Chromatin, SF-1, and CtBP Structural and Post-Translational Modifications Induced by ACTH/cAMP Accelerate CYP17 Transcription Rate A Dissertation Presented to The Academic Faculty By Eric B. Dammer In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Applied Biology Georgia Institute of Technology December 2008 Chromatin, SF-1, and CtBP Structural and Post-Translational Modifications Induced by ACTH/cAMP Accelerate CYP17 Transcription Rate Approved by: Dr. Marion B. Sewer, Advisor School of Biology Georgia Institute of Technology Dr. Kirill S. Lobachev Dr. Donald F. Doyle School of Biology School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Alfred H. Merrill, Jr. Dr. Edward T. Morgan School of Biology Department of Pharmacology Georgia Institute of Technology Emory University History is merely a list of surprises. It can only prepare us to be surprised yet again. -Kurt Vonnegut For Kate and Jeran, and Dad- ACKNOWLEDGEMENTS This work would not have been possible without the support and mentoring of my advisor, Dr. Marion Sewer. The amount of trust she extends and effort she expends on behalf of each of her students makes a tremendous difference not only in the life of each of us, but also in the lives we each have been enabled to touch through our continued efforts after our paths go separate ways. The environment in which scientific exchange occurs has been indispensible for my work to proceed; in particular, I am grateful for the collegial atmosphere of sharing and constructive interaction with fellow lab members, including Aarti Urs, Tuba Ozbay, Adam Leon, Anne Rowan, Dr. Donghui Li, Srinath Jagarlapudi, and Natasha Lucki. Each of our projects necessarily bridges our subjective interests with objective results, providing views of a world not yet fully realized. In addition, my friends and colleagues who reviewed parts of this dissertation, or inspired me with their own accomplishments, including Navin Elango,Srinath Jagarlapudi, and Natasha Lucki have my heartfelt gratitude. The contributions of time and guidance by my committee members have enabled my research by insuring organization and completeness, which are reflected on every page of the proceeding work. Finally, I thank each member of my family for their gifts of practical and emotional balance, enabling me to keep a healthy perspective throughout the process of realizing my dreams. Without love, we are nothing. Without the pursuit of passion, the heart grows listless. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ..............................................................................................v LIST OF TABLES .............................................................................................................x LIST OF FIGURES ......................................................................................................... xi LIST OF ABBREVIATIONS ....................................................................................... xiv SUMMARY ......................................................................................................................xv Chapter 1. Introduction....................................................................................................1 1.1 Causes and Effects of Induction of Adrenal Cortisol Synthesis ....................... 1 1.1.1 Steroid Precursor Mobilization and Induced Transcription of Steroidogenic Enzymes Are Hallmarks of Acute and Chronic Steroidogenesis ..................... 2 1.1.2 Cytochromes P450 Are Key Steroidogenic Enzymes ...................................... 3 1.1.3 How Is Cortisol Output Chronically Upregulated in Zona Fasciculata? .......... 6 1.1.4 Steroidogenic Factor-1 Directs Chronic Steroidogenesis in Adrenal Glands and Gonads, Enables Their Development, and Functions in the Hypothalamus and Pituitary ...................................................................................................... 7 1.2 Common Characteristics and Differences among Nuclear Receptors: NR Classes and Modules .................................................................................. 8 1.2.1 SF-1 Functional Architecture from Amino to Carboxy Terminus .................. 10 1.2.2 NR Ligands Induce Protein-Protein Interactions by Favoring Specific Modes of Coregulator Binding ................................................................................... 12 1.2.3 SF-1 Ligands Enable Corepressor and Coactivator Binding Modes .............. 15 1.3 Induced Transcription Overcomes Multiple Barriers through an Ordered Sequence of Events ......................................................................................... 17 1.3.1 The Central Role of Chromatin in Cyclic Transcription of Inducible Genes: An overview of progressive interactions of promoter chromatin, transcription factor(s), and coregulators ....................................................... 20 1.3.2 Transient Behavior of NR-Coregulator Complexes ....................................... 23 1.3.3 Coregulators Manipulate the Histone Code and Nucleosome-DNA Interaction ....................................................................................................... 25 1.3.4 The “Clearance Phase” of Cyclic Transcription: Corepressors enable a return to repressive chromatin structure ................... 32 1.4 Crosstalk between PTMs, Signaling and NR Transcriptional Mechanisms .................................................................................................... 37 1.4.1 SUMOylation / SUMOrylation ....................................................................... 37 1.4.2 Acetylation ...................................................................................................... 38 1.4.3 Phosphorylation of Factors with a Serine/Threonine-Proline Motif ............... 39 1.4.4 Other Modifications ........................................................................................ 44 vi Chapter 2. Materials and Methods................................................................................46 2.1 Reagents .......................................................................................................... 46 2.2 Cell Culture ..................................................................................................... 47 2.3 Plasmids and Mutagenesis .............................................................................. 47 2.4 Bacterial Expression of SF-1 .......................................................................... 49 2.5 In vitro kinase assays ...................................................................................... 49 2.6 Chromatin Immunoprecipitation (ChIP) ......................................................... 50 2.7 RNA Isolation, Real Time RT-PCR, and RNA Interference .......................... 51 2.8 Transient Transfection .................................................................................... 52 2.9 Mammalian Two-Hybrid ................................................................................ 53 2.10 Metabolic Labeling ......................................................................................... 54 2.11 In vitro Acetylation Assay .............................................................................. 54 2.12 Confocal Microscopy and Time Lapse Imaging ............................................. 54 2.13 Coimmunoprecipitation and Western Blotting ............................................... 55 2.14 Biomolecular Simulation ................................................................................ 56 2.15 Statistics .......................................................................................................... 56 Chapter 3. Results—Part I: Coregulator Exchange and Sphingosine-Sensitive Cooperativity of Steroidogenic Factor-1, GCN5, p54, and p160 Coactivators Regulate cAMP-Dependent CYP17 Transcription Rate ...........57 3.1 α-Amanitin, a Valuable RNA Polymerase II (Pol II) Inhibitor ...................... 57 3.2 cAMP Induces Cycles of SF-1 Binding to the CYP17 Proximal Promoter ... 59 3.3 Trans Activation of CYP17 Follows SF-1 Binding ........................................ 60 3.4 Histone H3 and H4 Acetylation Precedes Pol II Recruitment ........................ 62 3.5 Histone Acetyltransferase Recruitment Correlates with Histone Acetylation 64 3.6 Each Transcription Cycle Has a Unique Profile of p160 Binding .................. 65 3.7 GCN5 Interaction with SF-1 is Strengthened by p160 Coactivators .............. 65 3.8 GCN5 Acetyltransferase Activity Limits Interaction with SF-1 .................... 68 3.9 Class I HDACs Have Two Roles in Transcription Cycles Mediated by SF-1 68 3.10 Corepressor Recruitment Reciprocates SF-1 Loss from the CYP17 Promoter .......................................................................................................... 69 3.12 p54nrb Also Enables Assembly of a HAT/p160 Coactivator Complex ........... 72 3.13 CtBP Recruitment Corresponds with Exchange of Transcription Activators for Repressors on the CYP17 Promoter .......................................................... 73 3.14 CtBP1 Alters cAMP Dependent Activation of CYP17 and Basal Interactions among SF-1, GCN5, and SRC-1 ................................................. 77 3.15 Chromatin Remodeling Occurs before and after Pol II Interaction with the CYP17 Promoter ............................................................................................. 79 3.16 Activating Histone