Post-Translational Regulatory Mechanisms acting on KLF4 mediate Pluripotency Exit in Naïve Mouse Embryonic Stem Cells by Navroop Kaur Dhaliwal A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate department of Cell and Systems Biology University of Toronto © Copyright by Navroop Kaur Dhaliwal 2019 Post-Translational Regulatory Mechanisms acting on KLF4 mediate Pluripotency Exit in Naïve Mouse Embryonic Stem Cells Navroop Kaur Dhaliwal Doctor of Philosophy Graduate Department of Cell and Systems Biology University of Toronto 2019 Abstract Pluripotent embryonic stem (ES) cells have the potential to self-renew and differentiate to generate all adult tissues. Pluripotency is regulated by an interconnected network of transcription factors including OCT4, SOX2, NANOG and KLF4. This transcriptional network is integrated with extracellular signaling pathways regulating pluripotency and differentiation. Mouse ES cells can be maintained in a state known as naïve pluripotency by culture in leukemia inhibitory factor (LIF) and two signaling pathway inhibitors (LIF/2i). A previous study observed that a reduction in Klf4 transcript, due to LIF withdrawal, was the first change in pluripotency gene expression during differentiation. Based on this finding I hypothesized that removal of KLF4 protein from transcriptional network would be required for exit from pluripotency. To investigate this I examined the levels of pluripotency associated transcription factors in the nucleus of ES cells as they exit the pluripotent state. This investigation revealed that a reduction in KLF4 protein levels did not occur immediately following the reduction in Klf4 transcription; instead I identified nuclear export of KLF4 protein was required for the reduction in Klf4 transcription and pluripotency exit. Next, I investigated Klf4 gene and protein regulation and found that LIF/2i maintains Klf4 expression through both transcriptional and post-translational mechanisms. Specifically, KLF4 protein is highly stable in LIF/2i and this stability is maintained by physical interaction with active ii transcriptional complexes which ensure nuclear anchoring of the KLF4 protein. Surprisingly, KLF4 protein stability is so high (t½ >24 hr) that protein levels change by <2 fold when RNA levels are reduced by 17 fold. LIF/2i removal causes both nuclear export of KLF4 and reduced KLF4 stability which together lead to reduced gene expression of the other pluripotency transcription factors and exit from the pluripotent state. These mechanisms regulating KLF4 protein can inform the design of reprogramming and differentiation approaches for cellular therapies. iii Acknowledgments First and foremost, I would like to express my most sincere appreciation and gratitude to my supervisor, Professor Jennifer Mitchell for giving me opportunity to be a part of her lab. She is a true visionary representing a combination of leadership, scientific innovation and humanity from which I continue to learn. I am extremely obliged for receiving her tremendous support and guidance throughout the course of my research and especially during my maternity leave of absence. She has become a role model for me as a scientist, guide and working mother. I am grateful to her for providing me the opportunity to expand my academic horizons by presenting at national and international conferences. It has been my absolute honor to have been supervised by her over the past few years. I would also like to express my enduring gratitude to my advisory committee members, Professor Tony Harris and Professor Tim Westwood, for their insightful advice and suggestions throughout my PhD. I am very thankful to Professor Sue Varmuza for her guidance to carry out in vivo experiments. I sincerely acknowledge the support of Professor Eva Sapi by providing me letters of recommendation that helped me achieve success in my 2014, 2016 and 2017 OGS applications. I owe special thanks to Dr. Kamelia Miri for training and helping me out with mice handling, dissections and embryo staining. Thanks to Henry Hong for training me in confocal microscopy and for further assistance while imaging. I would also like to thank Yulia Katsman for being a good friend and guiding me through CRISPR/Cas9 protocols. I also thank Dr. Scott Davidson and Hala Tamim for their great help in carrying out site directed mutagenesis while I was away. Finally, I acknowledge the support of all of my previous and current lab members. I would like to thank my parents, Dr. Kulwant Dhillon and Dr. Guntejinderinderjit Dhillon for their unconditional love, support and guidance. Without their incredible inspiration, I would not be here today. Next, I would like to thank my favourite engineer and entrepreneur, my husband Himmat Dhaliwal who has brought joy, happiness and endless love in my life ever since we met. I thank him and my father in law, Harbans Dhaliwal, for understanding and allowing me to go back to school after marriage and one kid. I look forward to everything Himmat will help me accomplish and owe him more thanks that I can ever put in words. I would like to thank my mother in law, Ranjit Dhaliwal, the most. I admire her positive attitude. I could not have carried out my iv research with peaceful mind without her constant support, love, care and dedication at home. She has been the strongest pillar on which I ever leaned on during my hardest times. I would also like to thank rest of my family and friends to be patient and bearing with me during my PhD. Last but certainly not the least, I would like to thank my two little angels, Guransh and Jitkarn for being so supportive and patient during the whole course of my research. “If you wish to succeed in life, make perseverance your bosom friend, experience your wise counselor, caution your elder brother, and hope your guardian genius.” ― Joseph Addison September 2018 v Table of Contents Acknowledgments ......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations .................................................................................................................. xiv List of Appendices ..................................................................................................................... xvii Statement of publications .......................................................................................................... xviii Chapter 1: Literature Review ..........................................................................................................1 1.1 Cell potency ..............................................................................................................................2 1.2 Early stages of mammalian embryogenesis ...............................................................................3 1.2.1 The mouse as mammalian development model system .........................................3 1.2.2 Preimplantation phase .............................................................................................3 1.2.3 Postimplantation phase ..........................................................................................6 1.2.4 Regulative nature of early mammalian embryo .....................................................6 1.3 Embryonic stem (ES) cell derivation ........................................................................................7 1.4 In vitro culturing of mouse and human ES cells ......................................................................8 1.5 States of pluripotent ES cells ..................................................................................................10 1.5.1 Naïve pluripotent state ......................................................................................11 vi 1.5.2 Primed pluripotent state ..............................................................................12 1.6 Regulation of naïve pluripotent ES cells ............................................................................14 1.6.1 Transcriptional regulation .............................................................................14 1.6.1.1 RNA polymerase ....................................................................14 1.6.1.2 Transcription factors, enhancers and the regulatory landscape for gene transcription .............................................17 1.6.1.3 Nuclear transport mechanisms ...............................................19 1.6.1.4 Transcriptional networks regulating pluripotency .................20 1.6.2 Epigenetic regulation of pluripotency .........................................................22 1.6.3 Signaling pathways modulating naïve pluripotent state .............................24 1.6.3.1 LIF and JAK/STAT3 signaling ..............................................24 1.6.3.2 BMP4 signaling .....................................................................25 1.6.3.3 ERK signaling .......................................................................26 1.6.3.4 Wnt/β-Catenin signaling ........................................................27