MOLECULAR MECHANISMS OF NUCLEAR HORMONE RECEPTOR TRANSCRIPTIONAL SYNERGY AND AUTOINDUCTION by Pia D. Bagamasbad A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular and Developmental Biology) in the University of Michigan 2012 Doctoral Committee: Professor Robert J. Denver, Chair Professor Kenneth M. Cadigan Professor Cunming Duan Professor Audrey F. Seasholtz ©Pia D. Bagamasbad All rights Reserved 2012 To my father whose last wish was for me to pursue and finish my doctoral degree To my mother for her endless, unconditional love and support, and for teaching me the value of a good education and hardwork ii ACKNOWLEDGEMENTS I would like to thank my thesis advisor Dr. Robert J. Denver for his guidance, support and patience, not only in science but also through the personal obstacles that I have encountered through graduate school. He has been an excellent mentor and has greatly influenced my scientific growth and outlook. I thank my committee members, Dr. Kenneth Cadigan, Dr. Cunming Duan and Dr. Audrey Seasholtz for their insight and guidance. Their expertise in the different disciplines of biology was of significant importance in shaping my dissertation research. I thank the MCDB administrative staff, particularly Mary Carr and Kelly Campbell, for consistently helping me out with administrative paper works and concerns. I am grateful for the funding support provided by the Rackham Predoctoral Fellowship and the Edwin H. Edwards Scholarship given by the Department of Molecular, Cellular and Developmental Biology. I am lucky to have worked with people like Chris, Yoshi, Melissa, Joe and Rose who are always willing to help in experiments and have provided an intellectually stimulating and friendly lab environment. I am also grateful to Keith Williamson for his guidance in experimental techniques, and to Fang Hu and Ron Bonett for their contributions in my dissertation research. I thank Gizem Kalay, Anita Guidea and Karishma Collete for the friendship and emotional support through the past seven years I am grateful to Roche de Guzman for encouraging me to pursue my doctoral studies; Katrina Mae Carbreros and Ian Dinar Cuenco for the friendship and laughter; Papa family, Ng family, Ninna Yuson, Robert Diaz and especially Jomart and Gianna Rodriguez, Jeffrey Wesolowski and Gari Romano Vargas for the emotional support through the final stages of graduate school and for the sense of family they have provided for me in Ann Arbor. I would also like to thank Peng Ponce, Maya Medalla, Andre Ochoa, Angel Prudente, Notnot Gonzales and Frank Montano for the fun conversations, encouragement and friendship they provided through time and distance. Lastly, I thank Chito Fulgencio for his love, patience, understanding, and for being a source of inspiration. iii TABLE OF CONTENTS DEDICATION……………………………………………………………...ii ACKNOWLEDGEMENTS………………………………...…………….iii LIST OF TABLES…………………….……………………………….....vii LIST OF FIGURES……………………………………………………….ix LIST OF APPENDICES…………………………………………...…….xii ABSTRACT………………………………………………………………xv CHAPTER 1 INTRODUCTION TO THESIS……………………………….………………..1 1. Introduction………………………………………………...……...……..……..3 2. Nuclear Hormone Receptor Superfamily……………………………….……....4 3. Transcriptional Regulation by Nuclear Hormone Receptors…………………...6 3.1 Nuclear Hormone Receptor- DNA Interaction……………………….….8 3.2 Transcriptional Activation by Nuclear Hormone Receptors…………….…9 3.2.1 Histone Modifying Coactivators…………………………………….9 3.2.2 ATP-Dependent Chromatin Remodeling Complex………………..11 3.2.3 Mediator Complex…………………………………………………12 3.3 Transcriptional Repression by Nuclear Hormone Receptors …...…..…13 3.4 Other Modes of Transcriptional Regulation…………………………..…..17 4. Nongenomic Actions of Steroid and Thyroid Hormones…….………….……18 5. Modulation of Nuclear Hormone Receptor Signaling………….……….…….19 5.1 Nuclear Receptor Autoregulation…………………………………………20 5.1.1 Molecular Mechanisms of TR Autoregulation…………………….26 5.1.2 Physiological Significance of TR Autoregulation……………...….28 5.2 Nuclear Receptor Cross-regulation and Cooperativity…………..……….30 5.2.1 Molecular Mechanisms that Underlie Transcriptional Synergy by Nuclear Hormone Receptor…………………….………………….33 6. Synergistic Transcriptional Synergy of the Krüppel-like factor 9 Gene by iv Thyroid Hormone and Glucocorticoids……………………………….42 7. Thesis Summary………………………………………………………………46 8. References…………………………………………………………………….52 2 MOLECULAR BASIS FOR GLUCOCORTICOID INDUCTION OF THE KRÜPPEL-LIKE FACTOR 9 GENE IN HIPPOCAMPAL NEURONS …...64 Abstract ………………………………………………………………………….64 Introduction ……………………………………………………………………. .66 Materials and Methods …………………………….…………………………….68 Results………………………………………………...……………………….....75 Discussion ………………………………………..………...…………………...81 References………………………………………………………………………..96 3 DECIPHERING THE REGULATORY LOGIC OF AN ANCIENT, EVOLUTIONARILY CONSERVED NUCLEAR HORMONE RECEPTOR ENHANCER MODULE……………………………………………….………100 Abstract ………………………………………………………….……………100 Introduction ………………………………………………………...…………102 Materials and Methods ………………………………………………………...106 Results …………………………………………………………………...…….112 Discussion………………………………………………………………………120 References ……………………….…………………………………….………152 4 IDENTIFICATION OF GENES SYNERGISTICALLY REGULATED BY THYROID HORMONE AND GLUCOCORTICOIDS IN HIPPOCAMPAL NEURONS BY MICROARRAY ANALYSIS………………………………...157 Abstract .…………………….………………………………………………….157 Introduction ………………………………………………………………….....159 Materials and Methods ………………………..………………………………..161 Results ………………………………………………………………………….163 Discussion.………………………..………………………………….…….……165 References …………………………………….………………………………..189 5 A ROLE FOR KRÜPPEL-LIKE FACTOR 9 (KLF9) IN THE AUTOINDUCTION OF THYROID HORMONE RECEPTOR BETA……..192 Abstract …………………..…………………………………………………….192 Introduction ….……………………………..………….……………………….194 Materials and Methods ………………………………...……………………….198 Results…………………………………………………………………………..205 Discussion………………………………………………………………………211 References………………………………………………………………………229 v 6 CONCLUSIONS AND FUTURE DIRECTIONS…………………………...233 Conserved hormone-dependent regulation of the Klf9 gene…………………233 Molecular mechanisms of synergistic transcriptional regulation of the Klf9 gene by TR and GR…………………………………………………238 Functional Role of KLF9………………………………………………….…241 Concluding Remarks …………………………………………………….…..247 References…………………………………………….………………….…..249 APPENDICES ……………………………………………….…………………….252 vi LIST OF TABLES Table 2.1. Taqman assays used for quantitative real time PCR analysis of gene expression (RT-PCR) and chromatin immunoprecipitation (ChIP) assays…………………………………………………………….86 Table 2.2. Oligonucleotides used for construction of pGL4.23 promoter plasmids, DNAseI footprinting and electrophoretic mobility shift assay (EMSA)…………………………………………………………….…….87 Table 3.1. Taqman assays used for quantitative real time PCR analysis of gene expression (RT-PCR) and chromatin immunoprecipitation (ChIP) assays……………………………………….…………………………..135 Table 3.2 Oligonucleotides used for construction of pGL4.23 promoter plasmids and electrophoretic mobility shift assay (EMSA)…………………..137 Table 4.1. Top 25 genes up-regulated by 4h of T3 treatment in HT-22 ells……….168 Table 4.2. Top 25 genes down-regulated by 4h of T3 treatment in HT-22 ells……169 Table 4.3. Top 25 genes up-regulated by 4h of CORT treatment in HT-22 cells… 170 Table 4.4. Top 25 genes down-regulated by 4h of CORT treatment in HT-22 cells ……………………………………………………………..171 Table 4.5. Top 25 genes up-regulated by 4h of T3 plus CORT treatment in HT-22 cells……………………………………………………………………..172 Table 4.6. Top 25 genes down-regulated by 4h of T3 plus CORT treatment in HT-22 cells…………………………………………………………….. 173 Table 4.7 Summary of the representative number of genes independently and synergistically regulated by T3 and CORT treatment in HT-22 cells ….174 Table 4.8. Genes synergistically up-regulated by 4h of T3 plus CORT treatment in HT-22 cells……………………………………………………………...175 vii Table 4.9. Genes synergistically down-regulated by 4h of T3 plus CORT treatment in HT-22 cells……………………………………………………..……….179 Table 4.10. Gene ontology analysis of genes differentially regulated by T3…………….183 Table 4.11. Gene ontology analysis of genes differentially regulated by CORT…...184 Table 4.12. Gene ontology analysis of genes differentially regulated by T3 plus CORT………………………………………………………...……185 Table 4.13. Gene ontology analysis of genes synergistically up-regulated regulated by T3 plus CORT………………………………………………………..…186 Table 4.14. Gene ontology analysis of genes synergistically down-regulated regulated by T3 plus CORT……………………………………………………….187 Table 5.1. Oligonucleotides used for semi-quantitative and quantitative real time RT-PCR ………………………………………………………………..218 Table 5.2. Oligonucleotide primers used to generate truncated mutants and point mutations in the three zinc fingers of X. laevis TEB1…………………..219 Table 5.3. Oligonucleotide primers used for the analysis of the X. laevis TrA promoter……………………………………………………………..….220 Table 5.4. Short oligonucleotides corresponding to GC-rich regions (bold, underlined) of the X. laevis TrA promoter used as probes and competitors in EMSA………………………………………………………………..221 viii LIST OF FIGURES Fig.1.1. Nuclear hormone receptor superfamily………………………………………….5 Fig 1.2. Nuclear hormone receptor functional domains………………………………….7 Fig. 1.3. TRAP/DRIP/ Mediator complex………………………………………………16 Fig. 1.4. Molecular mechanisms for regulation of nuclear receptor (NR) expression….25 Fig. 1.5. Forms of nuclear receptor (NR) cross-regulation……………………………...37