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The Role of Taf Proteins During Early Zebrafish Development

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The Role of Taf Proteins During Early Zebrafish Development THE ROLE OF TAF PROTEINS DURING EARLY ZEBRAFISH DEVELOPMENT by ANDREAS ZAUCKER A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY College of Medicine & Dentistry The University of Birmingham April 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Replacement of the prototype promoter recognition factor (PRF) TFIID by alternative PRFs or changes in its subunit composition have recently been implicated in differentiation processes during development. TFIID is composed of TBP (TATA-binding protein) and 13 TAFs (TBP- associated factors). I comprehensively studied the roles of Tafs during vertebrate development using zebrafish model. Taf knock down (kd) phenotypes generated in an antisense morpholino oligonucleotide (MO) screen suggest differential functions of Tafs during zebrafish development. The kd phenotypes also propose a special requirement of DNA-binding Tafs during zebrafish development. In conjunction with zygotic mutant phenotype analysis of zebrafish taf8-mutants compared to taf6-mutants I investigated a potential coactivator function of Taf8 for Pparγ, which has been suggested by in vitro data. Oil Red O (ORO) stainings of 5 dpf (days post fertilisation) taf8- and taf6-mutant larvae revealed a specific lipogenesis defect in liver and intestine of taf8-mutant larvae. The results from treatments with a PPARγ inhibitor suggest that this lipogenic process is Pparγ-dependent. To convincingly establish a coactivator function of Taf8 for Pparγ during early zebrafish development, future work has to link the 5 dpf lipogenesis phenotype of taf8-mutants to defects in Pparγ-dependent transcription. I Acknowledgements At first I would like to thank my boss Dr. Ferenc Müller for his support and for his constant efforts in providing me opportunities to have a good start into my scientific career. I also would like to thank Laszlo Tora for his support and for the interesting PhD project, which is based on work by Mate Demeny. My PhD project was financially and scientifically supported by EUTRACC. I also would like to thank my second supervisor Eamonn Maher. Elke Ober and Roy Bicknell accepted to be examiners of my PhD thesis. During my PhD I had the pleasure to participate in interesting and successful collaborations with the Gissen-group and Zsolt Csenki. I really enjoyed working together with those wonderful people. Too many people contributed to the work presented in this thesis, so that I probably will forget to mention someone. First there are my current and former colleagues from the Mueller–group: Yavor Hadzhiev, Jennifer Roberts, Jochen Gehrig, Eva Kalmar, Irene Miguel and Nan Li. All of them were involved in the MO-screen. Especially Matthew Rawlings spent many hours preparing MO stock and injection solutions. Thanks for Irene for help with doing some qPCRs and RT- PCRs for me (and for the in situ song; should be OK that it is not in italics here). Nan Li performed Oil Red O stainings and always gave valuable advice. Turker Bodur helped with the optimisation a of a genotyping protocol. Lixin Yang from the Straehle lab at the ITG in the Karlsruhe Institute of Technology performed the microarray hybridisations and Remo Sanges (Stazione Zoologica Anton Dohrn, Napoli) performed a statistical analysis of the raw data. I had a great time in Birmingham and would like to thank the wonderful people from the Department of Medical & Molecular Genetics. I will surely miss you. Last but not least I want to thank my parents whose support I always can be certain. Special thanks to my mom who kept sending me parcels with sausage and wine (even bread), which have been so heavy that the old lady at the post office couldn’t lift them. They were always welcome. II Table of contents 1 Introduction ........................................................................................................................................... 1 1.1 Transcriptional regulation on the core promoter level.................................................................. 1 1.1.1 Overview of transcriptional regulation .................................................................................. 1 1.1.1.1 Chromatin as a regulator of transcription .......................................................................... 3 1.1.1.2 Proteins and complexes on enhancers .............................................................................. 5 1.1.1.3 Co-regulators and protein complexes linking enhancers to promoters ............................ 6 1.1.1.4 Promoter recognition factors ............................................................................................. 8 1.1.2 Diversity of the core Pol II promoter binding machinery ....................................................... 9 1.1.2.1 TBP family of proteins ......................................................................................................10 1.1.2.2 Structural function of TAFs in TFIID ..................................................................................14 1.1.2.3 Molecular and biological functions of TAFs .....................................................................17 1.1.3 TAF8 ......................................................................................................................................20 1.1.4 Hypothesis of transcriptional regulation on the core promoter ..........................................22 1.1.4.1 Developmental transcription on the core promoter level ...............................................22 1.2 Zebrafish Model ...........................................................................................................................25 1.2.1 General LOF-phenotype for cell-essential genes .................................................................26 1.2.2 MO-mediated knock down technology ................................................................................28 1.3 Potential coactivator function of TAF8 for PPARγ .......................................................................32 1.3.1 NRs (nuclear receptors) ........................................................................................................34 1.3.1.1 PPARs ................................................................................................................................38 1.3.1.2 PPARα ...............................................................................................................................39 1.3.1.3 PPARβ (PPARδ) .................................................................................................................40 1.3.1.4 PPARγ ...............................................................................................................................40 1.3.2 Transcriptional regulation by PPARγ ....................................................................................45 1.3.3 Zebrafish as model for lipid-metabolism and adipocyte biology .........................................48 1.3.3.1 PPARγ in zebrafish ............................................................................................................48 1.3.3.2 Zebrafish as a model for adipocyte research ...................................................................49 1.3.3.3 Zebrafish as model for hepatobiliary diseases .................................................................50 1.4 Objectives .....................................................................................................................................52 III 1.4.1 Distinct roles of TAFs in development..................................................................................52 1.4.2 Exploring the potential role of Taf8 in Pparγ-dependent gene regulation ..........................53 1.4.3 Zebrafish as a genetic model for studying metabolic disorders involved in ARC-syndrome 54 1.4.4 New methods to interfere with maternal effect genes .......................................................54 2 Material and Methods..........................................................................................................................56 2.1 Material ........................................................................................................................................56 2.1.1 Chemicals .............................................................................................................................56 2.1.2 Enzymes ................................................................................................................................58 2.1.3 Equipment and Consumables ...............................................................................................59 2.1.4 Kits ........................................................................................................................................61 2.1.5 Morpholino oligonucleotides ...............................................................................................62 2.1.6 PCR Primers (oligonucleotides)
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