REGULATION OF ACTIVATION PHASE OF ANGIOGENESIS BY TRANCRIPTION FACTORS ETS1 AND ETS2 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Sankha Ghosh, M.Sc. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2014 Dissertation Committee: Dr.Michael C. Ostrowski, Advisor Dr.Jeffrey Parvin Dr.Denis Guttridge Dr. Qianben Wang i Copyright by Sankha Ghosh 2014 i ABSTRACT Vascular remodeling is a necessary process not only for embryonic development but also for specific physiological and pathological conditions in adult. Several growth factors are required at specific stages in order for the vascular development to occur. Angiogenesis also involves complex crosstalk between several endothelial cell (EC) processes including cell cycle, cell survival and migration. Signaling pathways, stimulated by the growth factors and communication between endothelial cells and extra cellular matrix (ECM), coordinate these processes. Deregulation of any of the factors or pathways can lead to severe defects in vessel formation. Transcription factors ETS1 and ETS2 are required for EC functions necessary for embryonic angiogenesis. EC specific deletion of ETS2 with conventional deletion of ETS1 results in defective vascular branching. Additionally, the double mutant embryos are embryonic lethal. Owing to this lethal nature, the specific gene targets of these factors are yet to be identified. In the current study, we elucidate the effect of endothelial cell specific deletion of Ets1 and Ets2 on angiogenesis and characterize the downstream regulatory pathways. ii Inducible Cre-loxp technology was used to specifically delete both Ets1 and Ets2 in endothelial cells after birth. Deletion of Ets1 and Ets2, when restricted to endothelial cells in new born mice (P1-P3), reduced retinal angiogenesis. Similarly, EC infiltration and invasion into matrigel plugs subsided when matrigel admixed with mouse mammary tumor cells was injected into adult mice with inactivated Ets1 and Ets2 specifically in ECs. Gene expression array performed on RNA, isolated from cultured aortic endothelial cells, comparing the double knockout cells with controls revealed reduced expression of key cell cycle and cell survival regulators in double mutant cells. In addition, both these factors were found to occupy the enhancer regions of the target genes indicating these factors directly regulate expression of the target genes. Recruitment of coactivators CBP/p300 was diminished in the absence of ETS1 and ETS2. Deletion of Ets1 and Ets2 in cultured aortic EC resulted in altered cell cycle phases with a G2/M phase arrest and increased sensitivity to apoptosis in vitro. These results demonstrate that deletion of Ets1 and Ets2 in endothelial cells inhibits angiogenesis by blocking cell cycle progression and decreasing cell survival. iii DEDICATION This work is dedicated to my parents and my brother without their blessings and love I could not have accomplished this work. I also dedicate this work to my wife Tapahsama for her continuous support and encouragement through these years. iv ACKNOWLEDGMENTS I express my heartfelt gratitude to my advisor Dr. Michael C. Ostrowski, for his invaluable support, continuous guidance and especially his patience throughout this research work. His useful suggestion, moral support and encouragement to think independently enabled me to complete my work successfully. I am extremely grateful to Dr. Jeff Parvin, Dr. Dennis Guttridge, and Dr. Qianben Wang for their time, support and useful advice throughout this study. I thank all past and present members of Ostrowski Lab. I am extremely thankful to Dr. Haritha Mathsyaraja for being such a friend that she is. I thank Dr. Sudarshana Sharma for his extremely valuable intellectual inputs. I am grateful to Subhasree Balakrishnan for making my time in lab enjoyable. Many thanks go to Heaher Carrey, Jennifer Cabrera Anisha Mathur and Xin Liu for being such wonderful lab mates. I would also like to thank Dr. Lianbo Yu, Dr. Dias Kurmashev for their help with bioinformatics. I also thank the CCC Shared Resources and Cores for their essential role in this work. Last but not the least I thank my family, my wife Tapahsama and my wonderful group of friends here in Columbus for making graduate school and life in Columbus wonderful experience. v VITA 30 November 1980…………………………………… Born – Kolkata, India May 2005……………………………………………….B.Sc., Zoology University of Calcutta Kolkata, India July 2007………………………………………………..M.Sc., Zoology University of Calcutta Kolkata, India September 2007 – December 2009……………………..Teaching Assistant April 2010 – August 2014 The Ohio State University. January 2010 – March 2010…………………………….Research Assistant The Ohio State University. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology vi TABLE OF CONTENTS Page ABSTRACT…………………………………………………………………. ii DEDICATION………………………………………………………………. iv ACKNOWLEDGEMENTS………………………………………………… v VITA…………………………………………………………………………. vi LIST OF TABLES………………………………………………………….. xi LIST OF FIGURES………………………………………………………… xiii LIST OF ABBREVIATIONS………………………………………………. xiii CHAPTER 1 INTRODUCTION…………………………………………... 1 1.1. Development of Vascular Network…………………………………. 3 1.1.1. Evolution of Circulatory Network…………….……………… 3 1.1.2. Vasculogenesis………………………………………...……… 7 1.1.2.1. Mechanism of Vasculogenesis……….……...………... 7 1.1.2.2. Regulation of Vasculogenesis…..…….……...……….. 10 1.1.3. Angiogenesis…………………………………………….......... 13 1.1.3.1. Phases of Angiogenesis…………….....….…………… 13 1.1.3.2. Mediators of Vessel Branching.………………....…..... 21 1.1.3.3. Cellular Processes Involved in Angiogenesis….....…... 33 1.1.3.4. Angiogenesis in Development and Disease……........... 37 1.2. Ets Family of Transcription Factors...………………………………. 39 1.2.1. The ETS Domain………………………..…..………………….. 43 1.2.2. The PNT Domain……...….…………………………………… 44 vii 1.2.3. The Biological Role of Ets Factors…………………………… 47 1.2.3.1. Ets Factors in Embryonic Development...…...………... 47 1.2.3.2. Ets Proteins in Cancer………………...……...……….. 49 CHAPTER 2 MATERIALS AND METHODS…………………………... 51 2.1. Animal Husbandry………………………………………………….. 51 2.1.1. Transgenic Mice used……………………………....…………. 51 2.1.2. Animal Care…………………………………………………… 52 2.1.3. Mouse Genotyping……………………………………………. 52 2.1.3.1. Tail DNA Preparation………………………………… 52 2.1.3.2. Genotyping Primers and PCR Conditions……............. 52 2.1.4. Animal Procedures……………………………………………. 54 2.1.4.1. Induction of Gene Deletion ………………….……….. 54 2.1.4.2. Eye and Retina Dissection…..………………………… 54 2.1.4.3. Subcutaneous matrigel plug injection………………… 55 2.2. Tissue Culture and Viral Infections…………………………………. 55 2.2.1. Primary Cell Extraction and Culture………………………….. 55 2.2.1.1. Aortic Endothelial Cells………………………............ 55 2.2.2. Lentiviral Infections…………………………………………… 56 2.3. Microarray and Data Analysis……..………………………………… 56 2.4. cDNA Preparation…………………………………………………... 57 2.4.1. RNA Extraction……………………………………………….. 57 2.4.2. Reverse Extraction……………………………………………. 57 2.4.3. Primers Used for real-time PCR………………………............. 58 2.5. Chromatin Immunoprecipitation Assay (ChIP)……………………... 60 2.5.1. Chromatin Preparation………………………………………... 60 2.5.2. Primers Used for real-time PCR………………………............. 60 2.5.3 ChIP-Sequencing and Analysis………………………………... 61 2.6. Quantitative Real Time PCR (qRT-PCR)…………………………… 63 2.6.1. qRT-PCR Reaction Conditions……………………………….. 63 2.6.2. qRT-PCR Analysis……………………………………………. 63 2.7. Western Blot Analysis………………………………………………. 63 2.7.1. Protein Isolation………………………………………………. 63 2.7.2. Western Blot…………………………………………………… 64 2.8. Cell Based Assays…………………………………………………... 64 2.8.1. BrdU Proliferation Assay…………………………………….. 64 2.8.2. Apoptosis Assay………………………………………………. 64 2.8.3. Migration Assays……………………………………………… 65 2.8.3.1. Scratch Wound/Wound Healing Assay………………. 65 2.8.3.2. Single Cell Migration Track Assay……………........... 65 2.8.3.3. Matrigel Tube Formation Assay………………............ 65 2.9. Histology and Immunostaining..……………………………………. 66 viii 2.10. Imaging and Quantification…………………………………….….. 66 CHAPTER 3 Ets1AND Ets2 IN ACTIVATION PHASE OF ANGIOGENESIS…………………………………………… 67 3.1. Introduction……………...………………………………………….. 67 3.1.1. Ets Factors in Angiogenesis……………..………....…………. 69 3.1.2. Retinal Angiogenesis in Mice ……………….…....…………. 69 3.1.3. Trangenic Alleles Used…………………...………………..…. 73 3.2. Results…………………...………………………………………….. 77 3.2.1. Tie-2-Cre efficiently recombines Ets1/2fl alleles specifically in EC…………………………………………………………….. EC.. 77 3.2.2. Tie-2-Cre; Ets1fl/fl;Ets2fl/fl display defective retinal angiogenesis 79 3…..2.3. Tie-2-Cre; Ets1fl/fl;Ets2fl/fl demonstrate reduced tumor angiogenesis…………………………………………………... 85 3.2.4. Lentiviral-Cre efficiently deletes Ets1/2 in vitro 90 3………….…..…..2.5. Ets1 and Ets2 regulate endothelial cell cycle, cell survival and migration……………………………………………………... 92 3.2.5.1. EC Lacking Ets1 and Ets2 Undergo Increased Apoptosis 92 ……………….3.2.5.2. Loss Of Ets1 And Ets2 Leads To G2/M Phase Arrest... 93 3.2.5.3. EC Specific Ets1/Ets2 Ablation Results in Reduced Migration and Tube Formation In Vitro…….. 97 3.2.5.4. Deletion of Ets1/Ets2 Leads to Increased EC Adhesion. 97 3.3. Discussion.……………....………………………………………….. 102 CHAPTER 4 EC SPECIFIC REGULATORY NETWORK OF ETS1/2. 107 4.1. Introduction………...……………………………………………….. 107 4.1.1. Biological Role of ETS1 and ETS2 as Transcription Factors.... 108 4.1.2……………………………. Coactivators of Ets Factors