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Genetic and Epigenetic Modifiers of C9orf72 Expression in Neurodegenerative Disease University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 Genetic And Epigenetic Modifiers Of C9orf72 Expression In Neurodegenerative Disease Christopher Cali University of Pennsylvania Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Genetics Commons, Molecular Biology Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Cali, Christopher, "Genetic And Epigenetic Modifiers Of C9orf72 Expression In Neurodegenerative Disease" (2019). Publicly Accessible Penn Dissertations. 3631. https://repository.upenn.edu/edissertations/3631 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3631 For more information, please contact [email protected]. Genetic And Epigenetic Modifiers Of C9orf72 Expression In Neurodegenerative Disease Abstract Repeat expansion mutations in the gene C9orf72 are the most common cause of the fatal neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The molecular mechanisms that contribute to these diseases are still not fully understood. In this dissertation, we explore mechanisms associated with repeat expansions in C9orf72 that alter gene expression and contribute to disease. In chapter 2, we develop a novel method of targeted DNA methylation in order to study how epigenetic changes in C9orf72 expansion carriers contribute to disease pathways. We find that C9orf72 promoter hypermethylation is sufficiento t reduce gene expression and induce heterochromatin silencing of this locus in ALS patient derived cells. We also uncover a link between DNA damage repair pathways and DNA methylation where a double strand break in CpG islands can promote DNA methylation. Furthermore, we have taken advantage of these findings ot develop and optimize a novel targeted DNA methylation method that utilizes homology directed repair for precise methylation editing in the absence of off-target effects. In chapter 3, we ask whether intermediate length repeat expansions in C9orf72 are associated with a different neurodegenerative disease, corticobasal degeneration (CBD). CBD shares similar clinical symptoms with Parkinson’s disease but exhibits distinct tau pathology in neurons and glial cells. We find that intermediate C9orf72 repeat expansion carriers (17-30 repeats) are three times more likely to develop CBD than those with smaller repeats. While full expansion carriers with ALS/FTD tend to have reduced C9orf72 expression, we show that intermediate expansion carriers actually have increased C9orf72 mRNA and protein levels. This increase in C9orf72 expression drives aberrant gene expression profiles in vesicle trafficking, stress response and autophagy pathways. Furthermore, we find that autophagy initiated by nutrient starvation is deficient in cells that over-express C9orf72. In sum, this thesis contributes a novel method of targeted DNA methylation to the research community and shows how DNA methylation alters expression of a disease relevant gene. This work also highlights how variable lengths of the repeat expansion in C9orf72 can lead to distinctive underlying molecular mechanisms and ultimately drive risk for different neurodegenerative diseases. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Edward B. Lee Keywords C9orf72, DNA Methylation, HARDEN, Neurodegeneration Subject Categories Genetics | Molecular Biology | Neuroscience and Neurobiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3631 GENETIC AND EPIGENETIC MODIFIERS OF C9ORF72 EXPRESSION IN NEURODEGENERATIVE DISEASE Christopher P. Cali 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 2019 Supervisor of Dissertation ________________________ Edward B. Lee MD, Ph.D. Assistant Professor of Pathology and Laboratory Medicine Graduate Group Chairperson _______________________ Daniel S. Kessler, Ph.D. Chair, Cell and Molecular Biology Graduate Group Dissertation Committee Marisa S. Bartolomei, Ph.D. Perelman Professor of Cell and Developmental Biology Nancy M. Bonini, Ph.D. Florence R.C. Murray Professor of Biology Roger A. Greenberg, MD, Ph.D. J. Samuel Staub Professor of Cancer Biology F. Bradley Johnson, MD, Ph.D. Professor of Pathology and Laboratory Medicine GENETIC AND EPIGENETIC MODIFIERS OF C9ORF72 EXPRESSION IN NEURODEGENERATIVE DISEASE COPYRIGHT 2019 Christopher Paul Cali This work is dedicated to my grandparents for their courage in coping with devastating neurodegenerative diseases. Their experience has inspired me to pursue research that may one day help treat these terrible diseases. iii ACKNOWLEDGMENTS This work would not have possible without my co-workers and scientific mentors. I would like to thank my thesis advisor, Dr. Edward Lee, for his instrumental guidance and crazy (but often brilliant) ideas. I appreciate his mentorship in challenging me to do the highest quality science possible, but also knowing when to offer encouragement and confidence when things seemed bleak. I also want to specifically thank my thesis committee chair, Dr. Brad Johnson, for helping me cope with rejected papers, offering career advice and serving as an outside opinion on the quality of our work. I also thank the other members of my thesis committee, Dr. Marisa Bartolomei, Dr. Roger Greenberg and Dr. Nancy Bonini, for their enthusiasm, flexibility and helpful suggestions along the way. To my lab-mates, past and present, I thank you for your help, encouragement and advice over the years. The original lab members, Jenny and Elaine, helped teach me numerous scientific techniques and offered helpful career advice. The more recent members, Nabil, Jess, Ashley, Aivi and Josh, each helped me in different ways. Whether it was scientific guidance, life advice or just helping me to have fun during the work day, I couldn’t have gotten through the past six years without these friendships. I also want to thank the friends I have made in my graduate group here at Penn. Having such a great group of friends has made the graduate school process more manageable. I feel fortunate to be surrounded by such smart and fun classmates who have shared in the joy (and misery) of the PhD journey. I specifically want to thank Devin, Bobby, Steve B, Steve Z, David, Colleen, Somdutta, Terra and Christin, as well as my Genetics and Epigenetics friends Mikey P, Jonathan and Priya. I can’t express enough gratitude to my wife, Meghan, for helping me to have an enjoyable experience outside of the lab these past years, despite the long hours and many disappointments. She has never once doubted my ability to finish this work, and her confidence helped me through numerous roadblocks. To my parents, their encouragement and interest in my work was fundamental to my success. I can’t thank them enough for the values they’ve instilled in me and for the generosity they’ve shown me over the years. I could not have completed this work without their support. iv ABSTRACT GENETIC AND EPIGENETIC MODIFIERS OF C9ORF72 EXPRESSION IN NEURODEGENERATIVE DISEASE Christopher P. Cali Edward B. Lee Repeat expansion mutations in the gene C9orf72 are the most common cause of the fatal neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The molecular mechanisms that contribute to these diseases are still not fully understood. In this dissertation, we explore mechanisms associated with repeat expansions in C9orf72 that alter gene expression and contribute to disease. In chapter 2, we develop a novel method of targeted DNA methylation in order to study how epigenetic changes in C9orf72 expansion carriers contribute to disease pathways. We find that C9orf72 promoter hypermethylation is sufficient to reduce gene expression and induce heterochromatin silencing of this locus in ALS patient derived cells. We also uncover a link between DNA damage repair pathways and DNA methylation where a double strand break in CpG islands can promote DNA methylation. Furthermore, we have taken advantage of these findings to develop and optimize a novel targeted DNA methylation method that utilizes homology directed repair for precise methylation editing in the absence of off-target effects. In chapter 3, we ask whether intermediate length repeat expansions in C9orf72 are associated with a different neurodegenerative disease, corticobasal degeneration (CBD). CBD shares similar clinical symptoms with Parkinson’s disease but exhibits distinct tau pathology in neurons and glial cells. We find that intermediate C9orf72 repeat expansion carriers (17-30 repeats) are three times more likely to develop CBD than those with smaller repeats. While full expansion carriers with ALS/FTD tend to have reduced C9orf72 expression, we show that intermediate expansion carriers actually have increased C9orf72 mRNA and protein levels. This increase in C9orf72 expression drives aberrant gene expression profiles in vesicle trafficking, stress response and autophagy pathways. Furthermore, we find that v autophagy initiated by nutrient starvation is deficient in cells that over-express C9orf72. In sum, this thesis contributes a novel method of targeted DNA methylation to the research community and shows how DNA methylation alters expression of a disease relevant gene. This work also highlights
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