Rap1-Mediated Chromatin and Gene Expression Changes at Senescence
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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 Rap1-Mediated Chromatin And Gene Expression Changes At Senescence Shufei Song University of Pennsylvania Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Cell Biology Commons Recommended Citation Song, Shufei, "Rap1-Mediated Chromatin And Gene Expression Changes At Senescence" (2019). Publicly Accessible Penn Dissertations. 3557. https://repository.upenn.edu/edissertations/3557 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3557 For more information, please contact [email protected]. Rap1-Mediated Chromatin And Gene Expression Changes At Senescence Abstract ABSTRACT RAP1-MEDIATED CHROMATIN AND GENE EXPRESSION CHANGES AT SENESCENCE The telomeric protein Rap1 has been extensively studied for its roles as a transcriptional activator and repressor. Indeed, in both yeast and mammals, Rap1 is known to bind throughout the genome to reorganize chromatin and regulate gene transcription. Previously, our lab published evidence that Rap1 plays important roles in cellular senescence. In telomerase-deficient S. cerevisiae, Rap1 relocalizes from telomeres and subtelomeres to new Rap1 target at senescence (NRTS). This leads to two types of histone loss: Rap1 lowers global histone levels by repressing histone gene transcription and it also results in local nucleosome displacement at the promoters of the activated NRTS. Here, I examine mechanisms of site-specific histone loss by presenting evidence that Rap1 can directly interact with histone tetramers H3/H4, and map this interaction to a three-amino-acid-patch within the DNA binding domain. Functional studies are performed in vivo using a mutant form of Rap1 with weakened histone interactions, and deficient promoter clearance as well as blunted gene activation is observed, indicating that direct Rap1-H3/H4 interactions are involved in nucleosome displacement. In addition, I explore histone chaperones and chromatin remodelers that may function as Rap1 co-activators at senescence, and found that the histone H3/H4 chaperone Asf1 is required for full Rap1-mediated nucleosome displacement and gene activation. The epigenetic mark H3K4me3 is similarly involved, though the exact details of how this crosstalk occur remain to be elucidated. Remarkably, blunting NRTS activation, at least in the cases of the Rap1 mutant and Asf1 deletion, do not affect the pace of senescence-related cell cycle arrest, suggesting that negative aspects of senescence-related gene expression changes can be uncoupled from the tumor- suppressive properties of cell senescence. Furthermore, as features of the yeast and mammalian Rap1 proteins are conserved, I also explore roles of hRAP1 in human fibroblast senescence. Genome-wide ChIP-seq in senescent IMR-90s and RNA-seq in fibroblasts overexpressing hRAP1 reveal important roles for hRAP1, including relocalization to and regulation of histone genes and senescence-related genes. Direct H3/H4 interactions are also conserved, though more mapping studies are needed to determine the exact interaction surface on mammalian RAP1. Together, these studies illuminate mechanisms of Rap1-mediated senescence changes. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor F. Brad . Johnson Keywords cellular senescence, pace of senescence, pioneer transcription factor, Rap1 Subject Categories Biochemistry | Cell Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3557 RAP1-MEDIATED CHROMATIN AND GENE EXPRESSION CHANGES AT SENESCENCE Shufei Song A DISSERTATION in Biochemistry and Molecular Biophysics 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 _______________________ F. Brad Johnson Professor of Pathology and Laboratory Medicine Graduate Group Chairperson _______________________ Kim Sharp Associate Professor of Biochemistry and Biophysics Dissertation Committee Ben Black, Professor of Biochemistry and Biophysics Ronen Marmorstein, George W. Raiziss Professor and Vice-Chair, Professor, Department of Biochemistry and Biophysics Shelley Berger, Daniel S. Och Professor of Cell and Developmental Biology Eric Brown, Associate Professor of Cancer Biology ACKNOWLEDGMENT I recently went back to personal statements I had written when applying to graduate school, and perhaps in a moment of naivete I wrote “Not only do I want to push and test the known boundaries, I also hope to contribute something worthwhile to science and humanity as we battle with the secrets of life”. Grad school quickly disillusioned me when I realized that science, much like anything else in life, is composed of the nitty-gritty: the grand ideas yes, but also the endless troubleshooting, the frustrations of experiments not repeating, the uncertain interpretation of results and the effects of these on one’s happiness and self-worth. Looking back, there were certainly low points in my PhD that I couldn’t have moved past if not for the very special people in my life. Therefore, I really want to thank my advisor Brad for his constant support and his optimism, for explaining science in gentle and fun ways, for always being available with helpful suggestions, from big-picture directions on where my project is headed down to details of buffer components when experiments fail. He has modeled for me and others in the lab what it means to be a scientist with his extensive grasp of literature and keen sense for hot science topics and developments, but most importantly, with his character and integrity. He has also been one of my main sources of American political news, and though I still can’t keep who’s who straight in my head, Brad’s deep concern for the world, compassion for the less fortunate, and his staunch insistence on doing what’s right instead of what’s easy or beneficial has moved me to no end. I would also like to thank my family for their undying support. For my father, who first instilled in me a love for science and showed me what a scientist can achieve; for my mother and brother, who’ve travelled from China multiple times to visit me and constantly remind me that families are never separated by distance. For my parents-in-law, who supported my husband and I through a long-distance relationship, and has never pressured me or doubted me despite the many years it took to complete this thesis. ii To my past and current lab members, Becky Glineburg, Ting Yang, Qijun Chen, Jesse Platt, Rafael Fernandez and Javier Perez, thank you for always being ready to talk science and/or life. You have made work a comfortable and fun place to be, and your dedication, diligence, and intelligence inspire me. To my friends and church family, thank you for always being there, whether it’s for companionship or prayers. They say it’s good people that make good places. You have definitely made the city of brotherly love a warm and unforgettable place to spend my 20’s. To my cat Katniss, the only soul who has spent every day and night on this journey with me, and who is privy to my most intimate joys and grumblings. Your furry presence makes everything better. And finally, I would like to especially thank my husband Wouter. We embarked on this journey while he was working on his PhD in Australia, and over the years, we became engaged, married, and are now expecting our first child. He has scaled seas and mountains for me and given up a faculty position in Australia so that we can be together. He makes me laugh and think, he listens to my problems even though he’s an engineer and doesn’t understand biochemistry. He has spent at least one Thanksgiving Day and two Christmas Days in lab with me. It has been such a privilege sharing a life together, and I’m excited for where our adventures take us next. iii ABSTRACT RAP1-MEDIATED CHROMATIN AND GENE EXPRESSION CHANGES AT SENESCENCE Shufei Song F. Brad Johnson The telomeric protein Rap1 has been extensively studied for its roles as a transcriptional activator and repressor. Indeed, in both yeast and mammals, Rap1 is known to bind throughout the genome to reorganize chromatin and regulate gene transcription. Previously, our lab published evidence that Rap1 plays important roles in cellular senescence. In telomerase-deficient S. cerevisiae, Rap1 relocalizes from telomeres and subtelomeres to new Rap1 target at senescence (NRTS). This leads to two types of histone loss: Rap1 lowers global histone levels by repressing histone gene transcription and it also results in local nucleosome displacement at the promoters of the activated NRTS. Here, I examine mechanisms of site-specific histone loss by presenting evidence that Rap1 can directly interact with histone tetramers H3/H4, and map this interaction to a three-amino-acid-patch within the DNA binding domain. Functional studies are performed in vivo using a mutant form of Rap1 with weakened histone interactions, and deficient promoter clearance as well as blunted gene activation is observed, indicating that direct Rap1- H3/H4 interactions are involved in nucleosome displacement. In addition, I explore histone chaperones and chromatin remodelers that may function as Rap1 co-activators at senescence, and found that the histone H3/H4 chaperone Asf1 is required for full Rap1-mediated nucleosome displacement