FUNCTION OF CORE PROMOTERS IN DIFFERENTIAL GENE REGULATION DURING EMBRYOGENESIS By JOCHEN GEHRIG A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Department of Medical and Molecular Genetics School of Clinical and Experimental Medicine College of Medical and Dental Sciences The University of Birmingham January 2010 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The core promoter is the ultimate target of all transcriptional regulatory processes. The recently discovered diversity of core promoters and basal transcription factors suggests a regulatory role in differential gene expression. However, the direct contribution of the core promoter remains poorly understood. I investigated core promoters and their putative role in differential gene regulation using the zebrafish embryo as an in vivo model system. To analyse the functional requirement for the general transcription factor TATA‐box binding protein (TBP), a diverse set of promoters was tested for their TBP dependence. This analysis revealed a differential requirement of TBP for promoter activity. To further explore the roles of core promoters the ability of various core promoters to interact with tissue‐specific enhancers was investigated. A high‐throughput pipeline combining automated imaging with custom‐designed software for registration of spatial reporter gene activity in thousands of zebrafish embryos was developed. The technology was applied in a large‐scale screen analysing the tissue specific activities of 202 enhancer ‐ core promoter combinations. A variety of interaction specificities observed suggests an important role of the core promoter in combinatorial gene regulation. Overall, these findings indicate that the core promoter significantly contributes to differential transcriptional regulation in the vertebrate embryo. I Acknowledgements First I would like to thank my supervisor Dr Ferenc Müller for accepting me as a PhD student and for his continuous support, advice, encouragement and enthusiasm throughout my research. Thanks also go to all past and current members of the laboratory in Germany and in the UK for helpful discussions and the great working atmosphere: Marco Ferg, Yavor Hadzhiev, Eva Kalmar, Nan Li, Agnes Lovas, Matthew Rawlings, Jenny Roberts, Simone Schindler, Chengyi Song and Andreas Zaucker. I am especially grateful to all the people involved in the screening project: Dr Urban Liebel (Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology (KIT), Germany) for providing the screening platform and for his support and enthusiasm, Dr Markus Reischl (Institute for Applied Computer Sciences, KIT, Germany) for the development of the custom design software and the great collaboration despite the geographical distance, Eva Kalmar for her initial work and the good teamwork, and all the helping hands involved in the handling of thousands of zebrafish embryos: Marco Ferg, Andreas Zaucker, Simone Schindler, Yavor Hadzhiev, Chengyi Song and Nadine Gröbner (ITG, KIT). I would like to thank Dr Agnes Lovas for preliminary experiments and sharing 5’‐RACE data. Finally, I would like to thank my parents and my partner Silke for their continued emotional support and encouragement during this project. II Table of contents 1. GENERAL INTRODUCTION ............................................................................................................... 1 1.1. Regulation of RNA polymerase II dependent transcription .................................................... 1 1.1.1. A classical model of transcription initiation .................................................................................. 3 1.2. Diversity of core promoters .................................................................................................... 4 1.2.1. Promoter discovery in eukaryotic genomes ................................................................................. 4 1.2.2. Transcription start site distributions define promoter classes ..................................................... 6 1.2.3. Alternative promoters ................................................................................................................... 8 1.2.4. Bidirectional promoters .............................................................................................................. 10 1.2.5. Chromatin modifications at the core promoter .......................................................................... 10 1.2.6. Core promoter elements ............................................................................................................. 12 1.2.6.1. The TATA‐box .......................................................................................................................... 14 1.2.6.2. The initiator ............................................................................................................................ 15 1.2.6.3. The Downstream promoter element ...................................................................................... 16 1.2.6.4. The TFIIB recognition elements .............................................................................................. 17 1.2.6.5. Other core promoter elements............................................................................................... 18 1.2.7. CpG islands .................................................................................................................................. 19 1.3. Diversity of core promoter recognition factors .................................................................... 20 1.3.1. Variability in TAF composition of TFIID ....................................................................................... 20 1.3.2. The TATA‐box binding protein .................................................................................................... 23 1.3.3. The TBP related factor 1 .............................................................................................................. 26 1.3.4. The TBP like factor ...................................................................................................................... 26 1.3.5. The TATA‐box binding protein 2 ................................................................................................. 28 1.4. Core promoters in combinatorial gene regulation ............................................................... 30 1.4.1. Cis‐regulatory elements besides the core promoter .................................................................. 30 1.4.1.1. The proximal promoter ........................................................................................................... 31 1.4.1.2. Enhancers and Silencers ......................................................................................................... 32 1.4.1.3. Insulators ................................................................................................................................ 33 1.4.2. Transcription factors and enhancer activity................................................................................ 34 1.4.2.1. Activator mediated initiation of transcription ........................................................................ 36 1.4.3. Enhancer‐promoter interaction specificity ................................................................................. 38 1.4.3.1. Core promoter architecture and enhancer‐promoter specificity ........................................... 39 1.4.3.2. Promoter features of targets of long‐range regulation .......................................................... 40 1.5. Cis‐regulatory elements and human disease ........................................................................ 41 1.6. The maternal to zygotic transition ........................................................................................ 44 1.6.1. Regulation of transcription and mRNA levels at the MZT ........................................................... 45 1.6.2. The role of TBP‐family members in the mid‐blastula transition ................................................. 47 1.7. Zebrafish as a model organism ............................................................................................. 50 1.7.1. Studying gene regulatory function by transgenesis in zebrafish ................................................ 52 III 1.7.2. Large‐scale screening approaches .............................................................................................. 54 2. MATERIALS AND METHODS .......................................................................................................... 56 2.1. Materials ............................................................................................................................... 56 2.1.1. Chemicals .................................................................................................................................... 56 2.1.2. Enzymes .....................................................................................................................................