The Dynamic Role of Jumonji C Domain Containing Protein 6 in Placental Development and Disease by Sruthi Alahari A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Sruthi Alahari 2017 i The Dynamic Role of Jumonji C Domain Containing Protein 6 in Placental Development and Disease Sruthi Alahari Doctor of Philosophy Department of Physiology University of Toronto 2017 Abstract Perturbations in oxygen sensing are a defining feature of placental-associated pathologies such as preeclampsia, a serious disorder of pregnancy. Preeclamptic placentae have markedly elevated levels of Hypoxia Inducible Factor 1α (HIF1A), a master regulator of oxygen homeostasis. Mounting evidence implicates a family of Fe2+ and oxygen-dependent Jumonji C domain containing enzymes (JMJDs) as mediators of the epigenetic code and hypoxic gene expression. While several JMJDs are induced in hypoxia, their role in pregnancy remains unclear. The goal of this study was to characterize JMJD6 function in the placenta in physiological and pathological conditions, and unravel its regulatory relationship with von Hippel Lindau tumour suppressor (VHL), a key executor of the cellular hypoxic response. JMJD6 expression inversely correlated with changes in oxygen tension during placental development, while JMJD6 protein and mRNA were significantly elevated in low oxygen and in early-onset preeclamptic (E-PE) placentae. In vitro demethylation assays revealed that optimal JMJD6-dependent demethylation of its histone targets, H3R2me2s and H4R3me2s, occurred in normoxia, and this was impaired in E-PE placentae due to a hypoxia-iron imbalance. In cytotrophoblast cells, JMJD6 is a positive ii regulator of VHL gene expression in normoxia. Accordingly, JMJD6 histone targets in E-PE placentae showed marked reductions in their association with VHL promoter regions. Independent of VHL gene regulation, JMJD6 also controlled VHL protein stability through lysyl hydroxylation and promoting its SUMO1-dependent SUMOylation. Importantly, the Jumonji C catalytic domain was found to be indispensable in executing both functions of JMJD6. In summary, these data signify a novel function for JMJD6 as an oxygen sensor in the human placenta, exerting a dual role in regulating VHL gene and protein. Uncovering epigenetic oxygen sensing mechanisms controlling HIF1A is crucial for defining the unique molecular signature of preeclampsia, which may ultimately translate to molecular-based diagnosis and therapy. iii Acknowledgments First and foremost, I want to express my sincere gratitude to my supervisor, Dr. Isabella Caniggia, who has been both a professional and personal inspiration. She has guided me throughout the ups and downs of this journey and prepared me for a career in academia. I will always fondly look back on our long chats and shared excitement over new ideas. I would also like to acknowledge past and present members of the Caniggia lab who have made this journey that much smoother. Thank you to Sarah, Julia, Jon, Liane, Leo, Julien, Brandon and Abby for all the friendship, laughs and profound discussions on life and science. It was a pleasure working with you all. Special thanks to Andrea Tagliaferro for being a great friend, counsellor and constant support system. Thank you to my friends and mentors on the 6th floor of the Lunenfeld-Tanenbaum Research Institute, who have always provided a helping hand, some of who have been on this journey with me. I am grateful to the members of my supervisory committee: Dr. Theodore Brown, Dr. Daniel Drucker and Dr. Jaques Belik. I appreciate your invaluable feedback and encouragement throughout. Special thanks to Dr. Martin Post for his critical insights and appraisal of my research. Last but not the least, this work is also a reflection of the unwavering support of my family. Thank you to my loving husband, who has stood by me through thick and thin and encouraged me to do what I love. I am incredibly grateful to my wonderful parents who taught me valuable lessons in life, and my sister for always being there for me. They have worked selflessly to give me a better life and their love and guidance have made me the person I am today. iv Contributors The following people have contributed to the collection of material and generation of data reported in this thesis: Research Centre for Women’s and Infant’s Health (RCWIH) BioBank at Mount Sinai Hospital (Toronto, Canada) for supplying first trimester, normal and pathological human placental tissues and explants. Chapter 3: The data reported in this chapter are part of a manuscript submission to the journal PLOS Genetics. Dr. Isabella Caniggia and Dr. Jing Xu performed the experiments in Figure 3.1. Dr. Alessandro Rolfo performed the Western blotting for CP in preeclamptic placentae and sera, reported in Figure 3.5. Dr. Julia Garcia performed the experiments on VHL DNA methylation, reported in Figure 3.7. Chapter 4: The work presented in this chapter is part of a published manuscript in the journal, Endocrinology, in the form: Alahari, S., M. Post, and I. Caniggia, Jumonji Domain Containing Protein 6: A Novel Oxygen Sensor in the Human Placenta. Endocrinology, 2015. 156(8): p. 3012-25. Official permission was obtained from the journal. Dr. Alessandro Rolfo performed anti-sense oligonucleotide knockdown of HIF1A in placental villous explants, reported in Figure 4.4B (right panel). The work presented in this thesis was supported by a Canadian Institute of Health Research (CIHR) grant held by Dr. Isabella Caniggia. I am a recipient of an Ontario Student Opportunity Trust Fund (OSOTF) Award provided by Mount Sinai Hospital, and a Queen Elizabeth II Graduate Scholarships in Science & Technology (QEII-GSST) award. v Table of Contents Acknowledgments ........................................................................................................................ iv Contributors ...................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi Abbreviations ............................................................................................................................. xiv 1.1 Introduction ........................................................................................................................1 1.2 Mechanisms of Oxygen Sensing ........................................................................................1 1.2.1 Acute oxygen sensing responses by chemoreceptors ..............................................2 1.2.2 Chronic oxygen sensing responses by hypoxia-inducible factors ...........................5 1.2.3 The von-Hippel Lindau Tumour Suppressor (VHL) .............................................10 1.2.4 Hypoxia and the Epigenome ..................................................................................18 1.3 Epigenetic Regulation by the Jumonji C Family of Proteins .......................................19 1.3.1 Histone Modifications and the Epigenetic Code ...................................................20 1.3.2 Jumonji Domain Containing Family of Proteins ...................................................26 1.3.3 JmjC Domain Containing Protein 6 .......................................................................31 1.4 Oxygen Sensing in the Placenta ......................................................................................36 1.4.1 Early placental development ..................................................................................37 1.4.2 Oxygen changes in the developing placenta ..........................................................40 1.4.3 Oxygen sensing machinery in the developing placenta .........................................42 1.4.4 Placental pathologies characterized by disrupted oxygen sensing ........................43 1.4.5 Oxygen changes in preeclampsia ...........................................................................45 1.4.6 Epigenetics in the Placenta ....................................................................................48 vi 1.5 RATIONALE, HYPOTHESIS, OBJECTIVES ............................................................51 2 Materials and Methods ...........................................................................................................53 2.1 Placental Tissue and Sera from Human Subjects ..............................................................53 2.2 Mouse Strains.....................................................................................................................55 2.3 Primary Trophoblast Cell Isolation ....................................................................................55 2.4 Villous Explant Culture and antisense knockdown ...........................................................57 2.5 Human Cell Lines and Culture Conditions ........................................................................58 2.5.1 Pharmacological Treatments ..................................................................................59 2.6 Iron Assay