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Rap1 Relocalization Contributes to the Chromatin-Mediated Gene Expression Profile and Pace of Cell Senescence" (2014)

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Rap1 Relocalization Contributes to the Chromatin-Mediated Gene Expression Profile and Pace of Cell Senescence University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Rap1 Relocalization Contributes to the Chromatin-Mediated Gene Expression Profile and aceP of Cell Senescence Jesse Platt University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biology Commons, and the Family, Life Course, and Society Commons Recommended Citation Platt, Jesse, "Rap1 Relocalization Contributes to the Chromatin-Mediated Gene Expression Profile and Pace of Cell Senescence" (2014). Publicly Accessible Penn Dissertations. 1406. https://repository.upenn.edu/edissertations/1406 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1406 For more information, please contact [email protected]. Rap1 Relocalization Contributes to the Chromatin-Mediated Gene Expression Profile and Pace of Cell Senescence Abstract Cellular senescence is accompanied by dramatic changes in chromatin structure and gene expression. Using S. cerevisiae mutants lacking telomerase (tlc1Δ) to model senescence, we find that with critical telomere shortening the telomere binding protein Rap1 relocalizes to the upstream promoter regions of hundreds of new target genes. The set of new Rap1 targets at senescence (NRTS) are preferentially activated at senescence, and experimental manipulations of Rap1 levels indicate that it contributes directly to NRTS activation, potentially in conjunction with enzymes involved in H2B ubiquitylation (Bre1/ Lge1). A notable subset of NRTS includes the core histone-encoding genes; we find that Rap1 contributes to their repression and that histone protein levels decline at senescence. Rap1 and histones also display a target site-specific antagonism that leads ot diminished nucleosome occupancy at the promoters of upregulated NRTS. This antagonism apparently impacts the rate of senescence because under- expression of Rap1 or over-expression of the core histones delays senescence. Rap1 relocalization is not a simple consequence of lost telomere binding sites, but rather depends on the Mec1 checkpoint kinase. Rap1 relocalization is thus a novel mechanism connecting DNA damage responses (DDRs) at telomeres to global changes in chromatin and gene expression while driving the pace of senescence. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor F. Bradley Johnson Keywords cellular senescence, chromatin, Rap1, telomere Subject Categories Biology | Family, Life Course, and Society This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1406 RAP1 RELOCALIZATION CONTRIBUTES TO THE CHROMATIN-MEDIATED GENE EXPRESSION PROFILE AND PACE OF CELL SENESCENCE Jesse Platt 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 2014 Supervisor of Dissertation _____________________ F. Bradley Johnson, MD PhD Associate Professor of Pathology and Laboratory Medicine Graduate Group Chairperson _____________________ Daniel S. Kessler PhD, Associate Professor of Cell and Developmental Biology Dissertation Committee Eric Brown, PhD (Chair) Shelly Berger, PhD Associate Professor Daniel S. Och University Professor of of Cancer Biology of Cell and Developmental Biology Roger Greenberg, MD PhD Paul Lieberman, PhD Associate Professor Hilary Koprowski, M.D., Endowed Professor of Cancer Biology McNeil Professor of Molecular Medicine & Translational Research RAP1 RELOCALIZATION CONTRIBUTES TO THE CHROMATIN-MEDIATED GENE EXPRESSION PROFILE AND PACE OF CELL SENESCENCE COPYRIGHT 2014 Jesse Michael Platt This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ Dedication page To my parents and my family iii ACKNOWLEDGMENT My PhD was a formative experience and I would like to thank many people for their help and support. First and foremost, I would like to thank my parents and family for their support during my PhD. As always, they were there for me during the toughest times. In addition, I need to thank my amazing girlfriend, Yu-San Huoh, who was a wonderful sounding board and helped me get though some of the toughest times (including a thesis printing fiasco). I don’t think I can thank enough my outstanding advisor, Dr. F. Brad Johnson, who has helped me develop into the scientist I am today. It was an honor and a pleasure to get a chance to work with Brad, who continually blew me away with his smarts, patience, and ingenuity. Brad was always there for me answering my questions (at any hour of the day), helping me design better experiments, and constructively pointing out my weaknesses in the most kind and helpful ways. Though I still have much to learn, Brad has helped me take steps forward to become a more mature scientist. Working in the Johnson lab was some of the most fun I’ve had in a long time (and I hope to get some time to go back when I return to medical school). I also would like to thank the Johnson lab (in particular Alejandro Chavez) and my supportive committee. iv ABSTRACT RAP1 RELOCALIZATION CONTRIBUTES TO THE CHROMATIN-MEDIATED GENE EXPRESSION PROFILE AND PACE OF CELL SENESCENCE Jesse Platt F. Brad Johnson Cellular senescence is accompanied by dramatic changes in chromatin structure and gene expression. Using S. cerevisiae mutants lacking telomerase (tlc1Δ) to model senescence, we find that with critical telomere shortening the telomere binding protein Rap1 relocalizes to the upstream promoter regions of hundreds of new target genes. The set of new Rap1 targets at senescence (NRTS) are preferentially activated at senescence, and experimental manipulations of Rap1 levels indicate that it contributes directly to NRTS activation, potentially in conjunction with enzymes involved in H2B ubiquitylation (Bre1/Lge1). A notable subset of NRTS includes the core histone-encoding genes; we find that Rap1 contributes to their repression and that histone protein levels decline at senescence. Rap1 and histones also display a target site-specific antagonism that leads to diminished nucleosome occupancy at the promoters of upregulated NRTS. This antagonism apparently impacts the rate of senescence because under-expression of Rap1 or over-expression of the core histones delays senescence. Rap1 relocalization v is not a simple consequence of lost telomere binding sites, but rather depends on the Mec1 checkpoint kinase. Rap1 relocalization is thus a novel mechanism connecting DNA damage responses (DDRs) at telomeres to global changes in chromatin and gene expression while driving the pace of senescence. vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................ V LIST OF TABLES .................................................................................................................. XV LIST OF ILLUSTRATIONS................................................................................................XVI INTRODUCTION........................................................................................................................ 1 Telomere structure and function......................................................................................................................1 History and Background.......................................................................................................................................................1 Telomere Structure and Maintenance............................................................................................................................2 Senescence ................................................................................................................................................................3 Senescence-associated gene expression changes .....................................................................................................6 Senescence-associated chromatin changes .................................................................................................................7 Yeast System to Model Senescence...................................................................................................................8 The three models of yeast aging and senescence................................................................................................... 11 Rap1 ......................................................................................................................................................................... 12 vii CHAPTER 2: RAP1 RELOCALIZATION CONTRIBUTES TO THE CHROMATIN- MEDIATED GENE EXPRESSION PROFILE AND PACE OF CELL SENESCENCE .......35 INTRODUCTION .................................................................................................................................................... 35 RESULTS.................................................................................................................................................................. 39 Rap1 targets new loci in senescent cells .................................................................................................................... 39 NRTS are preferentially upregulated by Rap1 at senescence..........................................................................
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