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Role of Histone H1 in Neural Differentiation of Embryonic Stem Cells

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Role of Histone H1 in Neural Differentiation of Embryonic Stem Cells ROLE OF HISTONE H1 IN NEURAL DIFFERENTIATION OF EMBRYONIC STEM CELLS A Dissertation Presented to The Academic Faculty By Chenyi Pan In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the School of Biology Georgia Institute of Technology August 2015 Copyright © 2015 by Chenyi Pan i ROLE OF HISTONE H1 IN NEURAL DIFFERENTIATION OF EMBRYONIC STEM CELLS Approved by: Dr. Yuhong Fan, Advisor Dr. Alfred H. Merrill School of Biology School of Biology Georgia Institute of Technology Georgia Institute of Technology Dr. Francesca Storici Dr. Joseph M. Le Doux School of Biology Wallace H. Coulter Department of Georgia Institute of Technology Biomedical Engineering Georgia Institute of Technology Dr. Adegboyega "Yomi" Oyelere School of Chemistry and Biochemistry Georgia Institute of Technology Date Approved: February 10, 2015 i To my parents and my family for their love, faith, and support ii ACKNOWLEDGEMENTS I would like to express my sincerest thankfulness to my thesis advisor, Dr. Yuhong Fan, from whom I have been fortunate enough to receive patient guidance and constant support, not only in scientific research but also in personal life. Without her accepting me into her lab and training me on critical thinking, experimental skills, and scientific writing, I would never be able to accomplish all the work and make it this far. Her enthusiasm and perseverance on solving scientific problems have guided me through tough times and will continue to encourage me to strive for success. I want to show my appreciation to my committee members – Dr. Alfred Merrill, Dr. Francesca Storici, Dr. Joseph Le Doux, and Dr. Adegboyega (Yomi) Oyelere. Their guidance and help are valuable for my PhD study and future career. One group of people I’ll always remember and be grateful is the Fan lab at Georgia Tech including its past and current members. Dr. Yunzhe Zhang is a master in lab techniques and took care of everyone in the lab. She helped to derive a few H1 knockout ES cell lines used in my thesis, and also taught me a variety of techniques. I will never forget the scene that Dr. Yunzhe Zhang taught me lab techniques like preparing samples for PCR and running gel electrophoresis when I first joined the lab. Dr. Kaixiang Cao is such a wonderful buddy and helped me enormously in and outside research, especially on establishment of stable ES cell lines. I spent most of these years together with Dr. Yunzhe Zhang, Dr. Kaixiang Cao, and Dr. Magdalena Medrzycki. They are the ones to whom I always turn for help and advice, and I cannot thank them enough. I greatly appreciate Po-Yi Ho, Ting Wu, Zhiqiang Lin, Dr. Zheng Liu, Dr. iii Shiraj Panjwani, Samantha Lasater, and those helpful undergrads I’ve worked with, for all their help throughout these years. I am particularly grateful to Ting Wu for her help in research. This is a wonderful family filled with happiness, teamwork, and mutual support within and outside research. Because of these wonderful people, these years have become an unforgettable memory in my life. I thank Dr. Jessica Okosun and Dr. Jude Fitzgibbon at Queen Mary University of London and Dr. Hanjoong Jo at Georgia Tech/Emory University for fruitful collaborations. I want to thank the wonderful staffs in IBB at Georgia Tech for their technical support and advice including Steve Woodard, James "Allen" Echols, Nadia Boguslavsky, Andrew Shaw, and Aqua Asberry. They were always there to offer help whenever I needed. I am grateful to my friends at Georgia Tech community including Dr. Ziming Zhao, Dr. Jianrong Wang, Dr. Yu Zhang, Drs. Lee and Samantha Katz, Dr. Patrick Ruff, Dr. He Gong, Ziwei Sheng, Dr. Yongzhi Qiu, Dr. Ming Ruan, Dr. Danjue Chen, Dr. Yike Hu, and Li Zheng. I have been so fortunate to meet these amazing individuals and I appreciate their help along the way. Finally, to my parents, my sister, and members of my extended family, I cannot express how much I appreciate their support and encouragement. Their whole-hearted love and faith in me motivate me to overcome difficulties, to thrive, and to strive for success. I am forever indebted to them. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF FIGURES .......................................................................................................... vii LIST OF ABBREVIATIONS ............................................................................................. x SUMMARY ..................................................................................................................... xiv CHAPTER 1 ....................................................................................................................... 1 1.1 Linker Histone H1 Family ........................................................................................ 1 1.2 Histone H1 Depletion in Protists and Metazoans ..................................................... 5 1.3 The Role of Histone H1 in Chromatin Compaction and Gene Regulation ............ 10 1.4 Objectives ............................................................................................................... 15 1.5 References .............................................................................................................. 16 CHAPTER 2 ..................................................................................................................... 26 2.1 Abstract .................................................................................................................. 26 2.2 Introduction ............................................................................................................ 27 2.3 Materials and Methods ........................................................................................... 30 2.3.1 Generation of H1c/H1d/H1e/H10 quadruple knockout ESCs ......................... 30 2.3.2 Cell culture and growth curve assay ............................................................... 30 2.3.3 Construction of the inducible vector for H1a and H1b knockdown and generation of ESCs with an ultra-low H1 level (QKO/abi ESCs) ........................... 31 2.3.4 Histone extraction and HPLC analysis ........................................................... 31 2.3.5 In vitro neural differentiation of ESCs............................................................ 32 2.3.6 Antibodies ....................................................................................................... 33 2.3.7 Immunocytochemistry .................................................................................... 33 2.3.8 RNA extraction and quantitative RT-PCR ..................................................... 34 2.3.9 Generation of H1d rescue (QKO/abi/H1res ) ESCs ......................................... 34 2.3.10 Generation of ESCs with inducible Oct4 knockdown .................................. 35 2.3.11 Measurement of EB size ............................................................................... 35 2.3.12 BrdU incorporation assay of EBs ................................................................. 36 2.3.13 Senescence associated β-Galactosidase staining of EBs .............................. 36 2.3.14 Digestion of ESC genomic DNA with methylation-sensitive restriction enzymes ................................................................................................................... 36 2.3.15 Cell cycle analysis of ESCs .......................................................................... 37 2.4 Results .................................................................................................................... 38 2.4.1 Generation of H1c/H1d/H1e/H10 quadruple knockout ESCs ......................... 38 2.4.2 Generation of ESCs with an ultra-low H1 level ............................................. 42 v 2.4.3 Phenotypic analysis of ESCs with an ultra-low H1 level ............................... 45 2.4.4 Analysis of DNA methylation and histone modifications in bulk chromatin of ultra-low H1 ESCs ................................................................................................... 46 2.4.5 A dosage effect of histone H1 on neural differentiation of ESCs ................... 49 2.4.6 H1 depletion causes dysregulation of pluripotency-associated genes during neural differentiation ................................................................................................ 60 2.4.7 The total H1 level progressively increases during neural differentiation ....... 62 2.4.8 H1d overexpression restores the neural differentiation capacity of ESCs ...... 66 2.4.9 Severe H1 depletion causes a reduction in cell proliferation and cellular senescence in neural lineages................................................................................... 73 2.4.10 Oct4 knockdown rescues the defects in neural differentiation caused by H1 depletion ................................................................................................................... 76 2.5 Discussion .............................................................................................................. 83 2.6 References .............................................................................................................. 87 CHAPTER 3 ..................................................................................................................... 92 3.1 Abstract .................................................................................................................
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