REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS By Manal Hosawi A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology Committee Membership Dr. Amy Sprowles, Major Professor, Committee Chair Dr. Bruce A. O’Gara, Committee Member Dr. Edward Metz, Committee Member Dr. Jacob P. Varkey, Committee Member Dr. Erik S. Jules, Graduate Coordinator May 2016 ABSTRACT REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS Manal Hosawi Oct4 is a POU class V transcription factor that functions to maintain the pluripotent state of embryonic stem cells by activating or repressing the transcription of hundreds of target genes (Boyer et al. 2005). Aberrant expression of Oct4 has been reported in a number of cancers, suggesting that it may also play a role in cellular transformation (Tai et al. 2005). This study examines the role of the proto-oncogene cJun and its upstream kinase Jun N terminal kinase (JNK) in the regulation of Oct4 expression and the pluripotent state. Our laboratory identified a putative AP-1 binding site approximately 2500 bp upstream of the Oct4A transcription start site. We modulated the cJun/JNK pathway in two murine embryonic stem cell lines through chemical treatments and transient transfection of wild type and mutant GFP cJun constructs. Our results show mESCs overexpressing cJun forms or treated with 10 ng/ml, 50 ng/ml anisomycin and 50 µM JNK inhibitor SP600125 have an increase in the Oct4A isoform by immunocytochemistry and Western blot analysis, but not the total amount of Oct4. The potency of these cells is also affected. Increased JNK/cJun activity increased in alkaline phosphatase activity but resulted in a significant reduction of pancreatic islet like cluster size, number, insulin expression and the neuronal network between clusters. Loss of JNK/cJun activity had the opposite result: transfection of the transcriptionally inactive ii cJun mutant L40/42A or treatment with SP600125 resulted in increased number, size and neuronal network formation among the pancreatic islet like clusters. JNK modulation by chemical treatment also affected the formation of cardiomyocyte beating clusters. These results indicate the importance of the regulation of JNK/cJun pathway in stem cell potency, potentially through changes in the expression level of Oct4A. Future studies exploring the role of Oct4A in pluripotency and JNK/cJun activity in early embryonic development could provide insight into the role of this isoform in cellular transformation. iii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor Dr. Amy Sprowles who provided continues support during my Master study at Humboldt State University. Her motivation, patience and guidance helped me in all the time of research and writing thesis. I could not have imagined having a better advisor for my Master study. I also would like to thank my thesis committee members: Dr. Bruce A. O’Gara, Dr. Edward Metz, and Dr. Jacob P. Varkey, for their insightful comments, patience and advice. My sincere thanks also goes to my lab mates in Dr. Sprowles laboratory, Ms. Abigail Petersen, Ms. Akela Kuwahara, Ms. Haley E. Du Bois, Mr. Jack Guccione, Ms. Jacqueline Trzeciak, Mr. Johnny L. Castillo, Mr. Christopher H. Mendoza, Ms. Lauren Dahl and Mr. Michael Lopez, for their assistance and support. I want to express my special gratitude to my country Saudi Arabia for financial assistance during my stay in the United States and providing me with this opportunity to study abroad. I especially would like to express my eternal gratitude to my family; my father Mr. Mosa Hosawi, my mother Mrs. Sanaa Husayn and my brothers, for the endless support, encouragement, and love during my graduate education and throughout life. To my family, thank you for encouraging me in all of my pursuits and inspiring me to follow my dreams. iv TABLE OF CONTENTS REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS ................................................ i ABSTRACT ........................................................................................................................ ii REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS ............................................... ii ACKNOWLEDGEMENTS ............................................................................................... iv TABLE OF CONTENTS .................................................................................................... v LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF APPENDICES .................................................................................................... xi INTRODUCTION .............................................................................................................. 1 Transcription Factor Oct4 ............................................................................................... 5 The Regulation of Oct4 Expression ................................................................................ 6 The Transcription Factor cJun ........................................................................................ 9 Biological Roles of the Transcription Factor cJun and JNK .......................................... 9 Specific Aims: ............................................................................................................... 13 1. Determining the role of cJun activity on Oct4 expression in murine embryonic stem cells. .................................................................................................................. 13 2. Evaluating the role of cJun signaling on mESC potency. .................................... 14 3. Evaluating the role of JNK signaling on mESC potency. .................................... 14 MATERIALS AND METHODS ...................................................................................... 15 293T Cell Culture ......................................................................................................... 15 J1 and B6/BLU Murine Embryonic Stem Cell Culture ................................................ 16 Chemical Treatment of Mouse Embryonic Stem Cells and 293T ................................ 17 Protein Extraction and Western Blot ............................................................................ 17 Immunocytochemistry .................................................................................................. 20 Transient Transfection of 293T Cells ........................................................................... 21 Transient Transfection of Mouse Embryonic Stem Cells ............................................. 22 Differentiation of Mouse Embryonic Stem Cells ......................................................... 23 Embyoid Body formation ......................................................................................... 23 Directed differentiation from EBs to beating cardiomyocytes ................................. 24 Directed differentiation from EBs to Pancreatic islet-like insulin secreting clusters 24 Alkaline phosphatase .................................................................................................... 25 Statistical Analysis ........................................................................................................ 26 v RESULTS ......................................................................................................................... 27 The Role of the JNK/cJun Pathway on Oct4 Expression in Murine Embryonic Stem Cells .............................................................................................................................. 27 Examining the modulation of JNK/cJun pathway by chemical treatment in 293T .. 27 Examining the modulation of JNK/cJun pathway by chemical treatment on Oct4 expression in mESCs ................................................................................................ 35 Testing the Overexpression of cJun on Oct4 ................................................................ 55 The Role of CJun Signaling on Murine Embryonic Stem Cell Potency....................... 69 Testing the effects of cJun on alkaline phosphatase activity .................................... 69 Examining the effects of cJun on directed differentiation ........................................ 72 Directed differentiation into Pancreatic Islet Like Clusters and Cardiomyocytes .... 72 The Role of JNK Signaling on Murine Embryonic Stem Cell Potency ....................... 82 Testing the effects of modulating JNK signaling on alkaline phosphatase activity . 82 Testing the effects of modulating JNK on the differentiation of mESCs ................. 87 Directed differentiation into Pancreatic islet like clusters ........................................ 87 Directed differentiation into Cardiomyocytes .......................................................... 98 DISCUSSION ................................................................................................................. 101 CJun and JNK Signaling Affects Oct4 Expression....................................................