UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Mechanisms of Aryl Hydrocarbon Receptor-Mediated Regulation of Gene Expression and the Cell Cycle A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) In the Department of Environmental Health of the College of Medicine November 28, 2006 by Jennifer L. Marlowe B.S., Miami University, 1999 Committee Chair: Alvaro Puga, Ph.D. Professor Department of Environmental Health University of Cincinnati Committee: Dr. Timothy Dalton Dr. Mary Beth Genter Dr. Erik Knudsen Dr. Ying Xia The focus of this dissertation is the discovery of novel mechanisms and pathways of gene regulation by the aryl hydrocarbon receptor (AHR), primarily regarding the role of this protein in modulating cell cycle progression. The AHR is a member of the PAS (Per-Arnt-Sim) superfamily of receptors, which mediate responses to environmental stresses such as hypoxia and circadian rhythm, and control basic physiologic processes like vascular development, learning, and neurogenesis. The AHR protein was discovered by virtue of its high affinity interaction with the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and is now known as the primary mediator of the toxic effects of this and hundreds of other HAH and PAH ligands. The mechanisms by which the AHR acts to mediate toxicity of these compounds include the activity of the AHR as a potent transcriptional activator. The ligand-bound AHR, with its dimerization-binding partner ARNT, upregulates the expression of a battery of genes that function in the metabolism of PAH and HAH compounds. However, the diversity of toxic responses mediated by compounds such as TCDD are not adequately explained by the expression of this battery of genes. One of the primary roles of the AHR, both from a physiological and toxicological standpoint, is the control of cell cycle progression. The AHR may affect cell proliferation, differentiation, or apoptosis depending on the cell type examined, and the mechanisms of these effects remain unclear. Literally hundreds of genes have been implicated as being regulated either directly or indirectly by the AHR, and many of these genes are related to aspects of cell cycle control. The goal of this dissertation is to explore mechanisms by which the AHR modulates the cell cycle through the investigation of novel gene targets of the receptor. Chapter 2 summarizes the current body of knowledge regarding the AHR, its ligands, and perturbations of the cell cycle. Chapter 3 investigates a mechanism whereby the AHR is able to repress the expression of specific cell cycle-regulated genes through its interaction with iii the retinoblastoma protein, a tumor suppressor and major component of the G1/S checkpoint control mechanism. Chapter 4 explores an interaction between the AHR and E2F proteins, also major regulatory components of S-phase progression and DNA replication, and the constitutive activity of the AHR in maintaining basal expression levels of a large number of E2F-regulated genes. Finally, Chapter 5 outlines the identification of novel promoter targets of the AHR using chromatin immunoprecipitation and promoter tiling arrays. The results presented throughout this work show the diversity of AHR functions related both to toxicological endpoints and normal cell physiology, and illustrate the ability of this important transcription factor to regulate the expression of a large number of genes by a variety of distinctive mechanisms. iv v Acknowledgements My sincerest gratitude goes to Dr. Alvaro Puga for inviting me to join his lab as a graduate student, after hiring me as a technician with almost no experience in molecular or cell biology. He believed in me all those years ago, and the decision I made to complete my thesis work in his lab has led me to follow paths I only dreamed of going down before. I have learned almost everything I know about research, and a lot about life as well, from Alvaro, and am forever grateful for both the personal and professional experience of working in his lab. I am indebted to all of the members of my dissertation committee for their dedication, insight, and criticisms that have helped to produce this thesis. Many thanks to Drs. Timothy Dalton, Mary Beth Genter, Erik Knudsen, and Ying Xia for their help and encouragement. I would like to extend a special thanks to the classmates and lab partners who have helped me in countless ways over the years. Thank you to Xiaoqing Chang for both her friendship and willingness to help in any way I ever asked of her. From cell maintenance to western blots, from coimmunoprecipitations to PCR reactions, Xiaoqing was always there when I needed her. Thanks to Michael Schnekenberger for his tireless assistance and valuable scientific insight. Thank you as well to Mingya Huang, I have missed your companionship dearly since you left the lab, but have not forgotten your kind and generous spirit. I would also like to acknowledge the many terrific friends I have made through the years, those who have been there to commiserate with me on everything from the hardships of graduate school, relationships and family, to the drudgery of lab retreat planning. I am especially grateful for the support of Chris Curran, Scott Schneider, and Li Peng, always there when I needed to complain. I would like to acknowledge the contribution of all of the faculty members and students in the Department of Environmental Health’s Division of Toxicology. I feel I have received excellent training in this stimulating and critical environment, and I will undoubtedly use the skills I have learned here in many new and exciting ways. Thank you to my friends and family, past and present, for your invaluable perspective and support through this most trying of times. You have truly kept me from going off the deep end. Thanks to the many collaborators for providing various reagents. Their contribution is acknowledged within each chapter. Thanks to grant numbers 2R01 ES06273 and P30 ES06096 of the National Institutes of Environmental Health Sciences (NIEHS) for providing the funding for this research. Also thanks to the University of Cincinnati for support during my first two years of graduate school as a predoctoral Functional Genomics Fellow, and to The Ryan Foundation as well for fellowship support during my last years as a student. vi Table of Contents List of Tables…………………………………………………………………………………... 10 List of Figures………………………………………………………………………………….. 11 Abbreviations…………………………………………………………………………………... 13 Chapter I Introduction…………………………………………………………………………………….. 16 AHR Ligands, Exposure, and Human Health………………………………………………….. 17 TCDD and the AHR……………………………………………………………………………. 21 Physiological Roles of the AHR……………………………………………………………….. 23 The AHR Signaling Pathway…………………………………………………………………... 25 Expanding Roles for the AHR in Gene Expression and the Cell Cycle……………………….. 30 Chapter II Aryl Hydrocarbon Receptor: Cell Cycle Regulation, Toxicity and Tumorigenesis Introduction…………………………………………………………………………………….. 34 AHR ligand-dependent activation of signal transduction pathways…………………………… 35 AHR agonists activate immediate-early response genes………………………………………. 38 Ligand-independent cell cycle control through the AHR……………………………………… 39 AHR-mediated inhibition of apoptosis………………………………………………………… 40 Cell cycle arrest induced by AHR ligands……………………………………………………... 42 Conclusions…………………………………………………………………………………….. 45 References……………………………………………………………………………………… 48 7 Chapter III The Aryl Hydrocarbon Receptor Displaces p300 from E2F-Dependent Promoters and Represses S-Phase Specific Gene Expression Introduction…………………………………………………………………………………….. 61 Materials and Methods…………………………………………………………………………. 64 Results………………………………………………………………………………………….. 71 Discussion……………………………………………………………………………………… 80 References……………………………………………………………………………………… 85 Chapter IV Modulation of E2F-dependent gene expression by an interaction between the AHR and E2F proteins Introduction……………………………………………………………………………………. 105 Materials and Methods………………………………………………………………………… 108 Results…………………………………………………………………………………………. 118 Discussion……………………………………………………………………………………... 129 References……………………………………………………………………………………... 135 Chapter V ChIP-on-chip microarray analysis of AHR promoter binding sites in Hepa-1c1c7 cells Introduction……………………………………………………………………………………. 157 Materials and Methods………………………………………………………………………... 160 Results………………………………………………………………………………………… 164 Discussion……………………………………………………………………………………... 168 References……………………………………………………………………………………... 172 8 Chapter VI Conclusion…………………………………………………………………………………...... 192 References for Introduction and Discussion Sections………………………………………… 200 Appendix……………………………………………………………………………………… 212 9 List of Tables Chapter III Table 1 Gene-specific primer sets for real-time PCR analysis of relative mRNA expression levels……………………………………………………………….. 92 Table 2 Promoter-specific primer sets for chromatin immunoprecipitation analysis…... 92 Chapter IV Table 1 Gene-specific primer sets for real-time PCR analysis of relative