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Characterization of Length-Dependent GGAA-Microsatellites in EWS/FLI Mediated Ewing Sarcoma Oncogenesis

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Characterization of Length-Dependent GGAA-Microsatellites in EWS/FLI Mediated Ewing Sarcoma Oncogenesis Characterization of length-dependent GGAA-microsatellites in EWS/FLI mediated Ewing sarcoma oncogenesis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kirsten M. Johnson Graduate Program in Biomedical Sciences The Ohio State University 2018 Dissertation Committee: Stephen L. Lessnick, MD, PhD, Advisor Michael A. Freitas, PhD Denis C. Guttridge, PhD Charles E. Bell, PhD Copyright by Kirsten M. Johnson 2018 Abstract Objective: Ewing Sarcoma is a pediatric bone malignancy initiated by a t(11;22) chromosomal translocation that produces the EWS/FLI oncoprotein. EWS/FLI transcriptionally activates and represses its target genes to mediate oncogenic reprogramming. Expression of its up-regulated targets correlates with EWS/FLI binding to associated GGAA-microsatellites, which show length polymorphisms. These microsatellite polymorphisms may critically affect EWS/FLI-responsiveness of key gene targets. For example, NR0B1 is necessary for EWS/FLI mediated oncogenic transformation, and we found a “sweet-spot” of 20-26 repeat length as optimal for EWS/FLI mediated transcriptional activity at NR0B1 through clinical observations and in vitro studies. The mechanism underlying this optimal length, however, is unknown. Methods: We explored the stoichiometry and binding affinity of EWS/FLI for different GGAA-repeat lengths through biochemical studies, including fluorescence polarization, ChIP-seq, and RNA-seq, combined with bioinformatics analysis. Additionally, use of EWS/FLI deletion constructs has been critical for elucidating the particular binding behavior of EWS/FLI at different microsatellite repeat lengths. Luciferase reporter assays, anchorage-independent growth and proliferation assays, as well as CRISPR technology have extended our findings to the in vivo setting. Finally, microscopy studies including use of confocal and transmission electron microscopy (TEM) have contributed visual characterization of the specific biochemical mechanisms we are investigating. ii Results: CRISPR-mediated deletion of the NR0B1 GGAA-microsatellite in Ewing sarcoma cells provided our field with the first in vivo evidence for the necessity of EWS/FLI binding at GGAA-microsatellites for anchorage dependent growth. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion) demonstrate a stoichiometry of one monomer binding every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and was unmeasureable when the size of the microsatellite was increased to the “sweet-spot” length. In contrast, a fully- functional EWS/FLI mutant (Mut9, retaining approximately half of the EWS portion) showed low affinity for smaller GGAA-microsatellites, but instead significantly increased its affinity at “sweet-spot” microsatellite lengths. Single-gene ChIP and genome-wide ChIP-seq and RNA-seq studies extended these findings to the in vivo setting. Additionally, through bioinformatics analysis, we defined GGAA-microsatellites in a Ewing sarcoma setting, and showed GGAA-microsatellite length is predictive of EWS/FLI responsiveness (binding and transcriptional activation) at “promoter-like” EWS/FLI targets. Conclusion: Together, these data demonstrate the necessity for EWS/FLI binding at GGAA-microsatellites in Ewing sarcoma, and characterize their role in oncogenesis. These data also reveal an unexpected novel role for the EWS portion of the EWS/FLI fusion in DNA-binding. Overall, our results suggest a length-dependent biochemical mechanism for EWS/FLI binding and transcriptional regulation at GGAA-microsatellites. iii Dedication This work is dedicated to my family (Paul, Marianne, Quinn, Hayden, and Lauren Johnson), Randy Stokes, and my best friend Katelyn Sheffield, who encouraged me, put up with me, and served as my rocks throughout this process. I could never have done it without them. iv Acknowledgments I would like to especially acknowledge and thank my principle investigator and mentor, Dr. Stephen Lessnick, who accepted me into his lab and transformed me into a researcher. He never stopped believing in me, even when I gave up on myself. He is a truly talented mentor with a gift for seeing what is really important in research and in life—the reason I followed him and his lab to transfer from the University of Utah to The Ohio State University and Nationwide Children’s in Columbus, Ohio. I would like to thank the many people who supported this research with their time, talents, expertise, and advice. Thank you to Kathleen Pishas and Ryan Roberts for all of the support and advice. Special thanks to Nathan Callender, Jesse Crow, and Cenny Taslim for all of their hard work and for making my last year in lab memorable. I would also like to thank Dr. Charles Bell and Cynthia McCallister for their expertise and research support and help in this work. The National Cancer Institute (Grant F30 CA210588) provided financial support for my tuition fees, living expenses, research, and travel costs for presenting my research at conferences around the globe. I sincerely appreciate the support and invaluable opportunities this grant award has provided me in my training to become a physician scientist. v I would like to thank the MSTP and Biomedical Sciences Graduate Programs at The Ohio State University for accepting my transfer into their exceptional programs and the help and support they have provided along the way. vi Vita December 2011 ..............................................B.S. Molecular Biology, Brigham Young University 2012 to present ..............................................MSTP Trainee, The Ohio State University Publications Monument, Michael J., Johnson, Kirsten M., Grossman, Allie H., Schiffman, Joshua D., Randall, R. Lor, and Lessnick, Stephen L. Microsatellites with Macro-Influence in Ewing Sarcoma. Genes 2012, 3, 444-460. Monument, M., Johnson, K., McIlvaine, E., Abegglen, L., Watkins, W., & Jorde, L. et al. (2014). Clinical and Biochemical Function of Polymorphic NR0B1 GGAA- Microsatellites in Ewing Sarcoma: A Report from the Children's Oncology Group. Plos ONE, 9(8), e104378. doi:10.1371/journal.pone.0104378 Johnson KM, Mahler NR, Saund RS, Theisen ER, Taslim C, Callender NW, Crow JC, Miller KR, Lessnick SL. Role for the EWS domain of EWS/FLI in binding GGAA- microsatellites required for Ewing sarcoma anchorage independent growth. Proc Natl Acad Sci U S A. 2017 Aug 28. pii: 201701872. doi: 10.1073/pnas.1701872114. PubMed PMID: 28847958 Johnson KM* and Taslim C* (*co-first authors), Saund RS, Lessnick SL. Identification of two types of GGAA-microsatellites and their roles in EWS/FLI binding and gene regulation in Ewing sarcoma. PLOSOne U S A. 2017. doi: 10.1371/journal.pone.0186275 Fields of Study Major Field: Biomedical Sciences vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii Publications ....................................................................................................................... vii Fields of Study .................................................................................................................. vii Table of Contents ............................................................................................................. viii List of Tables ................................................................................................................... xiii List of Figures ................................................................................................................... xv Chapter 1: Introduction ...................................................................................................... 1 Ewing sarcoma ................................................................................................................ 1 EWS/FLI Transcriptional Regulation ............................................................................. 8 ETS factors & FLI binding............................................................................................ 11 EWS and its paralogs .................................................................................................... 21 Microsatellites ............................................................................................................... 27 Goals of thesis ............................................................................................................... 34 viii Chapter 2: Microsatellites with Macro-influence in Ewing Sarcoma ............................... 39 Abstract ......................................................................................................................... 39 Introduction ................................................................................................................... 40 ETS family of transcription factors ............................................................................... 42 EWS/FLI
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