MOLECULAR AND SYSTEMIC FUNCTIONS OF THE VERTEBRATE-SPECIFIC TATA-BINDING PROTEIN N TERMINUS by Olivier Lucas A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Veterinary Molecular Biology MONTANA STATE UNIVERSITY Bozeman, Montana March 2009 ©COPYRIGHT by Olivier Lucas 2009 All Rights Reserved I ii APPROVAL of a dissertation submitted by Olivier Lucas This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citation, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Edward E. Schmidt Approved for the Department of Veterinary Molecular Biology Dr. Mark Quinn Approved for the Division of Graduate Education Dr. Carl A. Fox I iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non- exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Olivier Lucas March 2009 I iv ACKNOWLEDGEMENTS First, I would like to thank my parents for providing me moral and financial supports during my studies. They have always encouraged me in my endeavors. In addition, I am lucky to have such a fantastic wife, encouraging me and supporting me through rough seas. I don’t know if I would have made it without her. I came to Bozeman determined to learn about the molecular biology toolbox. For this Ph.D. thesis, I felt that I had the most exciting possible project. I am thankful to my advisor, Dr. Ed Schmidt, to have handed me out such a fantastic study. Dr. Schmidt’s love and dedication for science have been contagious. That study could not have been performed without my labmates and collaborators. I am especially thankful to Dr. Justin Prigge and Dr. Elena Suvorova. Alla Bondareva was and will be a reference to me. Tedious genotyping required the help of Sonya Iverson, Marie-Clare Rollins, Ashley Siders, Stephanie Wallin and Carla Weisend. Gayle Callis has been of great help for histological procedures. My gratitude goes toward Jean Kundert for managing our mice colonies and helping me on whole animal procedures anytime I needed it. I feel honored to have generated mutant mice in collaboration with Dr. Mario Capecchi, Nobel Prize of Physiology or Medicine 2007. I am very thankful to Dr. Valérie Copié for her help, encouragements and availability. I am thankful to all my committee members: Dr. Valérie Copié, Dr. Mark Jutila, Dr. Adam Richman, Dr. Michael White and Dr. John Paxton, for their critical comments over the years. I v TABLE OF CONTENTS 1. MOLECULAR ORIGINS OF VERTEBRATE INNOVATIONS ................................ 1 Introduction .................................................................................................................... 1 Canonical Markers ......................................................................................................... 4 Overview ............................................................................................................ 4 Neural Crest Cells .............................................................................................. 4 Placodes.............................................................................................................. 7 Brain ................................................................................................................. 10 Vertebrate Specializations ............................................................................................ 14 Cartilage, Bone, and Teeth in Relation to Predation ........................................ 14 Ear .................................................................................................................... 15 Pharyngeal Arches ........................................................................................... 18 Pharynx Derived Organs .................................................................................. 19 Thyroid ........................................................................................................ 19 Pituitary ....................................................................................................... 20 Pancreas............................................................................................................ 21 Eye.................................................................................................................... 22 Multichambered Heart ..................................................................................... 23 Circulatory System and Endothelial Cells ....................................................... 26 The Adaptive Immune System ......................................................................... 28 Morphological Asymmetry .............................................................................. 30 Liver ................................................................................................................. 32 Novel Molecules .............................................................................................. 33 Steroid Hormones ........................................................................................ 33 Fibrillar Collagen ........................................................................................ 34 Genome Complexity .................................................................................................... 35 Evidence of New Vertebrate-Specific Domains and Proteins ......................... 35 Role of Duplication: .................................................................................... 36 Exonization: ................................................................................................ 38 Alternative and Atypical Splicing: .............................................................. 39 Transposition Events: .................................................................................. 39 Influence of Duplication on PPI Network ........................................................ 39 CRE Duplication and Subfunctionalization ..................................................... 40 Copy Number Variation ................................................................................... 41 MicroRNA ....................................................................................................... 41 Conclusion ................................................................................................................... 42 2. INTRODUCTION ....................................................................................................... 45 Evolutionary Perspectives on Transcriptional Regulation ........................................... 45 Intrinsic Protein Disorder and the TBP N terminus ..................................................... 48 I vi TABLE OF CONTENTS - CONTINUED Function of the TBP N Terminus ................................................................................. 52 Knowledge From the tbp ∆N/ ∆N Mice ............................................................................. 53 3. CO-EVOLUTION OF PITX TRANSCRIPTION FACTORS WITH TBP IN METAZOANS .................................................................................... 56 Introduction .................................................................................................................. 56 Materials and Methods ................................................................................................. 61 Yeast Two-Hybrid ............................................................................................ 61 Isolation of Orthologous cDNAs and Interaction Mapping Tests ................... 64 Mouse Embryonic Fibroblast Isolation and Transfection ................................ 65 RT-PCR and RNase Protection Assays ........................................................... 65 Whole Mount In Situ Hybridization ................................................................ 65 Results .......................................................................................................................... 66 Identification of TBP-Interacting Proteins ....................................................... 66 mPitx2 is a TBP-Binding Protein ..................................................................... 72 The TBP-Pitx Interaction Involves the Metazoan-Specific Domain of TBP ... 74 TBP-N Attenuates Pitx2-Dependent Transcription Activation........................ 76 The TBP Phyla-Specific Region is involved in Nppa Regulation in vivo ....... 79 Discussion .................................................................................................................... 82 Summary .......................................................................................................... 82 Implications .....................................................................................................