DREAM-MEDIATED REGULATION OF GCM1 IN THE HUMAN PLACENTAL TROPHOBLAST by Dorota Baczyk A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto © Copyright by Dorota Baczyk (2010) Abstract DREAM-MEDIATED REGULATION OF GCM1 IN THE HUMAN PLACENTAL TROPHOBLAST Degree: Master of Science Year of convocation: 2010 Name: Dorota Baczyk Graduate Department: Physiology University: University of Toronto The trophoblast transcription factor glial cell missing-1 (GCM1) regulates asymmetric division of placental cytotrophoblast to form the differentiated syncytiotrophoblast. Reduced GCM1 expression is a key feature of the hypertensive disorder preeclampsia. In-silico techniques identified a novel calcium-dependent transcriptional repressor – DREAM as a regulatory candidate for GCM1. The overall objective of this thesis was to determine if DREAM regulates GCM1 expression and therefore villous trophoblast turnover. siRNA-mediated DREAM silencing in both BeWo cells and floating villous explants significantly upregulated GCM1 causing reduced cytotrophoblast proliferation. Calcium-dependency was demonstrated in both BeWo cells and floating villous explants by contrasting the effects of ionomycin and nimodipine. A direct interaction between DREAM and the GCM1 promoter was demonstrated using EMSA and ChIP assay. DREAM is a negative upstream regulator of GCM1 expression in human placenta that participates in calcium-dependent trophoblast differentiation. ii Acknowledgements I would like to extend my gratitude to my supervisor Dr. Stephen Lye as well as a long-time mentor Dr. John Kingdom for providing me with the opportunity to undertake my training under their guidance and support. I would also like to thank my committee members Dr. Sue Quaggin, and Dr Ted Brown for their valuable contribution during my training. I would not have succeeded in this program without the remarkable support and assistance from Dr. Oksana Shynlova, Dr. Sascha Drewlo and Dr. Caroline Dunk. I would like to acknowledge the Canadian Institute of Health Research, the Samuel Lunenfeld Research Institute and the Department of Physiology for their funding and scholarships. Finally, I would like to thank my wonderful family, children Alexander and Natalia and husband Steve, for their enduring patience and support while I was pursuing my goal. iii TABLE OF CONTENTS iv Abstract ............................................................................................................................. ii Acknowledgements .......................................................................................................... iii TABLE OF CONTENTS ................................................................................................... iv List of tables ....................................................................................................................viii List of figures.................................................................................................................... ix List of abbreviations ......................................................................................................... xi INTRODUCTION .........................................................................................................1 1.1 The Placenta .............................................................................................2 1.1.1 Early stages of human placental development..........................................2 1.1.2 Mature placenta at term.............................................................................4 1.1.3 Placental Function .....................................................................................5 1.2 Placental trophoblast cell lineages............................................................6 1.2.1 Villous trophoblast layer.............................................................................6 1.2.2 Factors controlling villous trophoblast turnover .........................................8 1.2.3 Extravillous trophoblast cells ...................................................................10 1.2.4 Factors controlling EVT differentiation and invasion................................11 1.3 Glial Cell Missing 1 ..................................................................................12 1.3.1 GCM1 structure and target genes ...........................................................12 1.3.2 GCM1 regulation......................................................................................12 1.3.3 GCM1 function in placentation.................................................................13 1.4 Downstream regulatory element antagonist modulator (DREAM) ..........14 1.4.1 Structure ..................................................................................................14 1.4.2 Expression...............................................................................................14 1.4.3 Function...................................................................................................15 1.4.4 Calcium dependence...............................................................................16 1.4.5 Potential role of DREAM in pathophysiology of placental insufficiency syndromes ...............................................................................................18 1.5 Placental insufficiency syndromes...........................................................18 1.5.1 Preeclampsia...........................................................................................18 1.5.1.1 Pathophysiology of severe PE...........................................................19 1.5.1.2 Treatment ..........................................................................................20 1.5.1.3 Prevention of PE................................................................................20 1.5.2 Intrauterine Growth Restriction ........................................................21 1.6 Rational of the project..............................................................................21 1.7 Models used in the study.........................................................................22 1.8 Objective and aims ..................................................................................22 MATERIALS AND METHODS ................................................................................23 2.1 In vitro models .........................................................................................24 2.1.1 BeWo cells...............................................................................................24 2.1.1.1 Transfection of BeWo cells ................................................................24 2.1.1.2 Ionomycin and Nimodipine treatment ................................................24 2.1.2 First trimester placental explants.............................................................25 2.1.2.1 Floating first trimester placental explant model .................................25 2.1.2.2 Cytotoxicity assays............................................................................26 2.1.2.3 Placental villous explant immunohistochemistry................................26 2.2 Semi-thin sections...................................................................................27 2.3 TUNEL assay...........................................................................................28 2.4 BrdU incorporation and immunodetection ...............................................28 v 2.5 Quantitative real time-PCR......................................................................28 2.5.1 Tissue collection ......................................................................................28 2.5.2 RNA extraction.........................................................................................29 2.5.3 Reverse transcription...............................................................................29 2.5.4 Quantitative real time PCR ......................................................................30 2.5.5 Primers ....................................................................................................30 2.6 Image analysis and quantification of placental tissue sections................31 2.7 DNA/protein interactions..........................................................................32 2.7.1 Promoter analysis....................................................................................32 2.7.2 Chromatin Immunoprecipitation (ChIP) ...................................................32 2.7.2.1 PCR amplification of immunoprecipitated fragments.........................34 2.8 Electromobility Shift Assay (EMSA).........................................................34 2.8.1 Probe preparation....................................................................................34 2.8.2 Nuclear protein extraction........................................................................35 2.8.3 Probe/protein binding and electrophoresis ..............................................35 2.8.4 Transfer and detection.............................................................................36 2.9 Statistical analysis ...................................................................................36 RESULTS ................................................................................................................37 3.1 Investigation of DREAM expression pattern in the human placenta throughout pregnancy..............................................................................38