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Functions of DNA Damage Response Factors in Lymphocyte Development and Transformation

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Functions of DNA Damage Response Factors in Lymphocyte Development and Transformation University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2010 Functions of DNA Damage Response Factors in Lymphocyte Development and Transformation Bu Yin University of Pennsylvania School of Medicine, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cancer Biology Commons, Immunity Commons, and the Molecular Biology Commons Recommended Citation Yin, Bu, "Functions of DNA Damage Response Factors in Lymphocyte Development and Transformation" (2010). Publicly Accessible Penn Dissertations. 410. https://repository.upenn.edu/edissertations/410 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/410 For more information, please contact [email protected]. Functions of DNA Damage Response Factors in Lymphocyte Development and Transformation Abstract DNA double strand breaks (DSBs) can activate cell cycle checkpoints or apoptosis, and lead to genomic alterations that drive malignant transformation. The H2AX core histone variant is phosphorylated in chromatin around DSBs by kinases such as ATM and DNA-PKcs. However, how H2AX suppresses chromosome breaks and translocations in cells and prevents tumorigenesis in mice and humans is not well understood. V(D)J recombination is a genetically programmed DNA damage and repair process that assembles the variable region exons of antigen receptor genes in developing lymphocytes. Using an inducible V(D)J recombination system, I found that H2AX is phosphorylated along cleaved antigen receptor loci DNA strands, prevents their irreversible separation in G1 phase, and reduces chromosome breaks and translocations in subsequent cell cycles. Consistent with H2AX functions in DSB repair, I also demonstrated that conditional H2AX deletion results in accumulation of genomic instability in cells, but delays tumor onset in a mouse thymic lymphoma model, presumably due to increased death of cells with synthetic loss of multiple repair factors. To further test this possibility, I generated cells and mice deficient in both H2AX and TMA to examine whether ATM-independent H2AX functions downstream of other kinases are essential for proper DSB repair. I found that thymocyte-specific ablation of H2AX in ATM-deficient mice esultsr in a 50% reduction in thymus cellularity but does not accelerate or delay tumorigenesis. My results suggest that the outcomes of functional interactions between DNA damage response factors likely depend on the cellular context. Additionally, I discovered a novel function of ATM in regulating mono-allelic recombination at the immunoglobulin light chain locus. ATM could orchestrate the signaling pathways enforcing allelic exclusion to provide a time window to test whether the rearrangement on the first allele is productive. In summary, my work has provided novel mechanistic insights into how DNA damage and repair factors coordinately regulate V(D)J recombination, lymphocyte development and neoplastic transformation. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Craig H. Bassing, PhD Keywords DNA Damage and Repair, V(D)J Recombination, H2AX, ATM, Lymphoma, Allelic Exclusion Subject Categories Cancer Biology | Immunity | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/410 FUNCTIONS OF DNA DAMAGE RESPONSE FACTORS IN LYMPHOCYTE DEVELOPMENT AND TRANSFORMATION Bu Yin A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 Supervisor of Dissertation _______________________________ Craig H. Bassing, PhD, Assistant Professor, Pathology and Laboratory Medicine Graduate Group Chairperson ______________________________ Daniel S. Kessler, PhD, Associate Professor, Cell and Developmental Biology Dissertation Committee Shelley L. Berger, PhD, University Professor, Cell and Developmental Biology Tom Curran, PhD, FRS, Professor, Pathology and Laboratory Medicine Zissimos Mourelatos, MD, Associate Professor, Pathology and Laboratory Medicine Warren S. Pear, MD, PhD, Professor, Pathology and Laboratory Medicine M. Celeste Simon, PhD, Professor, Cell and Developmental Biology DEDICATION I dedicate this thesis to my parents, who raised me, cared for me, and had to get used to living without me being around, just because they love me and have always had faith in me. ii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Craig H. Bassing for his generous support, without which I would not have achieved what I have now. Thank you for your encouragement in my research, your guidance at and beyond the bench, and your efforts in helping me to become a better scientist and a better person in general. I am indebted to all current and past Bassing lab members, especially Andrea Carpenter, Velibor Savić and Beth Nuskey, for teaching me the essential techniques, Brenna Brady, Natalie Steinel, Marta Rowh and Angela Fusello for discussions, and Katherine Yang-Iott for making the lab feel like home. A lot of thanks also go to the Cancer Biology/Cell and Molecular Biology graduate group at Penn. The excellent faculty members and many exceptional graduate students have been tremendous role models. Among them I would like to acknowledge my thesis committee, Dr. Tom Curran, Dr. Shelley L. Berger, Dr. Warren S. Pear, Dr. Zissimos Mourelatos, and my chair Dr. M. Celeste Simon, for their support and scientific advice. Marisa Juntilla from Dr. Gary Koretzky’s lab was instrumental in the work described in Chapter II. Dr. Aaron Gitler has been very supportive in my job search. I would also like to acknowledge my collaborators, Dr. Barry P. Sleckman together with Grace Mahowald, Beth Helmink, Eric Gapud and Andrea Bredemeyer from Washington University at St. Louis. Without their pioneering work and sharing of expertise and reagents, most of my work would not have been possible. I’ve also enjoyed collaborations with Dr. Jane Skok’s lab at New York University, and with Dr. Larry Turka’s lab at Penn. I thank Xiaohe Liu from the Turka lab for being a close friend. iii ABSTRACT FUNCTIONS OF DNA DAMAGE RESPONSE FACTORS IN LYMPHOCYTE DEVELOPMENT AND TRANSFORMATION Bu Yin Advisor: Craig H. Bassing DNA double strand breaks (DSBs) can activate cell cycle checkpoints or apoptosis, and lead to genomic alterations that drive malignant transformation. The H2AX core histone variant is phosphorylated in chromatin around DSBs by kinases such as ATM and DNA-PKcs. However, how H2AX suppresses chromosome breaks and translocations in cells and prevents tumorigenesis in mice and humans is not well understood. V(D)J recombination is a genetically programmed DNA damage and repair process that assembles the variable region exons of antigen receptor genes in developing lymphocytes. Using an inducible V(D)J recombination system, I found that H2AX is phosphorylated along cleaved antigen receptor loci DNA strands, prevents their irreversible separation in G1 phase, and reduces chromosome breaks and translocations in subsequent cell cycles. Consistent with H2AX functions in DSB repair, I also demonstrated that conditional H2AX deletion results in accumulation of genomic instability in cells, but delays tumor onset in a mouse thymic lymphoma model, presumably due to increased death of cells with synthetic loss of multiple repair factors. To further test this possibility, I generated cells and mice deficient in both H2AX and ATM to examine whether ATM-independent H2AX functions downstream of other iv kinases are essential for proper DSB repair. I found that thymocyte-specific ablation of H2AX in ATM-deficient mice results in a 50% reduction in thymus cellularity but does not accelerate or delay tumorigenesis. My results suggest that the outcomes of functional interactions between DNA damage response factors likely depend on the cellular context. Additionally, I discovered a novel function of ATM in regulating mono-allelic recombination at the immunoglobulin light chain locus. ATM could orchestrate the signaling pathways enforcing allelic exclusion to provide a time window to test whether the rearrangement on the first allele is productive. In summary, my work has provided novel mechanistic insights into how DNA damage and repair factors coordinately regulate V(D)J recombination, lymphocyte development and neoplastic transformation. v TABLE OF CONTENTS Title Page i Dedication ii Acknowledgements iii Abstract iv Table of Contents vi List of Figures viii List of Tables x Chapter I – Introduction 1 DNA Damage and Repair Mechanisms Are Essential for Tumor Suppression 2 DNA Double Strand Break Response and Repair within Chromatin 3 H2AX Phosphorylation Is a Hallmark of Chromosomal DSB Response 9 V(D)J Recombination is a Physiological DNA Damage and Repair Process 15 Novel Cell Line Systems and Mouse Models to Study H2AX Functions 20 DNA Damage Signals and Mono-allelic V(D)J Recombination 26 Chapter II – Histone H2AX Stabilizes Broken DNA Strands to Suppress Chromosome Breaks and Translocations in V(D)J Recombination 35 ABSTRACT 36 INTRODUCTION 37 RESULTS 40 DISCUSSION 54 FIGURES AND FIGURE LEGENDS 59 Chapter III – Cellular Context Dependent Effects of H2ax and p53 Deletion Upon Development of Thymic Lymphoma 74 ABSTRACT 75 INTRODUCTION 76 RESULTS 80 DISCUSSION 91 FIGURES, FIGURE LEGENDS AND TABLES 96 Chapter IV – Functional Interactions Between ATM and
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