University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Function of Bromodomain and Extra-Terminal Motif Proteins (bets) in Gata1-Mediated Transcription Aaron James Stonestrom University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular Biology Commons, and the Pharmacology Commons Recommended Citation Stonestrom, Aaron James, "Function of Bromodomain and Extra-Terminal Motif Proteins (bets) in Gata1-Mediated Transcription" (2015). Publicly Accessible Penn Dissertations. 1148. https://repository.upenn.edu/edissertations/1148 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1148 For more information, please contact [email protected]. Function of Bromodomain and Extra-Terminal Motif Proteins (bets) in Gata1-Mediated Transcription Abstract Bromodomain and Extra-Terminal motif proteins (BETs) associate with acetylated histones and transcription factors. While pharmacologic inhibition of this ubiquitous protein family is an emerging therapeutic approach for neoplastic and inflammatory disease, the mechanisms through which BETs act remain largely uncharacterized. Here we explore the role of BETs in the physiologically relevant context of erythropoiesis driven by the transcription factor GATA1. First, we characterize functions of the BET family as a whole using a pharmacologic approach. We find that BETs are broadly required for GATA1-mediated transcriptional activation, but that repression is largely BET-independent. BETs support activation by facilitating both GATA1 occupancy and transcription downstream of its binding. Second, we test the specific olesr of BETs BRD2, BRD3, and BRD4 in GATA1-activated transcription. BRD2 and BRD4 are required for efficient anscriptionaltr activation by GATA1. Despite co-localizing with the great majority of GATA1 binding sites, we find that BRD3 is not equirr ed for GATA1-mediated transcriptional activation. However, exogenous BRD3 efficiently compensates for BRD2 loss, suggesting that BRD2 and BRD3 function redundantly. Third, we tested the role of BETs in mitotic bookmarking. We present evidence that mitotic binding by BRD4, the BET most strongly implicated in preserving transcriptional state during mitosis, is extensively remodeled during this phase of the cell cycle. Additionally, disruption of mitotic BET occupancy has no measurable impact on post-mitotic gene reactivation, calling into question the role of BETs in mitotic bookmarking. These results elucidate new factors critical for GATA1-mediated erythropoiesis. In addition to furthering understanding of the mechanisms underlying BET function, these findings have important consequences for the rational development of BET inhibitors. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Gerd A. Blobel Keywords BET proteins, Erythropoiesis, Hematopoiesis Subject Categories Molecular Biology | Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1148 FUNCTION OF BROMODOMAIN AND EXTRA-TERMINAL MOTIF PROTEINS (BETs) IN GATA1-MEDIATED TRANSCRIPTION Aaron James Stonestrom A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation ________________________ Gerd A. Blobel, M.D., Ph.D. Professor of Pediatrics Graduate Group Chairperson ________________________ Julie A. Blendy, Ph.D. Professor of Pharmacology Dissertation Committee Margaret M. Chou, Ph.D., Associate Professor of Pathology and Laboratory Medicine Mitchell A. Lazar, M.D., Ph.D., Professor of Medicine and Genetics Jianxin You, Ph.D., Associate Professor of Microbiology Kenneth S. Zaret, Ph.D., Professor of Cell and Developmental Biology FUNCTION OF BROMODOMAIN AND EXTRA-TERMINAL MOTIF (BET) PROTEINS IN GATA1- MEDIATED TRANSCRIPTION COPYRIGHT 2015 Aaron James Stonestrom 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/ ACKNOWLEDGMENTS I would first and foremost like express my gratitude to Gerd Blobel for the support and mentorship has provided. I am very lucky to have had the influence of his model of leadership, pragmatism, and skepticism on my scientific and personal development. In addition, I would like to thank the following: Blobel Lab University of Pennsylvania Caroline Bartman Narayan Avadhani Vivek Behera Skip Brass Christopher Edwards Abby Christian Wulan Deng Stephanie Cross Perry Evans Fevzi Daldal Jeremy Grevet Andrew Dancis Chris Hsiung Brian Keith Sarah Hsu Peter Klein Peng Huang Maggie Krall Kristen Jahn Stephen Liebhaber Stephan Kadauke Zissimous Mourelatos Janine Lamonica Vladimir Muzykantov Kiwon Lee Vikram Paralkar Jeremy Rupon Hetty Rodriguez Hongxin Wang Christopher Thom Ariel Yang Elizabeth Traxler Rena Zheng Mitch Weiss Ryan Wyanowanec Thesis Committee Xiaolu Yang Margaret M. Chou Mitchell A. Lazar Collaborators Jianxin You Ross C. Hardison, Penn State University Kenneth S. Zaret Cheryl A. Keller, Penn State University Belinda Giardine, Penn State University Christopher Vakoc, Cold Spring Harbor This work was supported by NIH grants: T32 DK007780 (Aaron Stonestrom), R01 DK054937 (Gerd Blobel) and R56 DK065806 (Gerd Blobel and Ross Hardison). iii ABSTRACT FUNCTION OF BROMODOMAIN AND EXTRA-TERMINAL MOTIF PROTEINS (BETs) IN GATA1-MEDIATED TRANSCRIPTIONAL ACTIVATION Aaron J. Stonestrom Gerd A. Blobel Bromodomain and Extra-Terminal motif proteins (BETs) associate with acetylated histones and transcription factors. While pharmacologic inhibition of this ubiquitous protein family is an emerging therapeutic approach for neoplastic and inflammatory disease, the mechanisms through which BETs act remain largely uncharacterized. Here we explore the role of BETs in the physiologically relevant context of erythropoiesis driven by the transcription factor GATA1. First, we characterize functions of the BET family as a whole using a pharmacologic approach. We find that BETs are broadly required for GATA1-mediated transcriptional activation, but that repression is largely BET-independent. BETs support activation by facilitating both GATA1 occupancy and transcription downstream of its binding. Second, we test the specific roles of BETs BRD2, BRD3, and BRD4 in GATA1-activated transcription. BRD2 and BRD4 are required for efficient transcriptional activation by GATA1. Despite co-localizing with the great majority of GATA1 binding sites, we find that BRD3 is not required for GATA1-mediated transcriptional activation. However, exogenous BRD3 efficiently compensates for BRD2 loss, suggesting that BRD2 and BRD3 function redundantly. Third, we tested the role of BETs in mitotic bookmarking. We present evidence that mitotic binding by BRD4, the BET most strongly implicated in preserving transcriptional state during mitosis, is extensively remodeled during this phase of the cell cycle. Additionally, disruption of mitotic BET occupancy has no measurable impact on post-mitotic gene reactivation, calling into question the role of BETs in mitotic bookmarking. These results elucidate new factors critical for GATA1-mediated erythropoiesis. In addition to furthering understanding of iv the mechanisms underlying BET function, these findings have important consequences for the rational development of BET inhibitors. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... IV LIST OF FIGURES ......................................................................................................................... X LIST OF TABLES .......................................................................................................................... IX CHAPTER 1. INTRODUCTION ...................................................................................................... 1 Transcriptional regulation ............................................................................................................ 1 Lineage-specific gene expression ........................................................................................... 1 Acetylation of histones and transcription factors ..................................................................... 2 Mitotic bookmarking: maintenance of transcriptional state through cell division ..................... 3 Bromodomain and Extra-Terminal motif proteins (BETs) ........................................................... 4 The BET family ........................................................................................................................ 4 Short BETs: BRD2 and BRD3 ................................................................................................. 6 BRD4 ....................................................................................................................................... 7 BET inhibitors .......................................................................................................................... 9 The erythroid transcription factor GATA1 .................................................................................. 10 Erythropoiesis .......................................................................................................................