MECHANISMS IN ASCL1-MEDIATED REPROGRAMMING OF FIBROBLASTS INTO NEURONS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOENGINEERING AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY QIAN YI LEE AUGUST 2017 © 2017 by Qian Yi Lee. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/hf795pb4608 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Marius Wernig, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Russ Altman I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Howard Chang I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Stephen Quake Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost for Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii Abstract Lineage reprogramming of somatic cells, i.e. the conversion of one cell type into another, unrelated cell type, has innovated the fields of stem cell research and translational medicine. The goal of my thesis is to understand the molecular mechanisms of induced lineage reprogramming to improve efficiencies and thereby enable its translation for clinical applications. Previous studies have shown that during reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) using transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), OSK act cooperatively as pioneering factors to first bind fibroblast enchancers to shut down the donor program before moving to activate the pluripotency circuit. It was also shown that iPSC reprogramming involves an initial stochastic phase to activate a subpopulation of cells that eventually enter a late hierarchical phase that activates the endogenous pluripotent circuitry. In contrast, studies in direct reprogramming of fibroblasts into induced neuronal (iN) cells and muscles by Ascl1 and Myod1 respectively have shown that Ascl1 and Myod1 bind immediately to their endogenous binding sites and activate their respective target programs. This leads us to hypothesize that the mechanism of direct reprogramming differs from iPSC reprogramming. Using single cell and bulk RNA sequencing (RNA-seq), Assay of Transposase Accessibly Chromatin with sequencing (ATAC-seq) and Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) to observe Ascl1 binding and changes in gene expression and chromatin accessibility during iN cell reprogramming, we found that in contrast to iPSCs, there is an initial homogenous response to Ascl1 within 2 days of transgene induction. There is a corresponding increase in accessibility of chromatin regions bound by Ascl1 within 12 hours, which leads to the up-regulation of a series of Ascl1 target genes that act in concert to facilitate a major cell fate transition between 2 – 5 days. Finally, a maturation phase between occurs after 5 days whereby the cells begin to activate neuronal and synapse maturation genes to become fully functional neurons. Surprisingly, we also found a small fraction of cells that got redirected to an alternate myogenic program, and showed that it can be attributed to unexpected similarities in Ascl1 and Myod1 DNA-binding affinities. Finally, we found that the myogenic program can by suppressed by pro-neuronal Myt1l to both increase Ascl1- mediated iN cell reprogramming efficiency and also redirect Myod1-expressing cells iv towards a neurogenic fate. These observations underscore the different molecular mechanisms between iN cell and iPSC reprogramming, reveal specific properties of transcription factors able to induce reprogramming, and also show the underlying the importance of endogenous co-factors in regulating cell fate during development. v Acknowledgements I am extremely grateful for the support and encouragement I have received through my PhD career, without which this thesis would not have been possible. I would first like to thank my mentor, Dr. Marius Wernig, for his guidance and support. He always has time to help me brainstorm new ideas, or troubleshoot persisting problems, His passion for science and insightful advice has also pushed me beyond my limits and helped me develop into the scientist I am today. I would like to thank my committee members – Dr. Howard Chang, Dr. Steve Quake and Dr. Russ Altman, as well as my committee chair Dr. Wing Wong, for their invaluable input and career advice. I especially would like thank Dr. Howard Chang for his close mentorship over the years as I collaborated closely with his lab. I would like to thank my other co-authors and collaborators, in particular Dr. Barbara Treutlein, Dr. Orly Wapinski and Dr. Moritz Mall, who have made invaluable contributions to this work, and without their insight and experimental support, this work would not have been possible. I am also thankful to my home department, Bioengineering, especially to Olgalydia and Justin, who had been very helpful and patient with me. Also, my graduate studies would not have been possible without my funding from the Agency of Science, Technology and Research (A*STAR). I would also like to thank everyone in the Wernig lab for their collaboration and support over the years. In particular, I would like to thank the members of our bay, Moritz, Cheen and Justyna for all our discussions, both scientific and not, that helped to push forward new ideas and also make life in lab so much more fun. Thanks also to Nan, Yihan, Sam, Soham and Tommy, who had been a lot of help through the years and been very patient with all my questions, especially when I was still trying to gain my bearings as a new graduate student. And also thank you to everyone (including Bahareh, Bo, Sarah, Lingjun vi and Katie) who was willing to humor me and go out for lunch or dinner or even for a hike to relax away from work. I am also thankful to my Singaporean friends, who formed an amazing and vibrant community here at Stanford. And I am grateful to my family, who have always been there for me and never wavered in their support. And last but not the least, a huge thank you to my boyfriend Winston Koh, who has been steadfast in his support, and has stayed by my side through this long road. vii Table of Contents Abstract ........................................................................................................................ iv Acknowledgements ...................................................................................................... vi Table of Contents ....................................................................................................... viii List of Figures ............................................................................................................... x Introduction .................................................................................................................. 1 REFERENCES ........................................................................................................... 4 CHAPTER 1: Dissecting direct reprogramming from fibroblast to neuron ......... 7 using single-cell RNA-seq ............................................................................................. 7 SUMMARY ................................................................................................................ 8 INTRODUCTION ...................................................................................................... 9 RESULTS ................................................................................................................. 10 EXPERIMENTAL PROCEDURES ......................................................................... 17 ACKNOWLEDGEMENTS ...................................................................................... 25 FIGURE LEGENDS ................................................................................................. 26 FIGURES .................................................................................................................. 30 EXTENDED DATA FIGURE LEGENDS .............................................................. 34 EXTENDED DATA FIGURES ............................................................................... 39 REFERENCES ......................................................................................................... 47 CHAPTER 2: Singular and prescient chromatin switch in the direct reprogramming of fibroblasts to neurons ................................................................ 52 SUMMARY .............................................................................................................. 53 INTRODUCTION .................................................................................................... 54