DISRUPTION OF NEURAL CREST ENHANCER LANDSCAPES AS AN ETIOLOGICAL MECHANISM FOR HUMAN NEUROCRISTOPATHIES I n a u g u r a l – D i s s e r t a t i o n zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Michaela Bartusel aus Koblenz Köln 2019 Berichterstatter/in: Prof. Dr. Brunhilde Wirth Prof. Dr. Mirka Uhlirova Tag der letzten mündlichen Prüfung: 06.12.2019 Table of Contents TABLE OF CONTENTS Table of Contents .................................................................................................................................................. 3 Kurzzusammenfassung .......................................................................................................................................... 7 Abstract ................................................................................................................................................................ 9 1 Introduction ............................................................................................................................................11 1.1 Transcriptional regulation of the genome .................................................................................................... 11 1.1.1 Enhancers as key cis-regulatory elements .............................................................................................. 12 1.1.2 Enhancers in human disease ................................................................................................................... 13 1.2 Neural Crest – the Fourth Germ Layer .......................................................................................................... 15 1.2.1 Neural crest development – a multistep process .................................................................................... 15 1.2.2 Cranial NCC and their regulation during formation of the vertebrate head ........................................... 16 1.2.3 Models to study NCC ............................................................................................................................... 17 1.3 Orofacial Clefts .............................................................................................................................................. 19 1.4 Genome Wide Association Studies (GWAS) .................................................................................................. 20 1.5 Novel candidate genes for non-syndromic OFC ............................................................................................ 23 1.6 Branchiooculofacial Syndrome (BOFS) and TFAP2A ..................................................................................... 26 1.7 Aims .............................................................................................................................................................. 29 2 Material & Methods ...............................................................................................................................30 2.1 Equipment ..................................................................................................................................................... 30 2.2 Chemicals and Reagents ............................................................................................................................... 31 2.3 Tissue Culture Procedures ............................................................................................................................ 34 2.3.1 Cell lines .................................................................................................................................................. 35 2.3.2 Tissue culture media and reagents ......................................................................................................... 35 2.3.3 hiPSC culture ........................................................................................................................................... 37 2.3.4 Derivation of patient-specific hiPSC ........................................................................................................ 37 2.3.5 Transfection of hiPSC .............................................................................................................................. 38 2.3.6 Generation of clonal hiPSC lines .............................................................................................................. 38 2.3.7 hNCC differentiation and short term maintenance ................................................................................ 39 2.3.8 Differentiation of NCC derived lineages .................................................................................................. 39 2.3.9 Alcian Blue staining ................................................................................................................................. 40 2.3.10 Scratch Assay ........................................................................................................................................... 40 2.3.11 CFSE Proliferation Assays ........................................................................................................................ 41 3 Table of Contents 2.3.12 Cell proliferation growth curves .............................................................................................................. 42 2.4 Clinical and genetic characterization of the BOFS patient inversion ............................................................ 42 2.4.1 Patient description .................................................................................................................................. 42 2.4.2 Characterization of chromosomal abnormalities .................................................................................... 43 2.4.3 Targeted Locus Amplification (TLA) ......................................................................................................... 44 2.5 General molecular biology methods ............................................................................................................. 46 2.5.1 Genomic DNA extraction ......................................................................................................................... 46 2.5.2 PCR for genotyping .................................................................................................................................. 47 2.5.3 RNA extraction ........................................................................................................................................ 48 2.5.4 cDNA synthesis ........................................................................................................................................ 48 2.5.5 RT-qPCR ................................................................................................................................................... 48 2.5.6 Allele-specific analysis of TFAP2A expression ......................................................................................... 49 2.5.7 Chemical Competent Cells ....................................................................................................................... 49 2.5.8 Molecular cloning .................................................................................................................................... 50 2.6 Enhancer reporter assays .............................................................................................................................. 51 2.6.1 Enhancer cloning ..................................................................................................................................... 51 2.6.2 In vitro reporter assays ............................................................................................................................ 52 2.6.3 In vivo reporter assays ............................................................................................................................ 53 2.7 Immunological Methods ............................................................................................................................... 53 2.7.1 Chromatin Immunoprecipitation (ChIP) .................................................................................................. 53 2.7.2 Immunofluorescence (IF) assays in cultured cells ................................................................................... 55 2.7.3 Flow Cytometry ....................................................................................................................................... 56 2.7.4 Western Blot ........................................................................................................................................... 57 2.8 Genetic engineering by Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR/Cas9) ....... 59 2.8.1 Design of guide RNAs (gRNA) .................................................................................................................. 59 2.8.2 Cloning of gRNA into the Cas9-Vector ..................................................................................................... 60 2.8.3 Delivery of pX330A-Cas9-gRNA vector into target cells .......................................................................... 60 2.8.4 Deletion detection by PCR ....................................................................................................................... 61 2.8.5 Rescue experiments ...............................................................................................................................