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Androgen Receptor and Histone Variant H2A.Z Interact to Regulate Fear Memory and Memory-Related Gene Transcription

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Androgen Receptor and Histone Variant H2A.Z Interact to Regulate Fear Memory and Memory-Related Gene Transcription Androgen Receptor and Histone Variant H2A.Z Interact to Regulate Fear Memory and Memory-Related Gene Transcription by Firyal Ramzan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy (Ph.D.) Psychology University of Toronto © Copyright by Firyal Ramzan 2019 Androgen Receptor and Histone Variant H2A.Z Interact to Regulate Fear Memory and Memory-Related Gene Transcription Firyal Ramzan Doctor of Philosophy (Ph.D.) Psychology University of Toronto 2019 Abstract Sex hormones are potent regulators of memory, but the role of androgen receptors in males is poorly characterized compared to the role of estrogens in females. Thus, the goal of my thesis is to characterize the role of AR in fear memory and the mechanistic basis of this effect in male mice. In Chapter 2, I utilized transgenic male mice that overexpress the androgen receptor (AR) to elucidate its effects on fear memory. I showed that AR overexpression impairs fear memory and that this effect is regulated by circulating androgens. Moreover, I showed that AR regulates the expression of several memory-related genes, including the epigenetic memory suppressor, histone H2A.Z. Our lab previously identified the histone variant H2A.Z as a negative modulator of fear memory through its effects on transcription. Given that AR is a transcription factor that interacts with H2A.Z in prostate cancer, I next characterized the functional and mechanistic interaction between the two. In Chapters 4 and 5, I used inducible-conditional H2A.Z knockout mice to delete the two genes (H2afz and H2afv) that encode H2A.Z in excitatory neurons in adult mice. I showed that simultaneous deletion of both H2A.Z-encoding genes enhanced fear memory, which extends our prior findings that virally-mediated depletion of H2afz alone enhances memory. Next, I showed that H2A.Z deletion blocked the effects of androgen receptor manipulation on fear memory, ii suggesting that AR and H2A.Z interact at a functional level. To test the mechanistic basis of this interaction, I utilized cultured hippocampal neurons to show that manipulating AR activation alters H2A.Z binding to DNA, suggesting that AR regulates behaviour through H2A.Z. Given its interaction with AR, I next investigated whether H2A.Z deletion produces unique effects in males and females. In contrast to males, H2A.Z deletion did not enhance fear memory in females, but it did serve as a protective factor against stress-enhanced fear memory (a PTSD-modeling paradigm). Overall, these data suggest that AR and H2A.Z interact at the chromatin level to regulate fear memory and that interactions with sex hormones may produce sex-specific effects of H2A.Z on fear memory. iii Acknowledgments Professional: Dr. Iva B. Zovkic: Thank you for believing in this collaborative study and being excited to take it on. Without your support and guidance, I would not have had the opportunity to do such an exciting study. Thank you also for your continuous push for me to be a better scientist, without which I would not have learned the meaning of hard work. Your excitement for research continues to inspire me and I can only hope to one day be as excited about science as you. Dr. D. Ashley Monks: Thank you for believing in me as a scientist and helping me get started as one. Your support and guidance from my undergraduate studies until now have been invaluable and have helped me build a foundation for future scientific endeavours. Dr. Alison Fleming and Dr. Veronica Afonso: the two people who really got me started on the path of scientific research by studying rats and changes in their sexual behaviour in response to stress during the Psychobiology Lab Course. Thank you for showing me that scientific research is a lot more than the dense articles we were otherwise made to read in undergraduate lectures. I would also like to thank Dr. Loren J. Martin, for his helpful comments and guidance through my Ph.D. committee meetings. I am truly grateful towards Dr. Ashlyn Swift-Gallant, a friend and former graduate student buddy, who taught me so much, from technical research skills to being a good lab mate. To Ilapreet Toor, fellow graduate student and sister from another mother but same dungeon, thank you for your constant support and positivity. I would also like to thank Dr. Melissa M. Holmes, for being the role model of how I aspire to be, and for making the basement lab space a welcoming and inclusive place throughout my time in the dungeons, from undergraduate thesis studies until now. iv Personal: I am especially grateful to my parents for being supportive and understanding of my choice to do a Ph.D. Without their sacrifices in immigrating to Canada, and their tireless support for their children to obtain more than them, I would not be where I am now. To Irshad, my husband, without your support throughout this journey I might have remained holed in the basement at UTM and remained unaware of the world around me. Thank you for sticking by me through the ups and downs of this rollercoaster ride. I am truly grateful for your constant love and support, it has helped me in more ways than you know. v Contributions Firyal Ramzan (author) solely prepared this thesis. All aspects of this body of work, including the planning, execution, analysis, and writing of all original research and publications was performed in whole or in part by the author. The following contributions by other individuals are formally and inclusively acknowledged: Dr. Iva B. Zovkic (Primary Supervisor and Thesis Committee Member) – mentorship; laboratory resources; guidance and assistance in planning, execution, and analysis of experiments as well as manuscript/thesis preparation Dr. D. Ashley Monks (Co-Supervisor and Thesis Committee Member) – mentorship; laboratory resources; guidance and assistance in planning, execution, and analysis of experiments as well as manuscript/thesis preparation Dr. Loren J. Martin (Thesis Committee Member) – mentorship; guidance in the interpretation of results as well as thesis preparation Dr. Ashlyn Swift-Gallant – teaching me the surgical skills required for experiments in Chapters 3 and 4 Amber B. Azam, M.Sc. – teaching me the behavioural testing skills and assistance in the execution of experiments for Chapter 3 Cindy Tao, M.A. – assistance in the execution of experiments for Chapter 4 Klotilda Narkaj – assistance in the execution of experiments for Chapters 4 and 5 Meaghan Hall – assistance in the execution of experiments for Chapter 4 Dr. Gilda Stefanelli – guidance in the execution of experiments for Chapter 4 Amanda Facciol – guidance in data analysis of experiments for Chapter 5 vi Table of Contents Acknowledgments.......................................................................................................................... iv Contributions.................................................................................................................................. vi Table of Contents .......................................................................................................................... vii List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii Chapter 1 Introduction and General Aims .......................................................................................1 1.1 Preamble ..............................................................................................................................1 1.2 Thesis Organisation and General Aims ...............................................................................2 1.2.1 Thesis Organisation .................................................................................................2 1.2.2 General Aims ...........................................................................................................3 Chapter 2 General Introduction .......................................................................................................4 2.1 Memory Formation and Consolidation ................................................................................4 2.1.1 Behavioural Paradigms to Study Memory ...............................................................5 2.2 Hormonal Effects on Learning and Memory .......................................................................7 2.2.1 Overview of Steroidogenesis ...................................................................................7 2.2.2 Behavioural effects of sex steroid hormones on learning and memory ...................9 2.2.3 Cellular effects of androgens on hippocampal structure and function ..................12 2.2.4 Androgen receptor (AR) and memory ...................................................................13 2.3 Epigenetic Effects on Learning and Memory ....................................................................15 2.3.1 Overview of Neuroepigenetics ..............................................................................15 2.3.2 Histone Post-translational Modifications ...............................................................16 2.3.3 Histone Variants.....................................................................................................19 2.3.4 Histone variant H2A.Z and transcription ...............................................................20 2.3.5 Histone variant H2A.Z and
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