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Literature Review and Scope of Work ESRP1 is a novel marker of germ cell development and gonadal cancer Shaghayegh Saeidi Doctor of Philosophy March 2018 Department of Anatomy and Neuroscience The University of Melbourne Australia Abstract: Alternative splicing (AS) plays critical roles in controlling developmental programs. To date, there is evident that many genes splice differently during gametogenesis. Since disruption in AS may result in various reproductive disorders such as cancer, regulation of this event would be crucial during gametogenesis. The regulation of alternative splicing occurs through a network of highly combinatorial molecular interactions and numerous RNA binding proteins (RBPs) and transcription factors have been implicated in this process. Among them, ESRP1 (Epithelial Splicing Regulatory Protein) is an important cell-type specific regulator that affects the splicing of various developmental genes. Given the high level of alternative splicing during gametogenesis and the association of ESRP1 with AS, I was interested in examining the expression of ESRP1 during germ cell development and gonadal cancer. In this study by droplet digital PCR (ddPCR), I identified that Esrp1 is expressed in developing murine male and female germ cells but not somatic cells. Esrp1 also showed a high level of expression in adult mouse spermatogonia. In addition, the result of immunofluorescence experiments showed that ESRP1 protein is most highly expressed in nuclei of pre-meiotic germ cells in adult testes and co-labeled with PLZF and c-KIT. However, it did not co-localized with SOX9 in somatic cells, indicating it is germ cell specific. Furthermore, no colocalization was detected between ESRP1 and SC35 (post-transcriptional splicing marker), which suggests a probable role for ESRP1 in co-transcriptional splicing. In addition, my studies on the expression of Esrp1 in gonadal cancers showed upregulation of Esrp1 in both serous and mucinous ovarian carcinomas, its correlation with the levels of E-cadherin (CDH1) expression and coincides with switches from mesenchymal to epithelial isoforms of CD44 and FGFR2. i In testicular cancer, my data showed that Esrp1 was also up-regulated in both seminoma and mixed-germ cell testicular cancers. However, it did not regulate splicing of FGFR2 and CD44 in testicular cancers. RNA interference-mediated knockdown of ESRP1 expression in the seminoma-derived TCam-2 cell line, followed by RNA sequencing resulted in the identification of 576 novel potential splicing targets for ESRP1 in germ cell tumor cell line. IPA analyses of the data demonstrated ESRP1 regulates alternative splicing of genes that are involved in directing critical pathways involved in cell migration, morphology, and behaviors that occur during EMT. My data also showed that four mitochondrial complexes of oxidative phosphorylation are affected by differential gene expression after silencing of ESRP1. Overall, these data suggest that ESRP1 is required for germ cell development and raises the possibility that ESRP1 plays a role in splicing of mitotic and premeiotic transcripts during spermatogenesis, particularly in spermatogonia. Furthermore, my findings reveal that ESRP1 plays an important role in gonadal cancer progression by regulation of AS of numerous genes that are related to EMT. Furthermore, differential gene expression after silencing of ESRP1 suggesting that ESRP1 expression in testicular germ cells may alter ATP production and thus affect energy metabolism of these cells. ii Declaration This is to certify that 1) This thesis comprises only of my original work towards the PhD; 2) Due acknowledgement has been made in the text to all other materials used; 3) The thesis is less than 100,000 words in length, exclusive of figures, tables, bibliographies and appendices. Shaghayegh Saeidi iii Preface According to the rules and regulations that govern the Doctor of Philosophy degree at the University of Melbourne, the assessment of my contribution to this thesis was made as follows; • Chapter 3: This chapter has been published in PLoS One “Saeidi S, Shapouri F, de Iongh RU, de Iongh RU, Casagranda F, Sutherland JM, Western PS, McLaughlin EA, Familari M, Hime GR. Esrp1 is a marker of mouse fetal germ cells and is upregulated in spermatogonia. PLoS ONE. 2018; 13(1): e0190925.” I am the lead author and contributed 80% of experimental work and wrote the first draft of the entire manuscript. Chapter 4: 100% Chapter 5: 100% Chapter 6: 100% iv Acknowledgments There are many friends, family, and faculty that supported me during my PhD career. Words cannot express how much their encouragement, inspiration, and motivation has meant to me. First and foremost, I would like to thank my supervisors, Prof. Gary Hime, Dr. Mary Familari and A/Prof. Robb De Iongh for their excellent guidance throughout my doctoral studies. They provided me with opportunities to take on challenging projects and experiments, allowing me to mature as a scientist, all the while being ever present to help me along the way when necessary. Besides my supervisors, I would like to thank my advisory committee, Dr Peter Kitchener for his insightful comments and encouragement. I would like to thank my previous supervisor, A/Prof Reza Aflatoonian, for all his support and encouragement during my academic career. I appreciate the role you played in helping me get here. My special and heartily thanks to Franca Casagranda for helping me learn new techniques. I really appreciate your kindness and support during my PhD. My lab members, Dr Nicole Siddall, Trisha, Aviv, Arjun, James, Yoshana, Elena and Andrew. Thank you for making the lab one of the happiest labs I’ve worked in. Working alongside you guys was truly a joy. I would like to express my gratitude to my lovely friends, Kiana, Akram, Mitra, Javad, Maryam and Farhad for their unconditional friendship, support and patience throughout these years. I would like to thank my best friend Farnaz. There is no one with whom I can share my tears and fears, if you were not here. Thanks for being by my side and always giving me reasons to cheer. v I am grateful for the financial support I have received during my studies as the recipient of the Melbourne International Research Scholarship, the Melbourne International Fee Remission Scholarship and the Department of Anatomy and Neuroscience Travel Scholarship. Finally, I would like to thank my family members: my parents, my sisters, my brothers in law, my nephews (Arvin and Parsa) and my aunts. I am 100% certain (p < 0.001) that I could not have completed this journey without your love and support. I cannot possibly express how grateful I am to all of you for putting up with the emotional roller coaster that I’ve been on over the past four years. vi Publications • Saeidi S, Shapouri F, de Iongh RU, de Iongh RU, Casagranda F, Sutherland JM, Western PS, McLaughlin EA, Familari M, Hime GR. Esrp1 is a marker of mouse fetal germ cells and is upregulated in spermatogonia. PLoS ONE. 2018; 13(1): e0190925. Conference presentations • Saeidi S, Shapouri F, de Iongh RU, Casagranda F, Western PS, McLaughlin EA, Sutherland JM, Familari M, Hime GR. The role of ESRP1 during gametogenesis. Published in clinical endocrinology. 2015. Volume 84: PP-48. • Saeidi S, Shapouri F, de Iongh RU, Casagranda F, Western PS, McLaughlin EA, Sutherland JM, Familari M, Hime GR. The role of ESRP1 in mammalian germ cell development. 2016. 19th Eroupean Testis Workshop. Saint-Malo, France. vii Table of Contents Preface................................................................................................................................ iv Chapter 1. Literature review and scope of work .........................................................1 1.1. Epithelial to Mesenchymal Transition (EMT) ............................................................2 1.2. Alternative splicing (AS) during EMT .......................................................................4 1.3. Regulatory splicing factors .........................................................................................6 1.4. ESRP1 as an epithelial-specific splicing regulatory factor .........................................7 1.5. Targets of ESRP1 ......................................................................................................10 1.5.1. FGFR2 .........................................................................................................11 1.5.2. CD44 ...........................................................................................................12 1.6. Regulation of ESRP1 expression ..............................................................................14 1.7. ESRP1 function, expression, and localization ..........................................................16 1.8. Germ cell development .............................................................................................18 1.9. Alternative splicing in germ cell development .........................................................20 1.10. ESRP1 and carcinogenesis ........................................................................................23 1.11. Hypothesis.................................................................................................................25 Chapter 2. Materials and methods ..............................................................................27 2.1. Molecular biology .....................................................................................................28 2.1.1. RNA extraction ............................................................................................28
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