AP-1-MEDIATED REGULATION OF HPV CHROMATIN TRANSCRIPTION by WEI-MING WANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Cheng-Ming Chiang Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2008 by Wei-Ming Wang All rights reserved TABLE OF CONTENTS Acknowledgements………………………………………………………………... 9 Glossary…………………………………………………………………………… 11 Abstract……………………………………………………………………………. 13 CHAPTER 1. GENERAL INTRODUCTION TO TRANSCRIPTIONAL REGULATION IN HUMAN PAPILLOMAVIRUSES………….. 15 Human Papillomaviruses………………………………………………... 15 Human Papillomaviruses and Human Diseases……………………. 15 HPV Biology……………………………………………………….. 17 Lessons Learned from Cervical Carcinogenesis…………………… 22 Regulation of HPV Oncogene Expression…………………………. 23 AP-1: a Ubiquitous Transcription Factor with Multiple Functions……... 26 Human AP-1 Complexes…………………………………………. 26 Regulation of AP-1 Activity……………………………………… 32 AP-1 and Skin Biology…………………………………………… 37 AP-1 Regulates HPV Oncogene Expression……………………….. 38 Mechanism of Transcriptional Initiation from Chromatin………………. 41 CHAPTER 2. EXPRESSION AND PURIFICATION OF FULL-LENGTH HUMAN DIMERIC AP-1 COMPLEXES USING A BACTERIAL POLYCISTRONIC EXPRESSION SYSTEM…... 46 Introduction……………………………………………………………… 46 Materials and Methods…………………………………………………... 50 Plasmid Constructions……………………………………………..... 50 Expression and Purification of Recombinant Full-Length Human AP-1 Complexes........................................................................... 52 Electrophoretic Mobility Shift Assay (EMSA)……………………... 55 In Vitro Transcription Assay………………………………………… 56 Results…………………………………………………………………… 57 Generation of Polycistronic Bacterial Expression Plasmids for Distinct Human AP-1 Complexes……………………………….. 57 Purification of Recombinant Human AP-1 Complexes…………….. 59 DNA Binding Activity of Purified Recombinant Human AP-1 Complexes………………………………………………………. 64 1 In Vitro Transcription Activated by Recombinant AP-1 Complexes Purified from the In Vitro or In Vivo Reconstitution Method…………………………………………………………... 70 Discussion……………………………………………………………….. 73 CHAPTER 3. CHARACTERIZATION OF PUTATIVE AP-1 BINDING 80 SITES IN HPV-11 URR…………………………………………. Introduction……………………………………………………………… 80 Materials and Methods…………………………………………………... 86 Plasmid Constructions………………………………………………. 86 Protein Expression and Purification ………………………………... 86 Electrophoretic Mobility Shift Assay (EMSA)……………………... 90 Calculation of the Fractional Occupancy…………………………… 91 Transient Transfection and Reporter Gene Assays……………….… 91 DNase I Footprinting………………………………………………... 92 Results…………………………………………………………………… 93 Five Putative AP-1 Sites in the HPV-11 URR Are Bound Differentially by Distinct Human AP-1 Complexes…………….. 93 AP-1 DNA Binding Activity Is Enhanced by Acetylation………….. 102 The Promoter-Proximal AP-1 Site Is Critical for HPV-11 E6 Promoter Activity in C-33A Cells…………………………….… 104 Discussion……………………………………………………………….. 107 Multiple AP-1 Binding Sites Are Well Conserved in HPV URRs….. 107 A Highly Conserved E6 Promoter-Proximal AP-1 Site Found in Genital HPVs……………………………………………………. 108 Redundant Occupancy of the Consensus-Like HPV E6 Promoter-Proximal AP-1 Site…………………………………… 110 The Promoter-Proximal AP-1 Site Is critical for HPV-11 E6 Promoter Activity in the Human C-33A Cell Line……………… 113 The Important Biolgical Role of Redundant Promoter Occupancy……………………………………………………..... 114 CHAPTER 4. MECHANISM OF TRANSCRIPTIONAL ACTIVATION OF THE HPV E6 PROMOTER MEDIATED BY HUMAN AP-1 COMPLEXES…………………………………………………… 116 Introduction……………………………………………………………… 116 2 Materials and Methods…………………………………………………... 121 Plasmid Constructions………………………………………………. 121 Protein Expression and Purification………………………………… 121 Chromatin Assembly and In Vitro Transcription Assay…………….. 121 Order-of-Addition Experiment……………………………………… 122 In Vitro HAT Assay…………………………………………………. 123 Immobilized AP-1 Pull-Down Assay……………………………….. 123 Reverse Transcription-PCR…………………………………………. 124 In Vivo Chromatin Immunoprecipitation (ChIP) Assay…………..… 125 Protein Detection in Cultured Cell Lines…………………………… 126 Antibodies…………………………………………………………... 126 Results…………………………………………………………………… 127 HPV Chromatin Transcription Can be Activated by Distinct Human AP-1 Complexes In Vitro…………………………………….. 127 Acetylation of HPV Chromatin Mediated by p300 Is an AP-1-Dependent Event……………………………………….. 131 HAT Activity of p300 Is Required for AP-1-Dependent HPV Chromatin Transcription In Vitro……………………………... 134 Direct Protein-Protein Interaction Is Required for the Recruitment of p300 to Support AP-1-Dependent HPV Chromatin Transcription………………………………………………….. 137 p300 is Recruited to the Activated HPV E6 Promoter to Perform Targeted Nucleosomal Acetylation in an AP-1-Dependent Manner In Vivo………………………………………………... 143 Discussion……………………………………………………………….. 150 Reconstituted HPV Chromatin Transcription Is Activated by Distinct AP-1 Complexes to Different Extents……………….. 150 p300 Is Essential for AP-1-Dependent HPV Gene Regulation.…….. 152 Possible Cofactors Involved in AP-1-Dependent HPV Gene Regulation…………………………………………………….. 154 Fra-1 May Act as a Competitive Inhibitor of c-Fos in AP-1-Dependent HPV Chromatin Transcription……………... 156 Fra-1 Is a Transcriptional Activator………………………………… 159 Molecular Insights for Positive and Negative Regulation of HPV Gene Expression……………………………………………… 161 3 CHAPTER 5. POST-TRANSLATIONAL MODIFICATION OF AP-1 COMPLEXES…………………………………………………… 165 Introduction……………………………………………………………… 165 Materials and Methods…………………………………………………... 174 Plasmid Constructions………………………………………………. 174 Protein Expression and Purification………………………………… 174 Electrophoretic Mobility Shift Assay (EMSA)……………………... 175 AP-1 In Vitro Acetylation Assay……………………………………. 176 Chromatin Assembly and In Vitro Transcription Assay…………..… 176 Results…………………………………………………………………… 177 The DNA Binding Activity of AP-1 Is Enhanced by p300-Mediated Acetylation……………………………………………………. 177 The DNA Binding Activity of AP-1 Is Enhanced by Human Ref-1.......................................................................................... 179 Discussion……………………………………………………………….. 183 CHAPTER 6. CONCLUSIONS AND FUTURE PERSPECTIVES……………. 192 Appendix A. Primers Used for Cloning of cDNAs Encoding Each Member of Human Jun and Fos Family Proteins…………………………... 196 Appendix B. Monocistronic Expression Plasmids of Human AP-1 Subunits…... 197 Appendix C. Polycistronic Expression Plasmids of Human AP-1 Complexes..... 199 Appendix D. Analysis of Purified Recombinant Human AP-1 Complexes…….. 201 Appendix E. In Vitro Chromatin Assembly and Transcription Using DNA Templates Containing HPV-11 URR AP-1 Site Mutateds in p7072-70G-Less/I+ (WT) Cassettes……………………………. 215 References…………………………………………………………………………. 218 4 LIST OF TABLES Table 1. Constructed plasmids expressing dimeric human AP-1 complexes........ 58 Table 2. Design of DNA fragments containing AP-1 binding sites for EMSA………………………………………………………………….. 85 Table 3. Primers used for mutagenesis of AP-1 binding sites in HPV-11 URR……………………………………………………………………. 87 Table 4. Primers used for generation of individual AP-1 binding site-containing DNA fragments for EMSA……………………….................................. 89 Table 5. Equilibrium binding constant (Kd) for individual AP-1 binding to each AP-1 site-containing DNA probe……………………………................ 98 5 LIST OF FIGURES Fig. 1. Schematic representation of the DNA genome of HPV-11…………… 18 Fig. 2. Sequence alignment of the conserved bZIP motifs within the Jun and Fos protein families…………………………………………………… 28 Fig. 3. Analysis of purified recombinant F:c-Jun/6His:FosB heterodimeric AP-1 complex…………………………………………………………. 61 Fig. 4. Coexpression enhances the stability of recombinant full-length human JunB protein expressed in E. coli……………………………………... 62 Fig. 5. Coomassie blue-stained gels of distinct purified recombinant human AP-1 complexes……………………………………………………….. 63 Fig. 6. Purified recombinant c-Jun/c-Fos binds to TRE in a sequence-specific manner………………………………………………………………… 66 Fig. 7. All the five c-Jun-containing AP-1 complexes exhibit robust DNA binding activity………………………………………………………... 67 Fig. 8. Heterodimeric AP-1 complexes exhibit stronger DNA binding activity than the homodimer…………………………………………………… 68 Fig. 9. Outline of the preparation of human AP-1 complexes from the initial construction of plasmids to the final step of purification……………... 69 Fig. 10. In vivo reconstituted c-Jun/c-Fos exhibits comparable transactivation activity as that reconstituted in vitro…………………………………... 72 Fig. 11. Plasmids used as templates for generation of DNA fragments containing individual wild-type or mutated AP-1 sites for EMSA…… 88 Fig. 12. Five putative AP-1 sites in the HPV-11 URR are differentially bound by distinct human AP-1 complexes…………………………………… 96 6 Fig. 13. Individual putative AP-1 sites exhibit different