Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2016 Investigating the Role of the Nucleosome Remodeling Factor INO80 in Development and NURF in Anti-Tumor Immunity zeinab abdallah elsayed Ms Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd © The Author Downloaded from https://scholarscompass.vcu.edu/etd/4663 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Investigating the Role of the Nucleosome Remodeling Factor INO80 in Development and NURF in Anti-Tumor Immunity A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. By ZEINAB ELSAYED Bachelor of Science, Al-Azhar University, Egypt, 2001 Master of Cytogenetics, National Research Center, Egypt, 2011 Advisor: Joseph W. Landry, Ph.D. Assistant Professor, Department of Human and Molecular Genetics School of Medicine Virginia Commonwealth University Richmond, Virginia December 2016 ACKNOWLEDGEMENT First and foremost, thanks to God since his will and grace is the main reason for my success. God blessed me with the ability to meet extraordinary people who helped me reach this point in my life. I would like to thank my advisor, Dr. Joseph W. Landry, for his patience during teaching me to finish my work, for giving me the opportunity to participate in such great projects and his support all of this time. I will be in his debt forever. I would like to thank my committee members: Dr. Jolene Windle for giving me a tremendous amount of her time, listening to me and sincerely helping me find my way out; Dr. Swati Palit Deb for her continuous encouragement and help whenever I asked for it; and Drs. Lynne Elmore and Jim Lister for their continuous help and support since they joined my committee. I would like to thank Dr. Gail Christie for accepting me into the MBG program and for her help in every step until now. In addition, I would like to thank Dr. Amal Mahmoud (head of the Cytogentics Department in Egypt), Dr. Nivin Helmy, and Dr. Hesham Fayek for their continuous support during my Ph.D. studies. I cannot find appropriate words to thank my parents for their unconditional love and continuous support and prayers for me. I am lucky to have such parents and my brother in my life. I am blessed to have my husband beside me during these four years and I am speechless for his support and help. I would like to thank my kids for being responsible and making me smile. Finally, I am very grateful and thankful for the Landry lab members for their contributions to the completion of this work. i Contributors Dr. Joseph Landry: Establishment of mouse lines and mouse dissection. Dr. Zhijun Qui: MEF and mESC isolation and characterization; ISH experiments, teratoma and embryoid body assays, mouse husbandry and genotyping. Veronica Peterkin: Mouse genotyping and RT-PCR experiments. Suehyb Alkhatib and Dorothy Bennett: NK cell depletion in vivo, Southern blot analysis and mouse genotyping. Aiman Alhazmi: Microarray experiments and data analysis. Kimberly Mayes: Data analysis, heparanse Western blot, RT-PCR and CHIP experiments, NK cell depletion in vivo. Mark Roberts and Kristen Peterson: Establishment of rADV and in vivo tumor studies. Nikhil Ailaney: Ncr1-Zeta chain reporter assay and qRT-PCR. ii Table of Content Acknowledgement i Contributors ii Table of Contents iii List of Figures vi List of Tables vii List of Abbreviations viii Abstract 1 PART I. Role of the Nucleosome Remodeling Factor INO80 in Development 3 1 Chapter 1. Background and Literature Review 3 1.1 DNA Packaging into Chromatin 3 1.2 Mechanisms of Chromatin Structure Modulation 4 1.2.1. Histone Modifications 4 1.2.2. Incorporation of Histone Variants 7 1.2.3. DNA Methylation 8 1.2.4. ATP-Dependent Chromatin Remodeling Complexes 9 1.3 Chromatin Remodeling and Development 11 1.3.1. SWI/SNF or BAF Complexes 11 1.3.2. ISWI Complexes 12 1.3.3. CHD Complexes 12 1.3.4. INO80 Family 13 1.4 The INO80 Complex 14 1.4.1. INO80 in DNA Damage and Replication 15 1.4.2. INO80 and Telomeres, Centromeres and Chromosomes 17 1.5 Embryonic Stem Cells 18 1.6 Mouse Early Development 21 1.7 Gastrulation 26 2 Chapter Two: Methods 29 2.1 Generation of INO80 Knockout Mice 29 2.2 DNA Extraction and Genotyping 29 2.3 Embryonic Cell Isolation and Maintenance 30 2.4 Detection of MEF Senescence by LacZ Staining 31 2.5 Alkaline Phosphatase Staining 31 2.6 Cell Doubling Time 31 2.7 Teratoma and Embryoid Body Formation 32 2.8 Whole Mount in Situ Hybridization 32 2.9 TUNEL Assay 34 iii 2.10 Chromatin Immunoprecipitation (ChIP) 35 2.11 Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) 37 2.12 2.12. qRT-PCR Assay 38 2.13 2.13. Statistics 40 3 Chapter Three: Results 41 3.1 Ino80 KO Embryos Fail to Develop after Implantation 41 Ino80 KO Embryonic Stem Cells Are Viable and Pluripotent Only 3.2 in the Presence of 2i 44 Ino80 KO ESCs Fail to Differentiate Using in Vitro Differentiation 3.3 Models 47 3.4 Ino80 KO Embryos Fail to Gastrulate 50 Ino80 Regulates Bmp4 Expression During Embryonic Stem Cell 3.5 Differentiation in Vitro 54 4 Chapter Four: Discussion and Future Directions 59 4.1 Discussion 59 4.1.1 Early Embryonic Lethality Due to Disruption of the Ino80 ATP- Dependent Chromatin Remodeling 59 4.1.2 The Differential Effect of Ino80 KO in MEFs and mESCs 60 4.1.3 Ino80 KO Effect on the Pluripotency of mESCs 62 4.1.4 The Reduced Size of the Epiblast in Ino80 KO Embryos 63 4.1.5 Ino80 KO Effect on the Proximal-Distal Axis Establishment 64 4.1.6 Ino80 Regulation of the Bmp4 Promoter 66 4.2 Future Directions 67 PART II. Role of the Nucleosome Remodeling Complex NURF in Anti-Tumor Immunity 69 Chapter 5. Background and Literature Review 69 5.1 The Immune System 69 5.2 NK Cell Development 69 5.3 Regulation of NK Cytotoxicity 72 5.4 Regulation of NK Cell Activity 74 5.4.1. Inhibitory Receptors 75 5.4.2. Costimulatory NK Receptors 77 5.4.3. Activating Receptors 77 5.5 NK Cells in Anti-Tumor Defense Mechanisms 81 5.6 Cancer Immunoediting 82 5.7 Breast Cancer and Immunoediting 87 5.8 The NURF Complex 89 5.8.1. The Three Main Subunits of the NURF Complex and their Roles in Cancer 89 5.8.2. NURF Remodeling Activity 90 5.8.3. The Biological Roles of the NURF Complex 91 iv 5.8.4. NURF and Cancer 93 Chapter six: Methods 9 5 6.1 Cell Culture 95 6.2 Western Blotting 95 BPTF KD in OT1 and NK Cells using Replication-Deficient 6.3 Adenovirus 97 6.4 Tumor Studies 97 6.5 Mouse NK Cell Purification 98 6.6 NK Cell Cytotoxicity Assay 99 6.7 Flow Cytometry 99 6.8 Enzyme Linked Immune-Absorbent Assay (ELISA) 100 6.9 Ncr1- ζ Chain Reporter Assay 101 6.10 qRT-PCR 101 6.11 Chromatin Immunoprecipitation (ChIP) 102 6.12 Statistics 10 2 7 Chapter Seven: Results 103 7.1 BPTF Depletion in Tumors Enhances NK Cell Antitumor Activity 103 BPTF Depletion in Cancer Cells Enhances NK Cell Cytotoxic 7.2 Activity In Vitro Through NCR Receptors 108 BPTF Regulates Cell Surface HS Abundance on HSPG and 7.3 Heparanase Expression 115 BPTF Depletion Enhances the Immune Response to Established 7.4 Tumors 118 NK Cytolytic Activity Toward BPTF KD Cells is Conserved in 7.5 Humans 122 8 Chapter 8. Discussion and Future Directions 125 9 Chapter 9. References 131 Vita 150 v List of Figures Figure 1.1: The Effect of ATP-Dependent Nucleosome Remodeling 10 Figure 1.2: Cartoon to Illustrate Stages in the Early Mouse Development 23 Figure 3.1: Ino80 is Crucial for Mouse Embryonic Fibroblast Survival and Early Embryonic Development 43 Figure 3.2: Ino80 KO ESC are Viable and Pluripotent Only in the Presence of 2i in the Culture Medium. 46 Figure 3.3: Ino80 is Essential for Embryonic Stem Cell Differentiation 49 Figure 3.4: Ino80 is Widely Expressed in Embryonic Tissues and is Essential for Establishing the Proximal-Distal Axis of the Post- Implantation Embryo 53 Figure 3.5: Ino80 Regulates Bmp4 Expression by Occupying its Promoter 56 Figure 3.6: The Bmp4 Promoter is More Accessible to Positive Regulatory Factors Upon Ino80 Deletion 57 Figure 3.7: Model of Ino80’s Effect on Bmp4 Gene Expression and its Consequences on the Establishment of the Proximal-Distal Axis in the Early Mouse Embryo 58 Figure 5.1: The Stages of NK Cell Development 71 Figure 5.2: The Three Phases of the Cancer Immune-Editing Process 85 Figure 7.1: NK Cells are the Key Effectors in BPTF KD 67NR and 66cl4 Tumor Weight Reduction 104 Figure 7.2: NK Cells Are More Active in the BPTF Depleted Tumor Microenvironments 107 Figure 7.3: A Cell Surface Factor Enhances Mouse NK and NK-92 Cel Activation Toward BPTF Depleted Tumor Cells 110 Figure 7.4: NKp30 is Required for BPTF KD Target Killing by NK-92 Cells in Vitro. 111 Figure 7.5: NK Cell Cytolytic Activity to BPTF KD Cells Requires NCR Receptors 114 Figure 7.6: BPTF Regulates Heparanase Expression 11 7 Figure 7.7: BPTF is Required for an Immune Suppressive Tumor Microenvironment 120 Figure 7.8: BPTF is not Required for NK or CD8+ T Cell Cytolytic Activity 121 Figure 7.9: NK Cytolytic Activity is Conserved in Human Cell Lines 123 Figure 7.10: A Model for BPTF Suppression of Anti-Tumor Activity 124 vi List of Tables Table 2.1: Primers Used for Ino80 Genotyping 30 Table 2.2: Primers Used for Bmp4 ChIP Analysis.