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Pitx1 Gene Regulation in Limb Development and Disease

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Pitx1 Gene Regulation in Limb Development and Disease A Dynamic Chromatin Architecture Modulates Pitx1 Gene Regulation in Limb Development and Disease Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by BJØRT KATRINARDÓTTIR KRAGESTEEN from Tórshavn, The Faroe Islands Berlin 2017 The present work was conducted from October 2013 to October 2017 at the Max Planck Institute for Molecular Genetics in Prof. Dr. Mundlos’ research group. 1st Reviewer: Prof. Dr. Stefan Mundlos 2nd Reviewer: Prof. Dr. Sigmar Stricker Date of defence: 29.01.2018 “There’s a crack, a crack, in everything, that’s how the light gets in.” Leonard Cohen Anthem, The Future 1992 Table of contents 1. INTRODUCTION ...............................................................................................................1 1.1. DEVELOPMENTAL GENE REGULATION .............................................................................. 1 1.1.1 Promoters .................................................................................................................... 1 1.1.2 Silencers and insulators .............................................................................................. 2 1.1.3 Enhancers and transcription factors .......................................................................... 3 1.1.4 Identification of enhancer elements .......................................................................... 5 1.2. 3D-ORGANISATION OF THE VERTEBRATE GENOME ......................................................... 6 1.2.1 Chromosome conformation capture .......................................................................... 6 1.2.2 Structure of chromatin domains ................................................................................ 8 1.2.3 Architectural proteins ................................................................................................. 9 1.2.4 Function of chromatin domains ............................................................................... 10 1.3 THE ROLE OF PITX1 IN VERTEBRATE LIMB DEVELOPMENT AND DISEASE ....................... 12 1.3.1 Limb development .................................................................................................... 12 1.3.2 Limb specification ..................................................................................................... 13 1.3.3 Skeletal formation of arms and legs ........................................................................ 15 1.3.4 Pitx1 is a master regulator of morphogenesis ......................................................... 16 1.3.5 Misregulation of PITX1 causes human limb malformations ................................... 18 1.3.6 Pitx1 regulation during development ...................................................................... 20 2. AIMS OF STUDY ............................................................................................................. 23 3. MATERIALS .................................................................................................................... 25 3.1 INSTRUMENTS ...................................................................................................................... 25 3.2 CHEMICALS .......................................................................................................................... 25 3.3 BUFFERS .............................................................................................................................. 25 3.4 ANTIBODIES ......................................................................................................................... 26 3.5 ENZYMES ............................................................................................................................. 27 3.6 KITS .................................................................................................................................... 27 3.7 BACTERIAL STRAINS ............................................................................................................... 27 3.8 VECTORS .............................................................................................................................. 27 3.9 PRIMERS .............................................................................................................................. 28 3.10 MOUSE LINES ..................................................................................................................... 31 3.11 SOFTWARE AND INTERNET RESOURCES ................................................................................... 32 4. METHODS ...................................................................................................................... 33 4.1 MOLECULAR BIOLOGY METHODS .................................................................................... 33 4.2 DNA ISOLATION ................................................................................................................ 33 4.2.1 Plasmid DNA ............................................................................................................. 33 4.2.2 Genomic DNA ............................................................................................................ 33 4.3 CLONING ........................................................................................................................... 34 4.3.1 LacZ Enhancer constructs.......................................................................................... 34 4.3.2 LacZ sensor constructs .............................................................................................. 34 4.3.3 pSilencer construct .................................................................................................... 34 4.3.4 CRISPR-Cas9 sgRNA design and cloning ................................................................... 35 4.4 CELL CULTURE AND MOUSE MODELS ............................................................................... 35 4.4.1 Preparation and culturing of feeder cells ................................................................ 35 4.4.2 Culturing of mouse embryonic stem cells ................................................................ 36 4.4.3 Transfection of G4 ESCs with CRISPR-Cas9 .............................................................. 36 4.4.4 Transfection and targeting of flippase mediated knock in of LacZ enhancers ....... 37 4.4.5 Screening of CRISPR-Cas9 targeted cells .................................................................. 38 4.4.6 Copy number analysis of mutant clones .................................................................. 39 4.4.7 Generation of transgenic mice using diploid and tetraploid aggregation ............. 39 4.4.8 Mouse crossings ........................................................................................................ 39 Table of contents 4.5 CHROMOSOME CONFORMATION CAPTURE ................................................................... 39 4.5.1 4C-seq ........................................................................................................................ 39 4.5.2 Capture Hi-C .............................................................................................................. 43 4.5.3 Hi-C Analysis .............................................................................................................. 45 4.5.4 3D Polymer Modelling .............................................................................................. 47 4.6 ANALYSIS OF MOUSE PHENOTYPES ................................................................................. 48 4.6.1 RNA extraction .......................................................................................................... 48 4.6.2 cDNA synthesis .......................................................................................................... 48 4.6.3 qRT-PCR ..................................................................................................................... 48 4.6.4 Whole-mount in situ hybridization .......................................................................... 48 4.6.5 LacZ Reporter Staining .............................................................................................. 50 4.6.6 Skeletal Preparation ................................................................................................. 50 4.6.7 Micro-computer Tomography .................................................................................. 51 5. RESULTS ........................................................................................................................ 53 5.1 PITX1 CIS-REGULATORY LANDSCAPE IN EMBRYONIC LIMBS ........................................... 53 5.1.1 Pitx1 cis-regulatory landscape contains a pan-limb cis-regulatory region ............ 53 5.1.2 Pitx1 is regulated by pan-limb enhancer, Pen ......................................................... 55 5.1.3 Distinct chromatin and epigenetic profiles in forelimb and hindlimb tissues ........ 57 5.2 3D REGULATION OF PITX1 IN EMBRYONIC LIMBS ........................................................... 59 5.2.1 Comparison of Capture-C and 4C-seq in forelimb and hindlimb buds .................... 60 5.2.2 cHi-C reveals a forelimb to hindlimb switch in chromatin architecture .................. 61 5.3 PERTURBATION OF PITX1 REGULATORY LANDSCAPE RESULTS IN LIMB MALFORMATIONS .................................................................................................................
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