1101-1103PubMed Google Scholar, 3Delattre O. Zucman J. Plougastel B. Desmaze C. Melot T. Peter M. Kovar H. Joubert I. de Jong P. Rouleau G. et al.Nature. 1992; 359: 162-165Crossref PubMed Scopus (1505) Google Scholar). Forced expression of EWS/FLI1 results in transformation and tumorigenesis in NIH (National Institutes of Health) 3T3 cells (4Thompson A.D. Teitell M.A. Arvand A. Denny C.T. Oncogene. 1999; 18: 5506-5513Crossref PubMed Scopus (89) Google Scholar). When either the EWS or FLI1 domains are deleted, transformation is lost (5May W.A. Gishizky M.L. Lessnick S.L. Lunsford L.B. Lewis B.C. Delattre O. Zucman J. Thomas G. Denny C.T. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 5752-5756Crossref PubMed Scopus (478) Google Scholar). Also, when EWS/FLI1 expression is reduced via RNAi, EFT cell lines typically undergo growth arrest (6Dohjima T. Lee N.S. Li H. Ohno T. Rossi J.J. Mol. Ther. 2003; 7: 811-816Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 7Matsunobu T. Tanaka K. Nakamura T. Nakatani F. Sakimura R. Hanada M. Li X. Okada T. Oda Y. Tsuneyoshi M. Iwamoto Y. Cancer Res. 2006; 66: 803-811Crossref PubMed Scopus (44) Google Scholar). Taken together, these data have bolstered the consensus view that EWS/ETS fusions are necessary both for EFT formation and for continued growth.Functions of normal EWS and ETS proteins have molded current views of how EWS/ETS fusions promote cellular transformation. ETS proteins belong to a well characterized family of winged helix-loop-helix transcription factors that are related through a highly conserved 85-amino acid ETS domain. They typically work by forming heteromeric complexes with other transcription factors and can act as either transcription activators or repressors. The ETS domain mediates site-specific DNA binding, recognizing the GGA(A/T) consensus sequence as well as facilitating protein-protein interactions (8Seth A. Ascione R. Fisher R.J. Mavrothalassitis G.J. Bhat N.K. Papas T.S. Cell Growth & Differ. 1992; 3: 327-334PubMed Google Scholar, 9Karim F.D. Urness L.D. Thummel C.S. Klemsz M.J. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Gunther C.V. Nye J.A. et al.Genes Dev. 1990; 4: 1451-1453Crossref PubMed Scopus (378) Google Scholar). The ETS proteins are involved in a wide variety of functions that include cellular proliferation, differentiation, cell cycle control, development, and tumorigenesis (8Seth A. Ascione R. Fisher R.J. Mavrothalassitis G.J. Bhat N.K. Papas T.S. Cell Growth & Differ. 1992; 3: 327-334PubMed Google Scholar, 10Papas T.S. Bhat N.K. Spyropoulos D.D. Mjaatvedt A.E. Vournakis J. Seth A. Watson D.K. Leukemia. 1997; 11: 557-566PubMed Google Scholar, 11Athanasiou M. Clausen P.A. Mavrothalassitis G.J. Zhang X.K. Watson D.K. Blair D.G. Cell Growth & Differ. 1996; 7: 1525-1534PubMed Google Scholar, 12Hart A. Melet F. Grossfeld P. Chien K. Jones C. Tunnacliffe A. Favier R. Bernstein A. Immunity. 2000; 13: 167-177Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 13Mélet F. Motro B. Rossi D.J. Zhang L. Bernstein A. Mol. Cell. Biol. 1996; 16: 2708-2718Crossref PubMed Scopus (97) Google Scholar). As a result of chromosomal translocation, the normal N terminus of FLI1 is replaced by N-terminal sequences from EWS. However, in all EFT fusions, the ETS domain is invariably present.EWS is a member of the ubiquitously expressed FET family of proteins, consisting of FUS (TLS), EWS, and TAF15 (hTAFII68). The N terminus of these proteins, which is retained in EFT fusion proteins, is a highly disordered peptide consisting of a series of degenerate repeats with a consensus sequence of NSYGQQS (14Ng K.P. Potikyan G. Savene R.O. Denny C.T. Uversky V.N. Lee K.A. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 479-484Crossref PubMed Scopus (82) Google Scholar). This domain can act as a potent transcriptional activator in heterologous model systems (15May W.A. Lessnick S.L. Braun B.S. Klemsz M. Lewis B.C. Lunsford L.B. Hromas R. Denny C.T. Mol. Cell. Biol. 1993; 13: 7393-7398Crossref PubMed Scopus (442) Google Scholar). The C terminus of this family, which is not present in EFT fusions, consists of an RNA-binding domain and several RGG-box domains. Although the functions of FET proteins have yet to be completely elucidated, evidence of interactions with TFIID, RNA polymerase II (Pol II) (16Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (309) Google Scholar, 17Bertolotti A. Melot T. Acker J. Vigneron M. Delattre O. Tora L. Mol. Cell. Biol. 1998; 18: 1489-1497Crossref PubMed Scopus (216) Google Scholar), and elements of the RNA splicing machinery (18Janknecht R. Gene. 2005; 363: 1-14Crossref PubMed Scopus (136) Google Scholar, 19Yang L. Embree L.J. Tsai S. Hickstein D.D. J. Biol. Chem. 1998; 273: 27761-27764Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) indicate that these proteins may play a role in transcription and/or mRNA splicing.Given this information, the prevailing theory is that EWS/FLI1 causes transformation by acting as an aberrant transcription factor. Much of this theory is on the basis of the replacement of the FLI1 activation domain with the more potent EWS activation domain, whereas the FLI1 DNA-binding domain remains intact and functional. EWS/FLI1 has been shown to bind directly to the promoters of several of its target genes and is also able to modulate gene expression in reporter assays (20Cironi L. Riggi N. Provero P. Wolf N. Suvà M.L. Suvà D. Kindler V. Stamenkovic I. PLoS ONE. 2008; 3: e2634Crossref PubMed Scopus (88) Google Scholar, 21Beauchamp E. Bulut G. Abaan O. Chen K. Merchant A. Matsui W. Endo Y. Rubin J.S. Toretsky J. Uren A. J. Biol. Chem. 2009; 284: 9074-9082Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Additionally, EWS/FLI1 retains the ability of native EWS to interact with Pol II (22Petermann R. Mossier B.M. Aryee D.N. Khazak V. Golemis E.A. Kovar H. Oncogene. 1998; 17: 603-610Crossref PubMed Scopus (125) Google Scholar). Despite this evidence, the explicit molecular mechanisms through which EWS/ETS fusions modulate gene expression remain ill defined.Previous microarray and ChIP experiments have identified several direct targets of EWS/FLI1 in both human and murine cell lines. Uridine phosphorylase (Upp) was identified as a direct target gene necessary for tumorigenesis that is up-regulated by EWS/FLI1 in NIH 3T3 cells (23Deneen B. Hamidi H. Denny C.T. Cancer Res. 2003; 63: 4268-4274PubMed Google Scholar). Although most research has focused on up-regulated genes, there are actually more genes repressed by EWS/FLI1 in microarray analysis. In NIH 3T3 cells, thrombospondin 2 (Tsp2) is a direct target gene of EWS/FLI1, and several of its binding sites are present in the Tsp2 promoter (24Potikyan G. Savene R.O. Gaulden J.M. France K.A. Zhou Z. Kleinerman E.S. Lessnick S.L. Denny C.T. Cancer Res. 2007; 67: 6675-6684Crossref PubMed Scopus (33) Google Scholar). Forced expression of thrombospondins antagonized EFT tumor growth in a murine model system, confirming the potential biologic impact of down-regulated target genes. In EFT cell lines, insulin-like growth factor binding protein 3 (IGFBP3), an important regulator of cell proliferation and apoptosis, has been shown to be directly down-regulated by EWS/FLI1 (25Prieur A. Tirode F. Cohen P. Delattre O. Mol. Cell. Biol. 2004; 24: 7275-7283Crossref PubMed Scopus (333) Google Scholar). Using these previously identified direct target genes in well characterized cellular model systems, we now find that EWS/ETS fusions can modulate target gene expression through both transcriptional and posttranscriptional mechanisms.DISCUSSIONFor the last 15 years, the hypothesis that EWS/FLI1 transforms cells by acting as an aberrant transcription factor has stood. However, trying to mechanistically dissect how these fusion proteins function at a molecular genetic level has been notoriously difficult. With this in mind, we began our investigation from a broad perspective by querying whether EWS/FLI1 regulated target genes at a transcriptional or posttranscriptional level. To do this, we compared the levels of mRNA and pre-mRNA of EWS/FLI1 direct target genes in both murine and human model systems. Our results in both systems fit the view that EWS/FLI1 can modulate target gene expression both at the level of transcript synthesis and at the level of mRNA degradation.The strategy of using pre-mRNA levels as a proxy for new transcript synthesis has been previously validated by other investigators. However, this approach has limitations. First, because pre-mRNA levels are assayed by qRT-PCR using site-specific primers spanning intron-exon borders, it assumes that EWS/FLI1 does not significantly alter splicing rates at each specific location. Second, it is well known that for each gene there is intron-to-intron variability in splicing rates. This makes it difficult to derive quantitative meaning from comparisons of pre-mRNA and mRNA levels. For example, EWS/FLI1 down-regulates IGFBP3 mRNA levels 40-fold more than pre-mRNA levels in A673 cells. However, this difference can only be interpreted qualitatively. Although both transcriptional and post-transcriptional regulation is occurring, the relative proportion of each mechanism cannot be gleaned from this assay.To gain further insight into transcript synthesis dynamics of EWS/FLI1 target genes, Pol II ChIP analyses were performed. The relative low level of enrichment of Pol II at the promoters of Upp and Tsp2 in NIH 3T3 cells and IGFBP3 in EFT cell lines when compared with GAPDH suggests that with or without EWS/FLI1, these genes are being transcribed at modest rates. Despite showing an almost 8-fold increase in Upp pre-mRNA in response to EWS/FLI1 in NIH 3T3 cells, there was no significant increase in Pol II occupancy. This suggests that in this cellular context, EWS/FLI1 may be exerting its effect on Upp at stages distal to recruitment of Pol II such as promoter escape, elongation rate, or movement through pause sites.In contrast to Upp, we found that for down-regulated target genes, EWS/FLI1 did alter promoter Pol II occupancy in both NIH 3T3 and EFT model systems. Although they were reproducible, the differences in Pol II occupancy were small, ∼3.5-fold for Tsp2 in NIH 3T3 cells and about 2-fold for IGFBP3 in EFT cell lines. EWS/FLI1 may also change the Pol II occupancy within the IGFBP3 coding region in A4573 cells, but the changes were near the limit of detectability. Because of the lower level of IGFBP3 Pol II ChIP enrichment in A673 cells, we were unable to determine the level of Pol II occupancy in the IGFBP3 coding region. Together with our pre-mRNA analyses, these data suggest that EWS/FLI1 down-regulates some target genes through a decrease in transcript synthesis.Although transcript synthesis may be playing a role in dictating the steady-state levels of EWS/FLI1 down-regulated targets, our pre-mRNA studies suggested that additional regulatory mechanisms might be at work as well. We confirmed this by finding that in A673 cells, EWS/FLI1 decreased the half-life of IGFBP3 transcripts by almost 3-fold. Although there was considerable experiment-to-experiment variation, the decrement in half-life was clearly statistically significant. The relatively high degree of variation is probably due to the serial manipulations needed in our model system, EFT cells first being lentivirally transduced with shRNA constructs and then treated with α-Amanitin. By sequentially coupling these two procedures, the net effect on overall variability of the assay is cumulative.When we tried to perform similar analyses with the A4573 EFT cell line, we were unable to obtain consistent results. This may be due to the fact that A4573 cells, like most EFT cell lines, undergo growth arrest in response to EWS/FLI1 knockdown. Factoring in this additional parameter could have increased the overall variability of the assay to the point that it was no longer interpretable. Alternatively, half-life modulation of EWS/FLI1-down-regulated target genes may take time to develop fully. Qualitatively comparing the change in IGFBP3 pre-mRNA and mRNA levels in response to EWS/FLI1 knockdown in the two EFT cell lines suggests that this might be true. Although the change in pre-mRNA in both cell lines was about 10-fold, IGFBP3 mRNA changed ∼30-fold in A4573 cells that by necessity were harvested 2 days after EWS/FLI1 knockdown, whereas an over 300-fold change was found in A673 cells that could be harvested 2 weeks after knockdown. In effect, the ability of A673 cells to survive in tissue culture with greatly reduced levels of endogenous EWS/FLI1, allowed us to uncover an additional mechanism of target gene modulation.On a mechanistic level, the loss of repression of IGFBP3 in A673 cells expressing an shRNA construct targeting endogenous EWS/FLI1 can be reestablished by transducing EWS/FLI1, which drives down IGFBP3 mRNA levels. However, neither transduction of EWS nor ETS EWS/FLI1 mutants were able to fully repress IGFBP3 to the extent seen with the wild-type fusion. This suggests that both these domains play functional roles in this process, perhaps by promoting protein-protein and DNA binding interactions through the EWS and ETS domains, respectively. The fact that IGFBP3 levels consistently increased in cells transduced with the EWS/FLI1(TPM) construct also suggests that this mutant might be acting in a dominant negative fashion by sequestering factors involved with EWS/FLI1 target gene repression.There are multiple ways through which cells can regulate transcript degradation rates, ranging from modulating components of the mRNA metabolism machinery to altering expression of specific microRNAs. For example, EWS/FLI1 has been shown to down-regulate miRNA145 in human pediatric mesenchymal stem cells (38Riggi N. Suvà M.L. De Vito C. Provero P. Stehle J.C. Baumer K. Cironi L. Janiszewska M. Petricevic T. Suvà D. Tercier S. Joseph J.M. Guillou L. Stamenkovic I. Genes Dev. 2010; 24: 916-932Crossref PubMed Scopus (217) Google Scholar). Although explicitly defining the components through which EWS/FLI1 modulates target gene half-life is beyond the scope of this manuscript, such investigations will depend on having well characterized EFT model systems that can exist in the absence of EWS/FLI1 for more than 1 or 2 days. This will be a challenge.Our work suggest that EWS/FLI1 may be able to induce large gene expression changes by causing smaller alterations in multiple stages of gene regulation. The regulation of casein by prolactin is a case in point of such synergy (40Guyette W.A. Matusik R.J. Rosen J.M. Cell. 1979; 17: 1013-1023Abstract Full Text PDF PubMed Scopus (305) Google Scholar). Prolactin was able to up-regulate casein mRNA 6–12-fold but only increased casein transcription by 2- to 4-fold. On further investigation, the authors also found that prolactin increased casein mRNA half-life and concluded that both mechanisms were necessary for proper regulation.EWS/FLI1 may work in a similarly multifaceted manner, at least for some down-regulated target genes. From a pragmatic point of view, this is not a welcoming prospect. Studying multiple regulatory mechanisms, each with a limited dynamic response to EWS/FLI1, will certainly be more challenging than investigating a single mode of action with a large dynamic range. If indeed EWS/FLI1 is working via multiple molecular mechanisms, effective therapeutic targeting of this fusion could prove to be difficult. The key to meeting these challenges will be to devise better EFT model systems in which EWS/FLI1 target gene modulation can be studied at multiple mechanistic levels. IntroductionThe Ewing family tumors (EFTs) 2The abbreviations used are: EFTEwing family tumorPol IIpolymerase IIUppuridine phosphorylaseTsp2thrombospondin 2IGFBP3insulin-like growth factor binding protein 3qRT-PCRquantitative RT-PCRU6 818U6 polymerase III promoterqPCRquantitative PCRUntra6mouse chromosome 6. are a family of malignant diseases that have traditionally been labeled Ewing's sarcoma, primitive neuroectodermal tumors, and Askin tumors (1Bernstein M. Kovar H. Paulussen M. Randall R.L. Schuck A. Teot L.A. Juergens H. Oncologist. 2006; 11: 503-519Crossref PubMed Scopus (338) Google Scholar). The genetic hallmark of EFTs is a reciprocal translocation between the N terminus of the EWS gene and the C terminus of one of five ETS transcription factor family members: FLI1, ERG, ETV-1, ETV4, or FEV (2Turc-Carel C. Philip I. Berger M.P. Philip T. Lenoir G. C. R. Seances Acad. Sci. III. 1983; 296: 1101-1103PubMed Google Scholar, 3Delattre O. Zucman J. Plougastel B. Desmaze C. Melot T. Peter M. Kovar H. Joubert I. de Jong P. Rouleau G. et al.Nature. 1992; 359: 162-165Crossref PubMed Scopus (1505) Google Scholar). Forced expression of EWS/FLI1 results in transformation and tumorigenesis in NIH (National Institutes of Health) 3T3 cells (4Thompson A.D. Teitell M.A. Arvand A. Denny C.T. Oncogene. 1999; 18: 5506-5513Crossref PubMed Scopus (89) Google Scholar). When either the EWS or FLI1 domains are deleted, transformation is lost (5May W.A. Gishizky M.L. Lessnick S.L. Lunsford L.B. Lewis B.C. Delattre O. Zucman J. Thomas G. Denny C.T. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 5752-5756Crossref PubMed Scopus (478) Google Scholar). Also, when EWS/FLI1 expression is reduced via RNAi, EFT cell lines typically undergo growth arrest (6Dohjima T. Lee N.S. Li H. Ohno T. Rossi J.J. Mol. Ther. 2003; 7: 811-816Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 7Matsunobu T. Tanaka K. Nakamura T. Nakatani F. Sakimura R. Hanada M. Li X. Okada T. Oda Y. Tsuneyoshi M. Iwamoto Y. Cancer Res. 2006; 66: 803-811Crossref PubMed Scopus (44) Google Scholar). Taken together, these data have bolstered the consensus view that EWS/ETS fusions are necessary both for EFT formation and for continued growth.Functions of normal EWS and ETS proteins have molded current views of how EWS/ETS fusions promote cellular transformation. ETS proteins belong to a well characterized family of winged helix-loop-helix transcription factors that are related through a highly conserved 85-amino acid ETS domain. They typically work by forming heteromeric complexes with other transcription factors and can act as either transcription activators or repressors. The ETS domain mediates site-specific DNA binding, recognizing the GGA(A/T) consensus sequence as well as facilitating protein-protein interactions (8Seth A. Ascione R. Fisher R.J. Mavrothalassitis G.J. Bhat N.K. Papas T.S. Cell Growth & Differ. 1992; 3: 327-334PubMed Google Scholar, 9Karim F.D. Urness L.D. Thummel C.S. Klemsz M.J. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Gunther C.V. Nye J.A. et al.Genes Dev. 1990; 4: 1451-1453Crossref PubMed Scopus (378) Google Scholar). The ETS proteins are involved in a wide variety of functions that include cellular proliferation, differentiation, cell cycle control, development, and tumorigenesis (8Seth A. Ascione R. Fisher R.J. Mavrothalassitis G.J. Bhat N.K. Papas T.S. Cell Growth & Differ. 1992; 3: 327-334PubMed Google Scholar, 10Papas T.S. Bhat N.K. Spyropoulos D.D. Mjaatvedt A.E. Vournakis J. Seth A. Watson D.K. Leukemia. 1997; 11: 557-566PubMed Google Scholar, 11Athanasiou M. Clausen P.A. Mavrothalassitis G.J. Zhang X.K. Watson D.K. Blair D.G. Cell Growth & Differ. 1996; 7: 1525-1534PubMed Google Scholar, 12Hart A. Melet F. Grossfeld P. Chien K. Jones C. Tunnacliffe A. Favier R. Bernstein A. Immunity. 2000; 13: 167-177Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 13Mélet F. Motro B. Rossi D.J. Zhang L. Bernstein A. Mol. Cell. Biol. 1996; 16: 2708-2718Crossref PubMed Scopus (97) Google Scholar). As a result of chromosomal translocation, the normal N terminus of FLI1 is replaced by N-terminal sequences from EWS. However, in all EFT fusions, the ETS domain is invariably present.EWS is a member of the ubiquitously expressed FET family of proteins, consisting of FUS (TLS), EWS, and TAF15 (hTAFII68). The N terminus of these proteins, which is retained in EFT fusion proteins, is a highly disordered peptide consisting of a series of degenerate repeats with a consensus sequence of NSYGQQS (14Ng K.P. Potikyan G. Savene R.O. Denny C.T. Uversky V.N. Lee K.A. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 479-484Crossref PubMed Scopus (82) Google Scholar). This domain can act as a potent transcriptional activator in heterologous model systems (15May W.A. Lessnick S.L. Braun B.S. Klemsz M. Lewis B.C. Lunsford L.B. Hromas R. Denny C.T. Mol. Cell. Biol. 1993; 13: 7393-7398Crossref PubMed Scopus (442) Google Scholar). The C terminus of this family, which is not present in EFT fusions, consists of an RNA-binding domain and several RGG-box domains. Although the functions of FET proteins have yet to be completely elucidated, evidence of interactions with TFIID, RNA polymerase II (Pol II) (16Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (309) Google Scholar, 17Bertolotti A. Melot T. Acker J. Vigneron M. Delattre O. Tora L. Mol. Cell. Biol. 1998; 18: 1489-1497Crossref PubMed Scopus (216) Google Scholar), and elements of the RNA splicing machinery (18Janknecht R. Gene. 2005; 363: 1-14Crossref PubMed Scopus (136) Google Scholar, 19Yang L. Embree L.J. Tsai S. Hickstein D.D. J. Biol. Chem. 1998; 273: 27761-27764Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) indicate that these proteins may play a role in transcription and/or mRNA splicing.Given this information, the prevailing theory is that EWS/FLI1 causes transformation by acting as an aberrant transcription factor. Much of this theory is on the basis of the replacement of the FLI1 activation domain with the more potent EWS activation domain, whereas the FLI1 DNA-binding domain remains intact and functional. EWS/FLI1 has been shown to bind directly to the promoters of several of its target genes and is also able to modulate gene expression in reporter assays (20Cironi L. Riggi N. Provero P. Wolf N. Suvà M.L. Suvà D. Kindler V. Stamenkovic I. PLoS ONE. 2008; 3: e2634Crossref PubMed Scopus (88) Google Scholar, 21Beauchamp E. Bulut G. Abaan O. Chen K. Merchant A. Matsui W. Endo Y. Rubin J.S. Toretsky J. Uren A. J. Biol. Chem. 2009; 284: 9074-9082Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Additionally, EWS/FLI1 retains the ability of native EWS to interact with Pol II (22Petermann R. Mossier B.M. Aryee D.N. Khazak V. Golemis E.A. Kovar H. Oncogene. 1998; 17: 603-610Crossref PubMed Scopus (125) Google Scholar). Despite this evidence, the explicit molecular mechanisms through which EWS/ETS fusions modulate gene expression remain ill defined.Previous microarray and ChIP experiments have identified several direct targets of EWS/FLI1 in both human and murine cell lines. Uridine phosphorylase (Upp) was identified as a direct target gene necessary for tumorigenesis that is up-regulated by EWS/FLI1 in NIH 3T3 cells (23Deneen B. Hamidi H. Denny C.T. Cancer Res. 2003; 63: 4268-4274PubMed Google Scholar). Although most research has focused on up-regulated genes, there are actually more genes repressed by EWS/FLI1 in microarray analysis. In NIH 3T3 cells, thrombospondin 2 (Tsp2) is a direct target gene of EWS/FLI1, and several of its binding sites are present in the Tsp2 promoter (24Potikyan G. Savene R.O. Gaulden J.M. France K.A. Zhou Z. Kleinerman E.S. Lessnick S.L. Denny C.T. Cancer Res. 2007; 67: 6675-6684Crossref PubMed Scopus (33) Google Scholar). Forced expression of thrombospondins antagonized EFT tumor growth in a murine model system, confirming the potential biologic impact of down-regulated target genes. In EFT cell lines, insulin-like growth factor binding protein 3 (IGFBP3), an important regulator of cell proliferation and apoptosis, has been shown to be directly down-regulated by EWS/FLI1 (25Prieur A. Tirode F. Cohen P. Delattre O. Mol. Cell. Biol. 2004; 24: 7275-7283Crossref PubMed Scopus (333) Google Scholar). Using these previously identified direct target genes in well characterized cellular model systems, we now find that EWS/ETS fusions can modulate target gene expression through both transcriptional and posttranscriptional mechanisms." @default.
• W2000639640 created "2016-06-24" @default.
• W2000639640 creator A5003605582 @default.
• W2000639640 creator A5044287026 @default.
• W2000639640 creator A5072278752 @default.
• W2000639640 creator A5089132706 @default.
• W2000639640 date "2011-07-01" @default.
• W2000639640 modified "2023-09-29" @default.
• W2000639640 title "Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms" @default.
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