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: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ RB-mediated Regulation of Transcription and Epigenetic Modifications 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 Cell Biology, Neurobiology, and Anatomy of the College of Medicine 2006 By Hasan Siddiqui B.S., University of South Alabama, 2000 Committee Chairman: Erik Knudsen, Ph.D ABSTRACT The retinoblastoma tumor suppressor protein (RB) is the ‘master regulator’ of cellular proliferation and is targeted for inactivation in the majority of human tumors. RB inhibits proliferation by repressing the transcription of genes essential for cell cycle progression. Although RB interacts with numerous chromatin modifying (e.g., histone deacetylases or HDACs, histone methyltransferases or HMTases) and remodeling (e.g., SWI/SNF chromatin remodeling complex) enzymes, the functional significance of these interactions is not apparent. Here we investigated the role of chromatin modifying and remodeling enzymes in RB- mediated transcriptional repression and epigenetic modifications. We find that active RB mediates histone deacetylation on endogenous RB target gene promoters. We also demonstrate that this deacetylation is HDAC dependent, since the HDAC inhibitor trichostatin A (TSA) prevented histone deacetylation at each promoter. However, TSA treatment blocked RB repression of only a specific subset of genes, demonstrating that the requirement of HDACs for RB-mediated transcriptional repression is promoter specific. Furthermore, we find that cell cycle inhibitory action of RB is not intrinsically dependent on the ability to recruit HDAC activity. The HDAC-independent repression was not associated with DNA methylation or gene silencing since it was readily reversible. We show that this form of repression resulted in altered chromatin structure and was dependent on SWI/SNF chromatin remodeling activity. Importantly, we demonstrate that SWI/SNF is required for histone deacetylation of RB target promoters. Furthermore, we show that loss of RB disrupts a specific epigenetic modification and heterochromatin protein 1 (HP1) dynamics in the chromatin. These studies highlight the influence of RB on transcriptional repression and epigenetic modifications. DEDICATION In Loving Memory of My Father S.B. Siddiqui, PhD (1942-2004) Acknowledgments I am greatly indebted to my advisor Dr. Erik Knudsen for his guidance and support throughout my graduate career. It was an exciting and memorable experience to have the opportunity to work under his supervision. Needless to say, I will always cherish my time in his laboratory. I am grateful to members of my thesis committee: Drs Robert Brackenbury, Sohaib Khan, Karen Knudsen and Bernard Weissman for their sincere suggestions, advice and help in both academic and personal matters. I want to thank all members of the Knudsens’ laboratories, past and present, for their help and suggestions during the course of my study. This work would not have been possible without the unconditional assistance from Ranjaka Gunawardena, Sejal Ranmal-Fox and David Solomon. I truly appreciate their help. I would also like to thank Emily Bosco, a great lab- colleague, who sacrificed valuable time on proof reading my manuscripts. I wish to express my heartfelt appreciations to my friends Moinul Islam, Andromeda Nauli, Saydur Rahman and Kazi Sayeed for their friendship, support, and humorous discussions during my stay in Cincinnati. I am immensely grateful to my wonderful parents S.B. Siddiqui and Firoza Begum for their endless love, support and encouragements, without which I would not have come this far. I would also like to thank my brother Raihan for his support. Finally, I wish to acknowledge my wife Rumana for her patience, love, support, understanding and companionship. For all that, I am truly grateful to her. TABLE OF CONTENTS Page Table of Contents………………………………………………………..1 List of Tables and Figures……………………………………………...3 Chapter I: Introduction……………………………………………….....5 A. Cancer and Cell Cycle…………………………………………5 B. Retinoblastoma Tumor Suppressor Protein (RB)………...8 C. Growth Suppression by RB…………………………………10 D. The E2F Family of Transcription Factors: Cellular target of RB……………………………………………………11 E. Chromatin Modifying and Remodeling Enzymes………..14 F. RB and Chromatin…………………………………………….18 G. Reference List…………………………………………………..21 Chapter II: Histone Deacetylation of RB-responsive Promoters: Requisite for Specific Gene Repression but Dispensible for Cell Cycle Arrest…………………………………………………………………....32 A. Abstract………………………………………………………...32 B. Introduction……………………………………………………34 C. Materials and Methods……………………………………....37 D. Results………………………………………………………….43 E. Discussion……………………………………………………..53 F. Reference List………………………………………………....59 Chapter III: Hierarchical Requirement of SWI/SNF in RB-mediated Repression of Plk1……………………………………………………..89 A. Abstract……………………………………………………......89 B. Introduction……………………………………………………91 1 C. Materials and Methods………………………………….......94 D. Results………………………………………………………….96 E. Discussion………………………………………………….…105 F. Reference List…………………………………………….…..109 Chapter IV: Loss of RB Compromises Specific Heterochromatin Modifications and Modulates HP1α Dynamics………………………..132 A. Abstract……………………………………………………...132 B. Introduction……………………………………………...….134 C. Materials and Methods………………………………........137 D. Results………………………………………………...……..140 E. Discussion…………………………………………………...145 F. Reference List……………………………………………....150 Chapter V: Summary and Conclusions………………………………....167 A. HDAC-dependent gene repression by RB……………168 B. Requirement of HDAC activity for RB-mediated cell cycle arrest…………………………………………….171 C. HDAC-independent gene repression by RB………....172 D. Cross talk between SWI/SNF activity and histone deacetylation…………………………………..…174 E. Regulation of Epigenetic Modification by RB………..175 F. Reference List……………………………………………...178 2 List of Tables and Figures Figure Page I-1 Functional inactivation of the RB pathway of cell cycle regulation………..7 I-2 Models of RB-mediated inhibition of E2F-regulated gene transcription....13 II-1 PSM-RB (active RB) represses a set of cell cycle genes and induces cell cycle arrest (A-B)………………………………………………………………72 II-1 PSM-RB (active RB) represses a set of cell cycle genes and induces cell cycle arrest (C-E)………………………………………………………………73 II-2 Active RB induces histone deacetylation at promoters of specific cell cycle genes……………………………………………………………………………75 II-3 RB induced deacetylation at specific promoters is mediated through histone deacetylases (HDACs)………………………………………………77 II-4 HDAC activity is dispensable for RB-mediated cell cycle arrest………….79 II-5 HDAC-dependent deacetylation is required by RB to repress specific promoters (A-C)………………………………………………………………..81 II-5 HDAC-dependent deacetylation is required by RB to repress specific promoters (D)…………………………………………………………………..83 II-6 The cyclin A promoter is not subjected to stable gene silencing (A-B)….84 II-6 The cyclin A promoter is not subjected to stable gene silencing (C)……86 II-7 RB-mediated repression of the cyclin A promoter involves chromatin remodeling (A)………………………………………………………………….87 II-7 RB-mediated repression of the cyclin A promoter involves chromatin remodeling (B)………………………………………………………………….88 3 III-1 RB pathway represses Plk1 expression (A-C)……………………………119 III-1 RB pathway represses Plk1 expression (D-E)……………………………120 III-2 SWI/SNF is required for RB-mediated repression of Plk1 (A-B)………..121 III-2 SWI/SNF is required for RB-mediated repression of Plk1 (C-E)………..122 III-3 SWI/SNF is dispensable for E2F chromatin/promoter association(A-B).124 III-3 SWI/SNF is dispensable for E2F chromatin/promoter association(C-E).125 III-4 SWI/SNF is not required for pocket protein association with the Plk1 promoter ……………………………………………………………………...127 III-5 SWI/SNF is required for histone deacetylase-mediated repression of the Plk1 promoter (A)…………………………………………………………….128 III-5 SWI/SNF is required for histone deacetylase-mediated repression of the Plk1 promoter (B-D)………………………………………………………….129 III-6 Schematic diagram of transcriptional regulation of Plk1…………………131 IV-1 Conditional loss of RB leads to target gene deregulation……………….159 IV-2 Acute RB loss results in specific loss of heterochromatin associated histone modifications (A-C)…………………………………………………160 IV-2 Acute RB loss results in specific loss of heterochromatin associated histone modifications (D-F)…………………………………………………161 IV-3 Acute RB loss does not relocate euchromatin markers into heterochromatin domains…………………………………………………..162 IV-4 Acute RB loss modulates HP1α dynamics (A)…………………………...164 IV-4 Acute RB loss modulates HP1α dynamics (B)…………………………...165 IV-4 Acute RB loss modulates HP1α dynamics (C)…………………………...166 4 Chapter I: Introduction Cancer and Cell Cycle Cancer refers to approximately 100 different diseases that arise in various tissues of the human body through distinct mechanisms. Each cancer has unique features yet the basic processes that lead to tumor formation appear to be similar. Tumor cells acquire the ability to