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Sp1) Regulates the Centrochromatin Landscape and Centromeric Transcription During Mitosis by Aislinn Rebecca Sowash Molinari

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Sp1) Regulates the Centrochromatin Landscape and Centromeric Transcription During Mitosis by Aislinn Rebecca Sowash Molinari Transcription Factor Specificity Protein 1 (Sp1) Regulates the Centrochromatin Landscape and Centromeric Transcription During Mitosis By Aislinn Rebecca Sowash Molinari July 2016 A Dissertation Presented to the Faculty of Drexel University College of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular and Cellular Biology and Genetics Chairperson Dr. Jane Clifford, Ph.D. Dr. Eishi Noguchi, Ph.D. Professor and Chair; Associate Associate Professor; Director, Dean for Medical Student Research Graduate Program in Molecular & Department of Biochemistry & Cellular Biology & Genetics Molecular Biology Department of Biochemistry & Molecular Biology Dr. Michael Bouchard, Ph.D. Dr. Elias Spiliotis, Ph.D. Director, Division of Biomedical Associate Professor; Director of the Science Programs; Associate Cell Imaging Center Professor Department of Biology Department of Biochemistry & Drexel University Molecular Biology Dr. Timothy Yen, Ph.D. Professor; Facility Director, Biological Imaging Facility Fox Chase Cancer Center Transcription Factor Specificity Protein 1 (Sp1) Regulates the Centrochromatin Landscape and Centromeric Transcription During Mitosis By Aislinn Rebecca Sowash Molinari July 2016 A Dissertation Presented to the Faculty of Drexel University College of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular and Cellular Biology and Genetics Copyright by Aislinn Rebecca Sowash Molinari 2016 DEDICATION To my brother Graham, who is a rock star and inspiration, and to my son Harrison, who helped me finish. i ACKNOWLEDGEMENTS I would like to begin by thanking my thesis advisor, Dr. Jane Azizkhan-Clifford, for the opportunity to work in her lab and to grow as a scientist under her mentorship. Dr. Clifford taught me how to be a critical, independent thinker, providing continued support and encouragement. She provided me with invaluable scientific advice, but only after I had exhausted all of my own resources. She also supported me while I navigated some complicated personal matters in a professional, yet comfortingly maternal manner. Dr. Clifford had continued faith in me, even when I didn’t have faith in myself, and for that I am deeply grateful. I would also like to thank the members of my thesis committee, including Dr. Eishi Noguchi, Dr. Michael Bouchard, Dr. Timothy Yen, and Dr. Elias Spiliotis, for your time, suggestions, critiques, and encouragements over the years. Finally, I would like to thank the office staff, including Jenny Sherwood, Kate Maum, and Lucia Boyer, for your resources, support, and friendship. To both current and past Clifford lab members – Thank you for your friendship and your support over the years, and for making the Clifford Lab an enjoyable place to work. I would especially like to thank Bill Donegan, who I have had the pleasure of working with for the majority of my time in graduate school. I have held your scientific suggestions and advice in the highest regard, and I have appreciated your willingness to listen to my grievances, both scientific and otherwise. I would also like to thank three of my mentees, Carol Stojinski, Sam Flashner, and Jacob Havens, for providing me with ii the opportunity to grow as a mentor and teacher. Finally, I would like to thank Yi Guo for your friendship through our greatest experiment yet. Lastly, I would like to thank my family for their love and support. Most importantly, thank you to my husband and best friend, Jonathan Molinari. You have loved me, supported me, and believed in me, and I could not have done any of this without you. iii TABLE OF CONTENTS Abstract…………………………………………………………………………………………xiii Chapter 1: Whole Chromosomal Instability……………………………………………….1 Introduction………………………………………………………………………………2 What is Numerical/Whole CIN (W-CIN)?…………..…………………………………3 Causes for Generation of W-CIN……………………………………………………...5 Centrosome Amplification……………………………………………………..5 Disengagement………………………………………………………...7 Procentriolar formation……………………………………………......8 Elongation………………………………………………………………8 Disjunction/Maturation and Movement……………………………....9 Causes for Centrosome Amplification……………………………….9 Centrosome Amplification and Chromosomal Instability…………10 Sister Chromatid Cohesion Defects………………………………………...10 Sister-Chromatid-Cohesin Complexes……………………………..11 Loading and Establishment of Sister Chromatid Cohesion……...12 Removal of Sister-Chromatid-Cohesin Complexes………………13 Sister Chromatid Cohesion Defects and Chromosomal Instability……………………………………………………………....13 Improper Kinetochore-Microtubule Attachment……………………………14 Aurora B Kinase and the Chromosomal Passenger Complex for Attachment Correction……………………………………………….16 iv Improper Kinetochore-Microtubule Attachment and Chromosomal Instability………………………………………………………………17 Weakening of the Spindle Assembly Checkpoint………………………….18 The Mitotic Checkpoint Complex…………………………………...19 Silencing the SAC……………………………………………….……20 Weakening of the SAC and Chromosomal Instability…………….21 Clinical Significance of W-CIN………………………………………………………..22 Functional Consequences of W-CIN………………………………………..23 Clinical Consequences……………………………………………………….25 Clinical Diagnosis of W-CIN………………………………………………….26 Exploitation of W-CIN for Anti-Cancer Therapies………………………….29 Conclusions…………………………………………………………………………….30 Chapter 2: Centromere Biology…………………………………………………………….37 Introduction……………………………………………………………………………..38 Human Centromeric DNA…………………………………………………………….40 Defining Centromeres: Centromeric Protein A (CENP-A)……………………… ..42 The human CENP-A Gene and Protein…………………………………….42 The CENP-A Nucleosome…………………………………………………...43 CENP-A Deposition at Centromeres………………………………………..46 Holliday junction recognition protein (HJURP)……………………47 The Mis18 Complex………………………………………………….48 CENP-C and CENP-I………………………………………….……..50 Other DNA Binding Proteins at Centromeres………………………………………51 Centromeric Protein B (CENP-B)……………………………………………51 Centromeric Protein C (CENP-C)………………………………………...…52 The CENP-T-W-S-X Heterotetramer………………………………………..54 v The Centrochromatin: Centromere-Specific Histone Modifications…………..….55 Histone Modifications at the Core Centromere…………………………….56 Histone Modifiers………………………………………………………….…..57 Transcription Through Core Centromeres…………………………………………..58 Evidence for Transcription at Core Centromeres in Human Cells……….58 Transcription Factors at Centromeres in Human Cells…………………...59 Timing of Core Centromere Transcription………………………………….59 Human Artificial Chromosomes (HACs)…………………………………….60 A Functional Requirement for Transcription at Core Centromeres……...62 Dysregulation of Centromeric Transcription………………………………..64 Conclusions…………………………………………………………………………….65 Chapter 3: Specificity Protein 1……………………………………………………….……71 Introduction………………………………………………………………………..……72 The Specificity Protein/Krüppel-like Factor (SP/KLF) Transcription Factor Family…………………………………………………………………………………...73 The Sp1 Gene and Protein(s)………………………………………………………..74 Sp1 and Gene Regulation…………………………………………………………….75 Sp1 as a General Transcription Factor……………………………………..75 Sp1 as a Specific Transcription Factor……………………………………..76 Post-Translational Modifications (PTMs) for Sp1…………………………………..78 Sp1 and Mitosis………………………………………………………………………..80 Sp1 and Whole Chromosomal Instability (W-CIN)…………………………………81 Sp1 and Centrosome Regulation……………………………………………82 Sp1 and Survivin………………………………………………………………82 Sp1 and Cancer………………………………………………………………………..84 Conclusions…………………………………………………………………………….85 vi Chapter 4: Transcription Factor Sp1 Regulates the Centrochromatin Landscape and Centromeric Transcription During Mitosis………………………………………….90 Introduction………………………………………………………………………….....91 Sp1 Localizes to Centromeres in Mitotic Cells……………………………………..92 Sp1 Binds to Centromeres and Pericentromeres in MCF 10A Cells…………….93 Sp1 Binding to Centromeres is Not Dependent on the Sp1 Zinc-Finger, DNA-Binding Domain………………………………………………………………....93 ATM Activity Is Required for Sp1 Localization to Centromeres…………………..94 Sp1 Knockdown Results in a Decrease in CENP-A at Centromeres…………….95 Decreased CENP-A at Centromeres is Not Due to Reduced Expression of CENP-A…………………………………………………………………………………96 Sp1 Knockdown Results in a Decrease in CENP-C at Centromeres……………97 Sp1 Contributes to Regulation of α-Satellite-Derived Long Non-Coding RNAs...98 Over-Expression of Sp1 Results in a Decrease in CENP-A at Centromeres….100 Sp1 Depletion Disrupts the Centrochromatin Landscape………………………..101 Conclusions…………………………………………………………………………...102 Chapter 5: Sp11-182 May Be Sufficient for Functional Centromeres…………………121 Introduction……………………………………………………………………………122 Sp11-182 Is Sufficient for DNA Double-Strand-Break Repair……………………...124 Sp11-182 Is Sufficient for Centrosome Regulation………………………………….124 Sp11-182 Rescues Centromere Distance Phenotype………………………………125 Sp11-182 Rescues CENP-A Intensity Phenotype by Immunofluorescence Imaging………………………………………………………………………………..126 Sp11-182 Partially Rescues CENP-A Binding by Chromatin Immunoprecipitation………………………………………………………………….126 Conclusion…………………………………………………………………………….127 vii Chapter 6: Discussion and Future Directions………………………………………….132 Introduction……………………………………………………………………………133 Discussion of Presented Results…………………………………………………...133 Transcription Factor Sp1 Regulates the Centrochromatin Landscape and Centromeric Transcription During Mitosis…………………………...134 Sp11-182 May Be Sufficient for Functional Centromeres………………….140 Current Model………………………………………………………………..142 Future Directions……………………………………………………………………..142 Identifying Sp1 Interacting
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