Condensin and Chromatin Mediated X Chromosome Architecture In
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Condensin and Chromatin Mediated X Chromosome Architecture in Caenorhabditis elegans by Alyssa C. Lau A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and Developmental Biology) in The University of Michigan 2016 Doctoral Committee: Associate Professor Gyӧrgyi Csankovszki, Chair Assistant Professor Laura Buttitta Professor Kenneth M. Cadigan Assistant Professor Sundeep Kalantry © Alyssa C. Lau 2016 This work is dedicated to my family. ii ACKNOWLEDGEMENTS I would like to take this opportunity to first and foremost thank my mentor Dr. Gyӧrgyi Csankovszki, who has always been extremely encouraging and supportive. All of her training and guidance has allowed me to grow as a scientist and has prepared me for the next stage of my career. With her support, I have also had the opportunity to pursue a Master’s degree in Bioinformatics and attend and present at scientific meetings. Her dedication to science, her students, and her family has been inspiring, and has motivated me throughout my Ph.D. career. I would also like to thank my committee members Dr. Ken Cadigan, Dr. Laura Buttitta, and Dr. Sundeep Kalantry. They have provided me with much kindness and their time, insights, and suggestions have helped develop my research. Next, I would like to thank all the members of the lab for their invaluable help over the years. They have made my time in Michigan a fun and less stressful one. I especially want to thank, Margarita Sifuentes, my fellow Graduate Student and friend, when I joined the lab she made me feel welcomed and she has always made the lab environment fun and exciting. I want to also thank our lab manager and my friend, Betsy Brouhard. She is hard working, extremely organized, and always very helpful and has been a great support. Thank you to all the other members of the lab past and present, Dr. Jianhao Jiang, Jessica Trombley, Mike Davis, Elaine Otchere, Netta Golenberg, iii Anna Cacciaglia and Dr. Laura Custer. Also I am glad I had the privilege to mentor such great undergrads, Jennifer Knister and Kevin Zhu. Additionally thanks to all the members of the Buttitta, Cadigan, Raymond, and Wierzbicki labs for borrowed regents and scientific input. Finally, I could not have made it to where I am now without the love and support of my friends and family. Thanks to my long time college friends who made cell biology and biochemistry lectures enjoyable and exciting. Thanks to my loving boyfriend, Christian Blanke, for always lifting me up and supporting my dream of getting a Ph.D. even when I had to work late or go into lab on the weekends. Most of all I want to thank my caring Mom, she has been my biggest fan and I cannot thank her enough. She has always been there to support me, encourage me, and to push me to my fullest. She really is the greatest, most loving, and crazy (in a good way) mom a person could ask for. I would like to thank John, for all the support and encouragement. I also want to thank my Dad, he always told me to do what I love and he's always believed in me. I can't forget my sister, Theresa, although she's my younger sister I truly look up to her and I know she is going to be a great PT. I am so lucky to have her as a sister and best friend. And thank you to my Grandmother, who is so proud of me no matter what. Lastly, thanks to Juniper and Summer for always welcoming me with a wagging tail whenever I come home from work! iv TABLE OF CONTENTS Dedication……………………………………………………………………………………......ii Acknowledgements…………………………………………………………………………......iii List of Figures………………………………………………………………………..................ix List of Tables……………………………………………………………………….................xiii Chapter 1: Introduction ……………………………………………………………………...1 Sex determination and dosage compensation……………………………………..…….1 X upregulation in Drosophila melanogaster………..…………………………………3 X chromosome hyperactivation and inactivation in mammals……………………...5 Up and downregulation of C. elegans X chromosomes…………………………….8 Condensin-mediated chromosome organization and gene regulation……………....12 Condensin complexes…………………………………………………………………12 Mitotic and meiotic defects in condensin mutants or knockdowns……………….13 Interphase defects in condensin mutants or knockdowns…………………………15 Condensin and chromatin mediated chromosome compaction…………………..17 Molecular mechanisms of condensin activity……………………………………….19 How does condensin regulate gene expression?................................................21 Chromatin structure and gene regulation………………………………………….……22 The nucleosome and histone modifications………………………....……………...23 Role of histone acetylation………………………...................................................24 v H4K16 acetylation……………………………………………………………………..25 MYST Family Histone Acetyltransferases…………………………………………..26 MOF containing complexes…………………………………………………..…..28 Tip60 complex…………………………………………………………………..….30 References…………………………………………………………….............................42 Chapter 2: The C. elegans dosage compensation complex mediates interphase X chromosome compaction…………………………………………………..…………..59 Abstract……………………………………………………………...................................59 Introduction…………………………………………………………….............................60 Results……………………………………………………………....................................65 Discussion……………………………………………………………..............................72 Materials and Methods………………………………………………………………........78 Acknowledgements…………………………………………………………..……...........82 References…………………………………………………………….............................95 Chapter 3: Anchoring of heterochromatin to the nuclear lamina helps stabilize dosage compensation-mediated gene repression…………..............................101 Abstract…………………………………………………………….................................101 Author Summary…………………………………………………………………….…...102 Introduction……………………………………………………………...........................103 Results……………………………………………………………..................................107 Discussion……………………………………………………………............................127 Materials and Methods…………………………………..………………………...........136 vi Acknowledgements……………………………………………………………...............142 References………………………………………………………………........................167 Chapter 4: An H4K16 histone acetyltransferase mediates decondensation of the X chromosome in C. elegans males……………………………………………..….172 Abstract…………………………………………………………….................................172 Author Summary…………………………………………………………………….…...173 Introduction……………………………………………………………...........................174 Results……………………………………………………………..................................177 Discussion……………………………………………………………............................192 Materials and Methods………………………………………………..…………...........202 Acknowledgements……………………………………………………………...............209 References……………………………………………………………...........................228 Chapter 5: A role for histone acetyltransferase activity in targeting the C. elegans dosage compensation complex to the X chromosomes………………...……...237 Abstract…………………………………………………………….................................237 Introduction……………………………………………………………...........................238 Results……………………………………………………………..................................240 Discussion……………………………………………………………............................245 Materials and Methods……………………………..……………………………...........247 Acknowledgements………………………………………………………………………249 References……………………………………………………………...........................256 vii Chapter 6: Conclusions and Future Directions……………………………………….260 Conclusions……………………………………………………………………………….260 Proposed Future Directions……………………………………………………………..266 References……………………………………………………………...........................270 viii LIST OF FIGURES Figure 1.1 Different dosage compensation strategies………………………………….33 Figure 1.2 Dosage compensation in Drosophila………………………………………...34 Figure 1.3 Stepwise model of mammalian X inactivation………………………………35 Figure 1.4 The dosage compensation complex (DCC) localizes to both X chromosomes in C. elegans hermaphrodite………………………………..36 Figure 1.5 Three condensin complexes………………………………………………….37 Figure 1.6 Condensin and chromatin mediated chromosome compaction…………..38 Figure 1.7 Molecular mechanisms of condensin activity……………………………….39 Figure 1.8 Schematic diagram comparing MYST family proteins and their protein domains…………………………………………...........................................40 Figure 1.9 Phylogenetic tree of proteins closely related to MYS-1……………………41 Figure 2.1 The absence of the DCC leads to enlarged X territories…………………..83 Figure 2.2 DCC depletion and mutations disrupt X chromosome compaction.……...85 Figure 2.3 The decondensed X chromatin structure in DCC-depleted worms is a result of defective compaction………………………………………………..86 Figure 2.4 X chromatin compaction is evident at all genomic distances examined…87 Figure 2.5 X chromosome compaction is not disrupted in condensin I or II depleted animals………………………………………………………………………….88 ix Figure 2.6 Depletion of the DCC-meditated histone modifiers leads to the loss of X chromosome compaction.…………………………………………………….89 Figure 2.7 X chromosome compaction is dependent on the DCC and the DCC- mediated histone modifications..……………………………………………..90 Figure 2.8 DCC depletion and mutants result in changes in the volume of the X chromosome.……………………..………………….…………………………91 Figure 2.9 No changes in X or chromosome I size in condensin I or II depleted animals.……………….………………………………………………………...92 Figure 2.10 Diploid condensin I and II depleted nuclei show no change in chromosome volume…………………………………………………………………………..93 Figure 2.11 Changes in