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100002369.Pdf “The Rbfox1 gene: expression analysis and study of the transcrip onal regula on” Disserta on zur Erlangung des Grades “Doktor der Naturwissenscha en” Am Fachbereich Biologie der Johannes Gutenberg-Universität Mainz Sònia Casanovas Palau geb. am 13.01.1982 in Reus-SPAIN Mainz 2018 Summary Summary The RBFOX1 gene, which is located on chromosome 16p13.2, encodes an RNA-binding protein that regulates pre-mRNA splicing events in specific cell types including neurons. In the mouse, the gene contains a large noncoding part in its 5' end with at least four alternative promoters, promoters 1B, 1C, 1D and 1E. Promoters 1B, 1C and 1D are brain specific and promoter 1E is muscle specific. The promoters drive the expression of the alternative Rbfox1 transcript isoforms, which differ in their 5'UTR but not in their coding exons. Copy number variants (CNVs) in the human RBFOX1 gene that are located in the 5'-noncoding part of the gene and that typically only affect some but not all RBFOX1 transcript isoforms, have been associated with a range of neurodevelopmental disorders such as autism spectrum disorder, intellectual disability, epilepsy, attention deficit hyperactivity disorder and schizophrenia. In this thesis, I analyzed the expression of the three brain-specific Rbfox1 transcript isoforms controlled by the promoters 1B, 1C and 1D during embryonic development of the cerebral cortex as well as in various brain regions of the juvenile mouse. Furthermore, through in silico analysis and luciferase assays, I characterized the alternative Rbfox1 promoters and demonstrated that the expression of Rbfox1 in primary cortical neurons is driven by promoters 1B and 1C. I identified three transcription factors (C-MYC, NEUROD2 and TCF4) that regulate the expression of Rbfox1 in cortical neurons. Using transcript-specific studies, I was able to show that TCF4 regulates the expression of transcripts 1B and 1C, whereas NEUROD2 only controls the expression of transcript 1B. -i- Zusammenfassung Zusammenfassung Das RBFOX1-Gen, das auf Chromosom 16p13.2 lokalisiert ist, kodiert für ein RNA-bindendes Protein, das Prä-mRNA-Spleißereignisse in bestimmten Zelltypen einschließlich Neuronen reguliert. In der Maus enthält das Gen einen großen nichtcodierenden Teil in seinem 5'-Ende mit mindestens vier alternativen Promotoren, den Promotoren 1B, 1C, 1D und 1E. Die Promotoren 1B-1D sind hirnspezifisch und der Promotor 1E ist muskelspezifisch. Die Promotoren steuern die Expression der alternativen Rbfox1-Transkript-Isoformen, die sich lediglich in ihren 5'UTR-Exons unterscheiden. Kopienzahlvarianten (CNVs) im humanen RBFOX1-Gen, die sich im 5'- nichtcodierenden Teil des Gens befinden und typischerweise nur einige, jedoch nicht alle RBFOX1-Transkript-Isoformen betreffen, sind mit einer Reihe von neurologischen Entwicklungsstörungen wie Autismus Spektrum Störung, geistige Behinderung, Epilepsie, Aufmerksamkeitsdefizit-Hyperaktivitätsstörung und Schizophrenie assoziiert. In dieser Arbeit analysierte ich die Expression der drei durch die Promotoren 1B, 1C und 1D gesteuerten Gehirn-spezifischen Rbfox1-Transkript-Isoformen während der Embryonalentwicklung des zerebralen Kortex der Maus sowie in verschiedenen Hirnregionen der juvenilen Maus. Darüber hinaus charakterisierte ich die alternativen Rbfox1-Promotoren und zeigte, dass die Expression von Rbfox1 in primären cortikalen Neuronen von den Promotoren 1B und 1C gesteuert wird. Ich identifizierte drei Transkriptionsfaktoren (C-MYC, NEUROD2 und TCF4), die die Expression von Rbfox1 in kortikalen Neuronen regulieren. Mittels transkriptspezifischer Untersuchungen konnte ich zeigen, dass TCF4 die Expression der Transkripte 1B und 1C reguliert, wohingegen NEUROD2 lediglich die Expression von Transkript 1B steuert. -ii- Abbreviations Abbreviations aCGH Array comparative EtBr Ethidium Bromide genomic hybridization Anti-DIG- Anti-digoxigenin EtOH Ethanol AP antibody ASD Autism Spectrum et al et alii Disorder ATP Adenosine triphosphate FBS Fetal bovine serum bHLH Helix-Loop-Helix FISH Fluorescence in situ hybridization bp Base pairs Fox gene Feminizing on X gene BS Binding site Gapdh Glyceraldehyde 3-phosphate dehydrogenas C-Box GWAS Genome-wide association studies cDNA Complementary DNA ISH In situ hybridization c-MYC Mouse IPCs Intermediate progenitor cells myelocytomatosis oncogene CNS Central nervous system KLF7 Krüppel Like Factor 7 CNV Copy number variants Kd Knockdown CP Cortical plate KO Knockout cRNA Complementary RNA LASR Large assembly of splicing regulators dH2O Distilled water LB lysogeny broth DIF Downstream intronic MZ Marginal zone flanking DIG Digoxigenin MAX MYC Associated Factor X DIV Days in vitro μl Microliters DMEM Dulbecco's Modified ml milliliters Eagle Medium miR302 MicroRNA 302 dNTP deoxynucleotide miR-980 Micro-RNA 980 triphosphate DPBS Dulbecco's phosphate mRNA Messenger Ribonucleic Acid buffered saline E Embryonic day mut Mutated E-box Enhancer box E. coli Escherichia coli MYCN v-myc myelocytomatosis viral related oncogene, neuroblastoma derived EST Expressed sequence tag NB Neuroblastoma -iii- Abbreviations NEUROD2 Neurogenic siRNAs Small interfering RNAs differentiation factor 2 NLS Nuclear localization SNPs Single-nucleotide polymorphism signal NMR Nuclear magnetic SNV Single-nucleotide variant resonance NPC Neural precursor cell NTC No template control SSC The Simons Simplex Collection P Post-natal day SVZ Subventricular zone PCR polymerase chain SP Subplate reaction PFA Paraformaldehyde PP Pre-plate TCF4 Transcription factor 4 Rbfox1 RNA-Binding Fox1 UIF Upstream intronic flanking RGCs Radial glial cells UPL Universal Probe Library RNA Ribonucleic Acid UTR Untranslated region rpm Revolutions per minute VZ Ventricular zone RRM RNA recognition motif ºC Celsius RT Room temperature % Percent RT-qPCR Real Time Quantitative PCR -iv- Contents Contents Summary......................................................................................................................................... i Abbreviations................................................................................................................................ iii 1 Introduction 1 1.1 Autism Spectrum Disorder (ASD)................................................................................. 1 1.2 The Rbfox family proteins……………………………………….………..………………………………..……. 7 1.2.1 Rbfox1 ….................................................................................................................. 11 1.2.1.1 The link between RBFOX1 and ASD………………………………………………….…... 12 1.2.1.2 The Rbfox1 gene ………………………….……………………….……………..……………....15 1.2.1.3 Regulation of Rbfox1 expression..……………….……………………..……………..…..17 1.2.1.4 RBFOX1 transcript isoforms…….……………………………….………………………..….18 1.2.1.5 The RBFOX1 protein……………………………………………….………..……………..…….20 1.2.1.5.1 Splicing functions of the RBFOX1 protein……………………….……..……21 1.2.1.5.2 Non-splicing functions of the RBFOX1 protein …….……………..….…..23 1.3 Candidate transcription factors………………………………………......................................... 25 1.3.1 The basic Helix-Loop-Helix (bHLH) protein family…………………………..……………………..25 1.3.2 c-MYC (Mouse myelocytomatosis oncogene)....………………………………….…..…………..27 1.3.3 MAX (MYC Associated Factor X)………………………………………………………………..…………28 1.3.4 MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived)...………………………………………………………………………………..…..…………….………………..28 1.3.5 NEUROD2 (Neuronal differentiation 2)……………………………………………………..….……...29 1.3.6 KLF7 (Krüppel Like Factor 7)………………………………………..………………….…………………...30 1.3.7 TCF4 (Transcription factor 4)………………………………………………………………………………..31 1.4 Purpose of the Thesis ................................................................................................34 2 Materials and Methods 35 2.1 Materials.................................................................................................................. 35 2.1.1 Reagents………………………......................................................................................... 35 2.1.2 Cell culture……………………………................................................................................ 38 Contents 2.1.3 Solutions and buffers …..…………………………………………………………..………………..….….. 39 2.1.4 Kits…………................................................................................................................ 46 2.1.5 Primers……………………………...................................................................................... 47 2.1.6 Vectors..................................................................................................................... 51 2.1.7 Bacteria.............................................................................................................….... 51 2.1.8 Enzymes…………………………………………….………………………………………………………..…..… 52 2.1.9 Cells……………………………….…………………………………..………………………………………..…….. 53 2.1.10 Small interfering RNA……………….…………………………………………..………….………….…….53 2.1.11 Mice…………………………….………………………………………………………………….…………….…. 54 2.1.12 Consumables......................................................................................................... 54 2.1.13 Laboratory equipment........................................................................................... 55 2.1.14 Software and Databases........................................................................................ 57 2.1.14.1 Programs…………………………………………………………..…………………….…………. 57 2.1.14.2 Online Tools……………………….…………….………………………..………………………. 58 2.2 Methods..................................................................................................................
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