EPAS1 REGULATION AND FUNCTION IN THE HUMAN TROPHOBLAST NORHAM ERLYANI BTE ABDUL HAMID B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS Writing this doctoral dissertation has been most challenging and it would not have been possible without the support of many people. First of all, my gratitude goes to A*STAR, NGS, and GIS for giving me the opportunity to pursue the Doctor of Philosophy. I would like to thank A/P Paul Robson for being a tremendous mentor, and for his invaluable guidance. Under his care, I was able to develop and grow into an independent researcher. His unyielding encouragement gave me confidence to pursue the work I have achieved today. For always pointing me in the right direction and having the student’s best interest at heart, I must thank the panel of advisors at GIS as well as my thesis advisory committee; A/P Neil Clarke, Asst. Prof. Tara Huber, Asst. Prof. Thilo Hagen, and Dr. Shyam Prabhakar. I owe special thanks to my co-workers, Dr. Wishva Herath and Dr. Li Juntao. Their indispensable expertise in Bioinformatics is deeply appreciated, without which much of the data would have been rendered unintelligible to me. To my peers Mehran Rahmani, Nani Djunaidi, and Tapan Mistri, I thank you all for the sharing of ideas as well as laboratory material. Together, we can conquer the PhD! Additionally, I would also like to extend my thanks to Sun Lili, Jameelah Sheikh, Woon Chow Thai, and all members of the lab for their support and cooperation, ensuring research in the Robson lab proceeds as smoothly as possible. To all colleagues at GIS, past and present, thank you for making this experience a memorable one for me. To Rani Ettikan, our dearest lab manager, we owe it to you for translating funding into laboratory reagents and consumables with great i efficiency. To Li Pin, my surrogate mother at work, thank you for the stimulating chats and discussions, and for always keeping me and Heather on track. To Heather Ang, my confidante and partner in Science, I thank you for the numerous discussions and exchange of ideas, as well as the long nights of thesis-writing and editing, staying up well into the witching hour. Our lively chats and inextinguishable discourse has not only challenged my outlook, but has most definitely fueled my development as a mature (maybe), thinking individual. Also to the creators of Fringe, thank you for validating my love for Science and the geek in me. I would like to thank Dr. Elisabeth Zielonka, Nadine Richter, and Syahirah Abd Karim for reading and editing my thesis, and for their valuable input and suggestions. To my closest friends; Norishikin, thank you for always having faith in me and for always pushing me to do better; Fadilah, Iskandar, and little Hayder, thank you for always being there for me and for keeping me sane. Specially to little Hayder, your toothy smiles and little steps have kept me grounded and looking to brighter days ahead. To Zulkarnain, my dearest husband, I thank you for your undying love and incredible support, no matter the nautical miles; I would not have made it without these wings. Most importantly, I would like to thank my parents for their endless support throughout my life. Mama and Daddy, you raised me, you guided me, and you supported me every step of the way. For that, I can never thank you enough. I hope the person I am today is a daughter you can be proud of. To them, I dedicate this thesis. ii TABLE OF CONTENTS SUMMARY vii LIST OF TABLES ix LIST OF FIGURES x LIST OF SYMBOLS xii CHAPTER 1: INTRODUCTION 1 1.1. Understanding placental development in humans. 2 1.1.1. From zygote to blastocyst. 3 • Human embryonic stem cells. 4 • The early trophoblasts; progenitor to the placenta. 5 1.1.2. Extraembryonic membranes and the placenta. 8 1.1.3. Implantation and placentation. 12 1.1.4. Molecular basis of trophoblast formation. 17 1.1.5. Identifying trophoblast cell types. 22 1.1.6. Modeling human placentation. 28 1.1.7. Preeclampsia: A multi-factorial pregnancy complication. 30 1.2. EPAS1 and the HIF family. 34 1.2.1. The canonical HIF pathway: Oxygen-dependent regulation. 38 1.2.2. HIF target genes. 42 1.2.3. HIF1α vs. HIF2α: Are they redundant? 45 1.3. Role of hypoxia and HIF in development. 48 1.3.1. Placentation begins in an environment of physiological hypoxia. 49 • EPAS1 is expressed most abundantly in the human placenta. 50 1.3.2. HIF activity impacts placental development. 54 1.3.3. EPAS1 as a candidate gene that confers reproductive success. 58 • Impact of SNP on gene expression. 61 1.4. Experimental hypothesis and approach. 67 iii CHAPTER 2: MATERIALS & METHODS 69 2.1. Cell culture. 70 2.2. Gene expression assay. 71 2.2.1. RNA extraction. 71 2.2.2. Complementary DNA synthesis. 72 2.2.3. Quantitative real-time polymerase chain reaction. 72 2.3. Assay for alternative transcripts. 73 2.4. Screen of human cell lines for presence of SNP. 75 2.5. Microarray. 76 2.6. Immunocytochemistry. 76 2.7. ChIP-Seq. 77 2.7.1. Chromatin immunoprecipitation. 77 2.7.2. Quality check of ChIP-DNA: Western blot and qPCR. 80 2.7.3. Sequencing. 82 2.7.4. Bioinformatics analysis of ChIP-Seq targets. 84 2.8. RNA-Seq. 84 CHAPTER 3: FUNCTIONAL CHARACTERIZATION OF THE TARGET EPAS1 SNP 86 3.1. Introduction. 87 3.2. Characterizing the locus of the EPAS1 SNP. 89 3.3. Alternative splicing between exon 2 and 7. 93 3.4. Screening of human cell lines for the target EPAS1 SNP. 99 3.5. Discussion. 101 3.5.1. Significance of the locus of the SNP. 101 3.5.2. Impact of cryptic splice sites in exon 6. 106 3.5.3. The SNP screening process. 110 3.6. Conclusion. 111 CHAPTER 4: IDENTIFICATION OF EPAS1 TARGETS 113 4.1. Introduction. 114 4.2. In vitro differentiation into trophoblast lineage at 20% and 3% oxygen. 117 iv 4.3. EPAS1 targets by ChIP-Seq. 123 4.4. In vitro & in vivo expression of EPAS1 binding targets. 133 4.5. Discussion. 137 4.5.1. Establishing the trophoblast lineage in 3% oxygen. 137 4.5.2. Verification of ChIP. 140 4.5.3. Assessment of EPAS1/HIF2α targets in the human trophoblast. 142 4.5.4. EPAS1 regulation and preeclampsia. 145 4.5. Concluding remarks. 147 CHAPTER 5: GLOBAL TRANSCRIPTOMIC ANALYSIS OF THE PRE- AND POST-FLOW PLACENTA 150 5.1. Introduction. 151 5.2 mRNA-seq data generation. 154 5.2.1. Human chorionic gonadotropin genes. 155 5.3. Differential analysis and gene ontology. 159 5.3.1. Hemoglobin genes. 163 5.3.2. Blood coagulation. 166 5.3.3. Complement system. 169 5.4. Genes most abundantly expressed in the placenta. 171 5.4.1. Chorionic somatomammotropin hormone 1. 171 5.4.2. Growth differentiation factor 15. 171 5.4.3. Peptidylprolyl isomerase B. 172 5.4.4. Epstein-Barr virus induced 3. 172 5.4.5. Ferritin light polypeptide. 173 5.5. Genes associated with preeclampsia and other pregnancy complications. 177 5.5.1. Leptin. 177 5.5.2. Inhibin genes. 177 5.5.3. Endoglin and fms-related tyrosine kinase 1. 178 5.5.4. Secreted phosphoprotein 1. 179 5.5.5. Hepatocyte growth factor. 179 5.6. Discussion. 181 5.6.1. Hemoglobin switching by the placenta. 181 v 5.6.2. Changing state of the placenta: Anti-coagulatory to pro-coagulatory. 184 5.6.3. Complement system and pregnancy. 186 5.6.4. Recognizing important placental genes. 188 5.6.5. Influence of the uterine environment on preeclampsia pathogenesis. 192 5.7. Concluding remarks. 199 CHAPTER 6: CLOSING REMARKS 203 BIBLIOGRAPHY 206 APPENDIX 241 vi SUMMARY Early development in humans occur in an environment of low oxygen. During the first trimester, maternal blood flow into the placenta is blocked by trophoblastic plugs; a phase we term ‘pre-flow’. Placental perfusion only begins upon the removal of these plugs, between 10 to 12 weeks of gestation. The sudden presence of blood at the end of the first trimester (i.e. post-flow) brings with it oxygenation, drastically modifying the uterine environment. We identified EPAS1 (also known as HIF2α), a transcription factor responding in low oxygen and which has been implicated in placental development and function, to be pivotal to the placental response to the changing oxygen conditions of its environment. Moreover, the EPAS1 gene recently emerged as the gene with the strongest signal of natural selection. A SNP was found in native Tibetan highlanders, a geographically-isolated population that has survived generations at reduced oxygen levels, which appeared to confer not only survivability, but also reproductive fitness. Given the rapid enrichment of this particular allele,, we reasoned that natural selection could likely be acting in utero, and that EPAS1 was not only instrumental in placental development, but that this SNP also effected a profound change in its role. Using a hESC-derived trophoblast culture maintained at 3% oxygen, I took a three- pronged approach to this study. I first characterized the SNP found in Tibetan people, assessing the impact of a nucleotide change in its position. Molecular analysis of EPAS1 transcripts uncovered two cryptic splice sites in close proximity to the SNP. Utilization of either cryptic splice site would lead to modification of the adjacent exon, likely resulting in attenuation of EPAS1 activity.