Robustness and Plasticity of Epithelial Cell State in Development and Malignancy Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophischen-Naturwissenschaftlichen Fakultät der Universität Basel Von Yoana Dimitrova, Aus Sofia, Bulgaria Basel, 2017 Originaldokument gespeichert auf dem Dokumentenserver der Universität Baseledoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Mihaela Zavolan (Dissertationsleitung) Prof. Dr. Gerhard M. Christofori (Korreferat) Basel, den 15.09.2015 (Dekan) Table of contents AKNOWLEDGEMENTS 4 ABSTRACT 5 INTRODUCTION 7 CHAPTER I. FUNCTIONS OF MIR-290 MICRORNAS IN EMBRYONIC STEM CELLS 9 EMBRYONIC STEM CELLS 9 DISCOVERY OF EMBRYONIC STEM CELLS 9 PLURIPOTENCY MAINTENANCE IN CELL CULTURE 11 PLURIPOTENCY IN DIFFERENT EMBRYONIC STATES 13 ESC DIFFERENTIATION 14 INDUCED PLURIPOTENCY 16 REGULATION OF PLURIPOTENCY 18 TRANSCRIPTIONAL CONTROL OF PLURIPOTENCY 18 Core Pluripotency Factors 19 Extended Network of Transcription Factors. 20 Function of MYC in Pluripotency Regulation 22 EPIGENETIC LANDSCAPE OF PLURIPOTENCY 23 RNA BINDING PROTEINS 25 MICRORNAS 26 RESULTS 30 “Em y n st m -specific microRNAs contribute to pluripotency by inhibiting regulators of mu t p d nt at n pat ways ” 30 CHAPTER II. TFAP2A IMPLICATION IN EPITHELIAL PLASTICITY IN BREAST CANCER AND DEVELOPMENT 45 EPITHELIAL PLASTICITY 45 EPITHELIAL PLASTICITY DURING EMBRYONIC DEVELOPMENT 46 EPITHELIAL PLASTICITY DURING CANCER PROGRESSION 47 GENE REGULATORY NETWORKS INVOLVED IN EMT 48 Transcription Level Regulation of Epithelial Plasticity 50 SNAIL Transcription Factors 53 ZEB Transcription Factors 54 bHLH Transcription Factors 55 microRNA Regulation of EMT 56 Splicing Factors in EMT 57 TFAP2A TRANSCRIPTION FACTOR 58 MODE OF ACTION AND CONTROL 59 AP-2 TRANSCRIPTION FACTORS IN DEVELOPMENT AND CANCER 60 RESULTS 62 “TFA 2A s a mp n nt t Z 1/2 n tw k t at u at s TGFβ1-induced epithelial to m s n yma t ans t n ” 62 DISCUSSION AND PERSPECTIVES 106 REFERENCES 110 Acknowledgements I would first like to thank my thesis advisor Prof. Dr. Mihaela Zavolan at the University of Basel for guiding me, advising me and supporting me as a PhD student in her group. I also thank the members of the jury: Prof. Dr. Gerhard M. Christofori for his helpful insights and comments on my research project, Prof. em. Walter Keller, who readily accepted to be the president of the Jury for being so friendly and optimistic, and Dr. Alexander Feuerborn whose remarks were particularly useful. Similarly, I am grateful to my former university research advisors, Prof. Laurent Paquereau, Prof. Anne-Catherine Pratz and Dr. Jefferey Shaw whose examples, as both scientists and mentors, influenced my decision to pursue a career in science. I am very thankful to Beatriz Dimitriades, William Aaron Grandy, Arnau Vilaseca-Vina, Andreas Gruber, Nitish Mittal, Daniel Mathow, Souvik Ghosh, Georges Martin and Xiaomo Wu for their help for the successful completion of this work. Especially, Aaron, Daniel and Xiaomo were implicated beyond their respective professional engagements and I am particularly appreciative of their support. A special thanks to Bea for our pleasant and motivating talks, and to George for sharing his experience and the incredible knowledge he has with the rest of us. Aaron is a great leader and biologist whose encouragement was very important for my survival as a graduate student. Likewise, X a m ’s d d at n and pass n have always been an important example to me. I had the chance to learn a lot from her, and our lunch conversations have always been full of content. Andreas Gruber’s help in proofreading and organizing data on top of his robust scientific input and funny jokes was critical. Thanks to Nitish for sharing great dinners, a beautiful wedding and most importantly his wide knowledge. Dominique Jedlinski and Afzal Pasha were both great colleagues and I am happy we could share our PhD time. I would like to thank the rest of my colleagues, to name some of them, Biter Bilen, Christoph Rodak, Jean Hausser, Jérémie Breda, Alexander Kanitz, Foivos Gypas, Ralf Schmidt, Rafal Gummienny, Anneke Brümmer, Joao C. Guimaraes, and Andrzej Jerzy Rzepiela as they have been actively involved in transforming our lab into a friendly and stimulating environment. I also thank the people who were not in Zavolan lab but helped me out. From fear of missing out I will not name them separately but among them are members of the D-BSSE sequencing facility, t B z nt um’s microscopy and proteomics facility, and the chemistry department. I keenly enjoyed the time, conversations and social activities shared with members of the Heinrich Reichert and Erick Van Nimwengen Groups, and in fact with all of my colleagues from the 6th floor of the Biozentrum. I also show gratitude for my friends’ and family’s support. I particularly thank Kate for being always there no matter the distance and time. I thank my mom, dad, my sister and my uncle. I have the greatest mother and sister and love them deeply. My family gave me unconditional love and care and I am thankful that I can count on them in tough times. I still miss my father a lot and wish that he could have been here to share those precious moments with me. I especially thank my husband Mitko. Without his love, patience and understanding this work would have never happened. His precious advices and thoughtful comments, combined with extensive proofreading, profound suggestions and great ideas that he shared are only a negligible part of his everlasting support. There have been plenty of difficult moments and he stayed next to me unconditionally. Thank you! Last but not least, I want to thank my daughter, Kassia for being the cheerful and great kid she is. Although she joined this adventure at the end, I feel she was a key element to my success. Together with her father they are the most important people in my life. I love you! Abstract A better understanding of the molecular mechanisms that control pluripotency, differentiation and epithelial phenotypical plasticity is crucial for the development of the current knowledge in many general processes such as cell identity maintenance and cell fate decision-making. Embryonic stem cells (ESC) pluripotency maintenance and differentiation are of key importance to the embryonic development, as well as to the progress in stem cells technologies. The role of miR-290-295 cluster members in preserving the pluripotent state and differentiation potential of mouse ESC is well established. Nevertheless, the precise list of targets translating the microRNAs functionality is incomplete. In our study we, firstly, identified and validated miR-290 targets with high confidence. We further confirmed the expression variation of IRF2 in response to miRNAs’ depletion in ESC. Moreover, we revisited the involvement of nuclear factor kappa-B (NF-kB) pathway in the miRNA-dependent regulation in mESCs. Hence, our results provided new understanding of the role and mechanistic of miR-290-295 microRNAs involvement in ESC pluripotency and differentiation. In a similar fashion to ESC pluripotency and differentiation mechanisms, a global analysis-approach that compares and combines data from different epithelial to mesenchymal transition (EMT) models enabled us to construct a more detailed network of regulatory entities implicated in epithelial plasticity. The maintenance and plasticity of the epithelial cell phenotype are important events not only during normal embryonic development, but also to cancer progression and metastasis formation. Comparing this network between mouse and human, we identified a new transcription factor (TF) motif TFAP2A/C that is consistently involved in EMT. When applying the NMuMG cellular model of TGFβ-induced EMT, we found that the predicted activity of the TFAP2A/C is inversely correlated to the Tfap2a mRNA expression during the process. We have confirmed that TFAP2A directly binds to the promoter of Zeb2, a TF central to EMT. Thus, it regulates the expression of this gene. Furthermore, the TFAP2A overexpression in NMuMG cells modulates the cells’ epithelial phenotype and induces changes in cell adhesion and morphology. This Page 5 of 124 overexpression was followed by increased mRNA levels of EMT master regulator TFs, together with an elevated expression of genes involved in cellular adhesion. Therefore, we identified a potentially new role of TFAP2A transcription factor, which suggests that elements of its regulatory function during neural crest development might operate in mechanisms controlling epithelial plasticity in normal breast and tumor tissues. Overall, we characterized another facet of microRNAs’ function in pluripotency and differentiation in ESC, as well as a new aspect of the implication of TFAP2A in epithelial cell state integrity and plasticity. Therefore we contributed to expanding our insight of how are regulated at molecular level the cell identity homeostasis and the unfolding of cellular phenotypical plasticity. Page 6 of 124 Introduction Molecular Basis of Cellular Specialization The development of vertebrate organisms follows a strictly defined program that gives rise to a multitude of cell types and tissues from a single cell. A complex network of signaling cues, gene expression regulators and epigenetic factors define the fate of individual cells within the developing organism. Thus, even though all cells in an organism carry the same genetic information, they assume cell fates with little overlap in functionality. Terminally differentiated cells generally maintain their identity across various conditions and stimuli, but exogenously-driven changes in gene expression can reverse, or even drastically change cell fate. In the late eighties a pioneering study by Walter Gehring introduced the concept of a selector transcription factor, which governs a particular developmental decision (Schneuwly, Klemenz et al. 1987). In an ingenious experiment, he showed that the exogenous expression of a transcription factor, Antennapedia, promotes the development of legs at the place where antennae would normally develop in the fly Drosophila melanogaster.