ERG; Alternative splicing; RBFOX2; EWS-FLI1; Ewing sarcoma
Abstract :
[en] ERG family proteins (ERG, FLI1 and FEV) are a sub- family of ETS transcription factors with key roles in physiology and development. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 regulates both transcription and alternative splicing of pre- messenger RNAs. However, whether wild-type ERG family proteins might regulate splicing is unknown. Here, we show that wild-type ERG proteins asso- ciate with spliceosomal components, are found on nascent RNAs, and induce alternative splicing when recruited onto a reporter minigene. Transcriptomic analysis revealed that ERG and FLI1 regulate large numbers of alternative spliced exons (ASEs) en- riched with RBFOX2 motifs and co-regulated by this splicing factor. ERG and FLI1 are associated with RB- FOX2 via their conserved carboxy-terminal domain, which is present in EWS-FLI1. Accordingly, EWS-FLI1 is also associated with RBFOX2 and regulates ASEs enriched in RBFOX2 motifs. However, in contrast to wild-type ERG and FLI1, EWS-FLI1 often antag- onizes RBFOX2 effects on exon inclusion. In particu- lar, EWS-FLI1 reduces RBFOX2 binding to the ADD3 pre-mRNA, thus increasing its long isoform, which represses the mesenchymal phenotype of Ewing sarcoma cells. Our findings reveal a RBFOX2-mediated splicing regulatory function of wild-type ERG family proteins, that is altered in EWS-FLI1 and contributes to the Ewing sarcoma cell phenotype.
✱ These authors have contributed equally to this work.
Language :
English
Title :
ERG transcription factors have a splicing regulatory function involving RBFOX2 that is altered in the EWS-FLI1 oncogenic fusion
Publication date :
01 May 2021
Journal title :
Nucleic Acids Research
ISSN :
0305-1048
eISSN :
1362-4962
Publisher :
Oxford University Press, United Kingdom
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
The authors wish it to be known that, in their opinion, the first two and last three authors should be regarded as Joint First and Joint Last Authors, respectively.
Sharrocks, A.D. (2001) The ETS-domain transcription factor family. Nat. Rev. Mol. Cell Biol., 2, 827-837.
Hollenhorst, P.C., McIntosh, L.P. and Graves, B.J. (2011) Genomic and biochemical insights into the specificity of ETS transcription factors. Annu. Rev. Biochem., 80, 437-471.
Rambout, X., Detiffe, C., Bruyr, J., Mariavelle, E., Cherkaoui, M., Brohée, S., Demoitié, P., Lebrun, M., Soin, R., Lesage, B. et al. (2016) The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression. Nat. Struct. Mol. Biol., 23, 663-672.
Tomlins, S.A., Rhodes, D.R., Perner, S., Dhanasekaran, S.M., Mehra, R., Sun, X.-W., Varambally, S., Cao, X., Tchinda, J., Kuefer, R. et al. (2005) Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science, 310, 644-648.
Ichikawa, H., Shimizu, K., Hayashi, Y. and Ohki, M. (1994) An RNA-binding protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with t(16;21) chromosomal translocation. Cancer Res., 54, 2865-2868.
Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., de Jong, P. and Rouleau, G. (1992) Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature, 359, 162-165.
Boulay, G., Sandoval, G.J., Riggi, N., Iyer, S., Buisson, R., Naigles, B., Awad, M.E., Rengarajan, S., Volorio, A., McBride, M.J. et al. (2017) Cancer-specific retargeting of BAF complexes by a prion-like domain. Cell, 171, 163-178.
Gangwal, K., Sankar, S., Hollenhorst, P.C., Kinsey, M., Haroldsen, S.C., Shah, A.A., Boucher, K.M., Watkins, W.S., Jorde, L.B., Graves, B.J. et al. (2008) Microsatellites as EWS/FLI response elements in Ewing's sarcoma. Proc. Natl. Acad. Sci. U.S.A., 105, 10149-10154.
Knoop, L.L. and Baker, S.J. (2000) The splicing factor U1C represses EWS/FLI-mediated transactivation. J. Biol. Chem., 275, 24865-24871.
Sanchez, G., Bittencourt, D., Laud, K., Barbier, J., Delattre, O., Auboeuf, D. and Dutertre, M. (2008) Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer. Proc. Natl. Acad. Sci. U.S.A., 105, 6004-6009.
Selvanathan, S.P., Graham, G.T., Erkizan, H.V., Dirksen, U., Natarajan, T.G., Dakic, A., Yu, S., Liu, X., Paulsen, M.T., Ljungman, M.E. et al. (2015) Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing. Proc. Natl. Acad. Sci. USA, 112, E1307-E1316.
Knoop, L.L. and Baker, S.J. (2001) EWS/FLI alters 5'-splice site selection. J. Biol. Chem., 276, 22317-22322.
Dutertre, M., Sanchez, G., De Cian, M.-C., Barbier, J., Dardenne, E., Gratadou, L., Dujardin, G., Le Jossic-Corcos, C., Corcos, L. and Auboeuf, D. (2010) Cotranscriptional exon skipping in the genotoxic stress response. Nat. Struct. Mol. Biol., 17, 1358-1366.
Paronetto, M.P., Bernardis, I., Volpe, E., Bechara, E., Sebestyén, E., Eyras, E. and Valcárcel, J. (2014) Regulation of FAS exon definition and apoptosis by the Ewing sarcoma protein. Cell Rep., 7, 1211-1226.
Braunschweig, U., Gueroussov, S., Plocik, A.M., Graveley, B.R. and Blencowe, B.J. (2013) Dynamic integration of splicing within gene regulatory pathways. Cell, 152, 1252-1269.
Rambout, X., Dequiedt, F. and Maquat, L.E. (2018) Beyond transcription: roles of transcription factors in pre-mRNA splicing. Chem. Rev., 118, 4339-4364.
Han, H., Braunschweig, U., Gonatopoulos-Pournatzis, T., Weatheritt, R.J., Hirsch, C.L., Ha, K.C.H., Radovani, E., Nabeel-Shah, S., Sterne-Weiler, T., Wang, J. et al. (2017) Multilayered control of alternative splicing regulatory networks by transcription factors. Mol. Cell, 65, 539-553.
Kiang, K.M.-Y. and Leung, G.K.-K. (2018) A review on adducin from functional to pathological mechanisms: future direction in cancer. BioMed Res. Int., 2018, 3465929.
Lykke-Andersen, J., Shu, M.D. and Steitz, J.A. (2000) Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell, 103, 1121-1131.
Carrillo, J., Garcia-Aragoncillo, E., Azorin, D., Agra, N., Sastre, A., González-Mediero, I., Garcia-Miguel, P., Pestaña, A., Gallego, S., Segura, D. et al. (2007) Cholecystokinin down-regulation by RNA interference impairs Ewing tumor growth. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res., 13, 2429-2440.
Sun, S., Zhang, Z., Fregoso, O. and Krainer, A.R. (2012) Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2. RNA, 18, 274-283.
Cassonnet, P., Rolloy, C., Neveu, G., Vidalain, P.-O., Chantier, T., Pellet, J., Jones, L., Muller, M., Demeret, C., Gaud, G. et al. (2011) Benchmarking a luciferase complementation assay for detecting protein complexes. Nat. Methods, 8, 990-992.
Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M. and Gingeras, T.R. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinforma. Oxf. Engl., 29, 15-21.
Shen, S., Park, J.W., Lu, Z., Lin, L., Henry, M.D., Wu, Y.N., Zhou, Q. and Xing, Y. (2014) rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc. Natl. Acad. Sci. U.S.A., 111, E5593-E5601.
Anders, S., Pyl, P.T. and Huber, W. (2015) HTSeq-a Python framework to work with high-throughput sequencing data. Bioinforma. Oxf. Engl., 31, 166-169.
Love, M.I., Huber, W. and Anders, S. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol., 15, 550.
Park, J.W., Jung, S., Rouchka, E.C., Tseng, Y.-T. and Xing, Y. (2016) rMAPS: RNA map analysis and plotting server for alternative exon regulation. Nucleic Acids Res., 44, W333-W338.
Goeman, J.J. and Solari, A. (2011) Multiple testing for exploratory research. Stat. Sci., 26, 584-597.
Durand, G., Blanchard, G., Neuvial, P. and Roquain, E. (2020) Post hoc false positive control for spatially structured hypotheses. Scand. J. Stat., 47, 114-1148.
Nagai, N., Ohguchi, H., Nakaki, R., Matsumura, Y., Kanki, Y., Sakai, J., Aburatani, H. and Minami, T. (2018) Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition. PLoS Genet., 14, e1007826.
Langmead, B. and Salzberg, S.L. (2012) Fast gapped-read alignment with Bowtie 2. Nat. Methods, 9, 357-359.
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R. and 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinforma. Oxf. Engl., 25, 2078-2079.
Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nusbaum, C., Myers, R.M., Brown, M., Li, W. et al. (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol., 9, R137.
Quinlan, A.R. and Hall, I.M. (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinforma. Oxf. Engl., 26, 841-842.
Tirode, F., Surdez, D., Ma, X., Parker, M., Le Deley, M.C., Bahrami, A., Zhang, Z., Lapouble, E., Grossetête-Lalami, S., Rusch, M. et al. (2014) Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations. Cancer Discov., 4, 1342-1353.
Neugebauer, K.M. (2019) Nascent RNA and the coordination of splicing with transcription. Cold Spring Harb. Perspect. Biol., 11, a03227.
Damianov, A., Ying, Y., Lin, C.-H., Lee, J.-A., Tran, D., Vashisht, A.A., Bahrami-Samani, E., Xing, Y., Martin, K.C., Wohlschlegel, J.A. et al. (2016) Rbfox proteins regulate splicing as part of a large multiprotein complex LASR. Cell, 165, 606-619.
Snel, B., Lehmann, G., Bork, P. and Huynen, M.A. (2000) STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res., 28, 3442-3444.
Stark, C., Breitkreutz, B.-J., Reguly, T., Boucher, L., Breitkreutz, A. and Tyers, M. (2006) BioGRID: a general repository for interaction datasets. Nucleic Acids Res., 34, D535-D539.
Siddique, H.R., Rao, V.N., Lee, L. and Reddy, E.S. (1993) Characterization of the DNA binding and transcriptional activation domains of the erg protein. Oncogene, 8, 1751-1755.
Anderson, E.S., Lin, C.-H., Xiao, X., Stoilov, P., Burge, C.B. and Black, D.L. (2012) The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B. RNA, 18, 1041-1049.
Ray, D., Kazan, H., Cook, K.B., Weirauch, M.T., Najafabadi, H.S., Li, X., Gueroussov, S., Albu, M., Zheng, H., Yang, A. et al. (2013) A compendium of RNA-binding motifs for decoding gene regulation. Nature, 499, 172-177.
Conboy, J.G. (2017) Developmental regulation of RNA processing by Rbfox proteins. Wiley Interdiscip. Rev. RNA, 8, e1398.
Nakagaki-Silva, E.E., Gooding, C., Llorian, M., Jacob, A.G., Richards, F., Buckroyd, A., Sinha, S. and Smith, C.W. (2019) Identification of RBPMS as a mammalian smooth muscle master splicing regulator via proximity of its gene with super-enhancers. eLife, 8, e46327.
Jangi, M., Boutz, P.L., Paul, P. and Sharp, P.A. (2014) Rbfox2 controls autoregulation in RNA-binding protein networks. Genes Dev., 28, 637-651.
Zhang, C., Zhang, Z., Castle, J., Sun, S., Johnson, J., Krainer, A.R. and Zhang, M.Q. (2008) Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2. Genes Dev., 22, 2550-2563.
Remy, I. and Michnick, S.W. (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat. Methods, 3, 977-979.
Braeutigam, C., Rago, L., Rolke, A., Waldmeier, L., Christofori, G. and Winter, J. (2014) The RNA-binding protein Rbfox2: an essential regulator of EMT-driven alternative splicing and a mediator of cellular invasion. Oncogene, 33, 1082-1092.
Venables, J.P., Brosseau, J.-P., Gadea, G., Klinck, R., Prinos, P., Beaulieu, J.-F., Lapointe, E., Durand, M., Thibault, P., Tremblay, K. et al. (2013) RBFOX2 is an important regulator of mesenchymal tissue-specific splicing in both normal and cancer tissues. Mol. Cell. Biol., 33, 396-405.
Chaturvedi, A., Hoffman, L.M., Welm, A.L., Lessnick, S.L. and Beckerle, M.C. (2012) The EWS/FLI oncogene drives changes in cellular morphology, adhesion, and migration in Ewing sarcoma. Genes Cancer, 3, 102-116.
Franzetti, G.-A., Laud-Duval, K., van der Ent, W., Brisac, A., Irondelle, M., Aubert, S., Dirksen, U., Bouvier, C., de Pinieux, G., Snaar-Jagalska, E. et al. (2017) Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells. Oncogene, 36, 3505-3514.
Tirode, F., Laud-Duval, K., Prieur, A., Delorme, B., Charbord, P. and Delattre, O. (2007) Mesenchymal stem cell features of Ewing tumors. Cancer Cell, 11, 421-429.
Yang, Y., Park, J.W., Bebee, T.W., Warzecha, C.C., Guo, Y., Shang, X., Xing, Y. and Carstens, R.P. (2016) Determination of a comprehensive alternative splicing regulatory network and combinatorial regulation by key factors during the epithelial-to-mesenchymal transition. Mol. Cell. Biol., 36, 1704-1719.
Hegele, A., Kamburov, A., Grossmann, A., Sourlis, C., Wowro, S., Weimann, M., Will, C.L., Pena, V., Lührmann, R. and Stelzl, U. (2012) Dynamic protein-protein interaction wiring of the human spliceosome. Mol. Cell, 45, 567-580.
Huang, S.-C., Ou, A.C., Park, J., Yu, F., Yu, B., Lee, A., Yang, G., Zhou, A. and Benz, E.J. (2012) RBFOX2 promotes protein 4.1R exon 16 selection via U1 snRNP recruitment. Mol. Cell. Biol., 32, 513-526.
Li, Y., McGrail, D.J., Xu, J., Mills, G.B., Sahni, N. and Yi, S. (2018) Gene regulatory network perturbation by genetic and epigenetic variation. Trends Biochem. Sci., 43, 576-592.
de Bruin, R.G., Shiue, L., Prins, J., de Boer, H.C., Singh, A., Fagg, W.S., van Gils, J.M., Duijs, J.M.G.J., Katzman, S., Kraaijeveld, A.O. et al. (2016) Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression. Nat. Commun., 7, 10846.
Hayakawa-Yano, Y., Suyama, S., Nogami, M., Yugami, M., Koya, I., Furukawa, T., Zhou, L., Abe, M., Sakimura, K., Takebayashi, H. et al. (2017) An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling. Genes Dev., 31, 1910-1925.
Wang, Y., Ma, M., Xiao, X. and Wang, Z. (2012) Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules. Nat. Struct. Mol. Biol., 19, 1044-1052.
Kato, M. and McKnight, S.L. (2018) A solid-state conceptualization of information transfer from gene to message to protein. Annu. Rev. Biochem., 87, 351-390.
Ying, Y., Wang, X.-J., Vuong, C.K., Lin, C.-H., Damianov, A. and Black, D.L. (2017) Splicing activation by Rbfox requires self-aggregation through its tyrosine-rich domain. Cell, 170, 312-323.
Chong, S., Dugast-Darzacq, C., Liu, Z., Dong, P., Dailey, G.M., Cattoglio, C., Heckert, A., Banala, S., Lavis, L., Darzacq, X. et al. (2018) Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science, 361, eaar2555.
Im, Y.H., Kim, H.T., Lee, C., Poulin, D., Welford, S., Sorensen, P.H., Denny, C.T. and Kim, S.J. (2000) EWS-FLI1, EWS-ERG, and EWS-ETV1 oncoproteins of Ewing tumor family all suppress transcription of transforming growth factor beta type II receptor gene. Cancer Res., 60, 1536-1540.
Riggi, N., Knoechel, B., Gillespie, S.M., Rheinbay, E., Boulay, G., Suvà, M.L., Rossetti, N.E., Boonseng, W.E., Oksuz, O., Cook, E.B. et al. (2014) EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell, 26, 668-681.
Guillouf, C., Gallais, I. and Moreau-Gachelin, F. (2006) Spi-1/PU.1 oncoprotein affects splicing decisions in a promoter binding-dependent manner. J. Biol. Chem., 281, 19145-19155.
Selvanathan, S.P., Graham, G.T., Grego, A.R., Baker, T.M., Hogg, J.R., Simpson, M., Batish, M., Crompton, B., Stegmaier, K., Tomazou, E.M. et al. (2019) EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex. Nucleic Acids Res., 47, 9619-9636.
Shapiro, I.M., Cheng, A.W., Flytzanis, N.C., Balsamo, M., Condeelis, J.S., Oktay, M.H., Burge, C.B. and Gertler, F.B. (2011) An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet., 7, e1002218.
Riggi, N., Cironi, L., Provero, P., Suvà, M.-L., Kaloulis, K., Garcia-Echeverria, C., Hoffmann, F., Trumpp, A. and Stamenkovic, I. (2005) Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res., 65, 11459-11468.
Guillon, N., Tirode, F., Boeva, V., Zynovyev, A., Barillot, E. and Delattre, O. (2009) The oncogenic EWS-FLI1 protein binds in vivo GGAA microsatellite sequences with potential transcriptional activation function. PloS One, 4, e4932.
