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DISSERTATION INFLUENCE AND REGULATION OF PCBP2 AND YTHDF2 RNA-BINDING PROTEINS DURING SELF-RENEWAL AND DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS Submitted by Adam M. Heck Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2019 Doctoral Committee: Advisor: Carol J Wilusz Co-Advisor: Jeffrey Wilusz Erin Osborne Nishimura Tai Montgomery Wen Zhou Copyright by Adam M. Heck 2019 All Rights Reserved ABSTRACT INFLUENCE AND REGULATION OF PCBP2 AND YTHDF2 RNA-BINDING PROTEINS DURING SELF-RENEWAL AND DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS Embryonic stem cells (ESCs) are able to self-renew or differentiate into any cell type in the body, a property known as pluripotency that enables them to initiate early growth and development. However, the ethical implications of harvesting and manipulating ESCs hinders their use in basic research and the clinical applications. Thus, the discovery that somatic cells can be exogenously reprogrammed into induced pluripotent stem cells (iPSCs) offers new and exciting possibilities for gene therapy, personalized medicine and basic research. However, more research is needed into the mechanisms involved in regulating pluripotency in order for iPSCs to reach their full potential in the research lab and clinic. To maintain a state of self-renewal, yet also be able to rapidly differentiate in response to external signals, pluripotent stem cells need to exert tight control over gene expression through transcriptional and post-transcriptional mechanisms. There are several notable transcriptional networks that regulate pluripotency, but the post-transcriptional mechanisms remain poorly characterized. mRNA decay is one form of post-transcriptional regulation that can help to both maintain the steady-state of a transcriptome or facilitate its rapid remodeling. To this end, degradation rates are influenced by the elements contained in an mRNA and the RNA-binding proteins (RBPs) they associate with. Previous reports have indicated the RNA modification N6- methyladenosine (m6A) and C-rich sequence elements (CREs) can affect mRNA decay in pluripotent stem cells. Therefore, we sought to further understand the roles of m6A and CREs in ii mRNA decay in stem cells by characterizing the expression and mRNA targets of two RBPs that recognize these elements, YTHDF2 and PCBP2, respectively. In this thesis, I report YTHDF2 is differentially regulated in pluripotent and differentiated cells and that YTHDF2 contributes to pluripotency by targeting a group of mRNAs encoding factors important for neural development. The down-regulation of YTHDF2 during neural differentiation is consistent with increased expression of neural factors during this time. Moreover, YTHDF2 expression is regulated at the level of translation via elements located in the first 300 nucleotides of the 3’ untranslated region of YTHDF2 mRNA. Based on these results, I propose that stem cells are primed for rapid differentiation by transcribing low levels of mRNAs encoding neural factors that are subsequently targeted for degradation, in part by YTHDF2, until differentiation is induced. On the other hand, PCBP2 is up-regulated upon differentiation of pluripotent stem cells and regulates several mRNAs associated with pluripotency and development, including LIN28B. Notably, expression of long non-coding RNAs (lncRNAs) that contain human endogenous retrovirus element H (HERV-H) is influenced by PCBP2. HERV-H lncRNAs are almost exclusively expressed in stem cells and play a role in maintaining a pluripotent state, although their functions are not fully understood. Intriguingly, some HERV-lncRNAs can also regulate PCBP2 expression, as altering the expression of LINC01356 or LINC00458 effects PCBP2 protein levels. Based on these results, I propose the reciprocal regulation of PCBP2 and HERV- H lncRNAs influences whether stem cells maintain a state of self-renewal or differentiate. Taken together, these findings demonstrate that YTHDF2 and PCBP2 post-transcriptionally regulate gene expression in stem cells and influence pluripotency. iii ACKNOWLEDGEMENTS The saying ‘it takes a village’ is not far off when it comes to the number of people who have helped and supported me on this journey. To those whom I do not mention by name, and there will be many, know that I still greatly appreciate what you did for me during my graduate career. I would like to start off by thanking my advisor Dr. Carol Wilusz whose guidance and support have been instrumental to my success as a researcher. In addition, our back and forth during the writing process of my thesis and other published works, painful as it was at times for both us, helped to vastly improve my written communication skills. I would also like to thank my co- advisor Jeff Wilusz for his advice regarding my projects, scientific research and life in general. These discussions often occurred over beers and/or bowling, which I always appreciated. I want to thank my graduate committee members Drs. Erin Osborne Nishimura, Tai Montgomery and Wen Zhou for their active involvement in my dissertation project and contributions to my scientific progress. A special thanks to Dr. Osborne Nishimura who was my go-to for any and all computational questions I had during my sequencing analysis. I am thankful for research training support from the GAUSSI computational training program at CSU and the Graduate Research Fellowship Program both through the National Science Foundation. I would like to than Drs. Thomas Chen, Carol Wilusz and Asa Ben-Hur for giving me the opportunity to participate in the GAUSSI computational training program. I would also like to thank Dr. Stu Tobet and my fellow members of his Fall 2015 grant writing course who helped me hone my application for the Graduate Research Fellowship Program. These programs provided research funding and educational experiences, which greatly enhanced my graduate student experience. Throughout my graduate school journey, members of the Wilusz Lab have provided me with advice, protocols, reagents, laughs and, most importantly, friendship. I will also be thankful for their help and support. Dr. Aimee Jalkanen helped show me the ropes when I first began and iv also taught me how to culture iPS and HFF cells. Dr. Annie Zhang Bargsten shared many a laugh with me and provided me with numerous reagents and protocols. John Anderson taught me several techniques, helped me track down supplies, reagents and protocols and answered countless questions. Dr. Donnie Evans was my co-pilot for the latter portion of the PCBP2 project and provided expertise and reagents. Dr. Phillida Charley helped me setup and run several experiments. Dr. Dan Michalski provided valuable insights, particularly in the design of the RNA pulldown experiment. John Brown, Erica Reith, Cary Mundell and Lyv Quillen all provided valuable assistance during their summer or semester research sessions and I was proud to mentor all of them. Saving the best for last, I want to give a shout out to Dr. Joe Russo who is my science ‘big brother’. He provided me with invaluable advice and support as well as reagents shared labor and good times in lab, for all of which I am truly thankful. I am also extremely grateful for the amazing and supportive community of graduate students and post docs in the CMB program, particularly the 2014 cohort I came in with, as well as the BMS department. A special thanks to Dr. Mark Stenglein and Dr. Justin Lee and the Next Generation Sequencing Core at CSU, who not only helped me navigate the sequencing process, but also generously provided a critical enzyme for my library preparation. I would also like to thank Dr. Brian Geiss who provided me with training and access to a luciferase plate reader and fluorescence microscope As well as Dr. Alan Schenkel for allowing me to use his fluorescence microscope. Thanks to Dr. Dan Sloan for providing access to his TapeStation2200 machine and insightful advice during the design and analysis of my RNA sequencing data. Finally, I appreciate all the love and support I have received from family, friends, the Small Council, roommates and colleagues throughout this time. I would especially like to thank my parents Kim and Jon Heck as well as my sister Kari and brother Andy for their love and always being there for me all of these years. Most of all, I want to thank my wife, Ashley, who is my biggest supporter. She has been with me step for step during this journey; from late night study v sessions during our first year, to writing our prelim proposals at the same desk in our third year, to defending within a week of each other. I am forever grateful that she came into my life. vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................................ iv Chapter 1: Introduction .............................................................................................................. 1 1.1 Stem cells ....................................................................................................................... 1 1.1.1 Embryonic stem cells .............................................................................................. 1 1.1.2 Induced pluripotent stem cells .....................................................................................
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