Investigation and comparison of the regulation of ATOH1 in the mammalian and avian inner ear Miriam Gómez Dorado Submitted for the degree of PhD University College London 2017 Declaration “I, Miriam Gómez Dorado, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Where work has been conducted by other members of our laboratory, this has been indicated by their initials or an appropriate reference” SD = Dr. Sally Dawson JG = Dr. Jonathan Gale ND = Dr. Nicolas Daudet NH = Mrs. Naila Haq CG = Miss Ana Cláudia Gonçalves 1 Abstract Atonal homolog 1 (Atoh1) is a basic helix-loop-helix (bHLH) transcription factor required for the formation of sensory hair cells in the inner ear (Bermingham et al. 1999). Understanding the Atoh1 regulatory network is crucial for the development of new therapies to treat hearing loss. However, to date, little is known about the mechanisms controlling ATOH1 expression. The loss of sensory hair cells can cause deafness and balance disorders. In the mammalian inner ear, the loss of ATOH1 expression in adults has been linked with a limited capacity to regenerate hair cells (Wang et al. 2010). However, in non- mammalian vertebrates, ATOH1 expression re-activates spontaneously after hair cell damage and new hair cells can be formed throughout life (Cafaro et al. 2007) (Daudet et al. 2009). In this thesis, I aimed to identify regulatory elements responsible for ATOH1 expression through a comparative analysis of avian and mammalian ATOH1 gene loci. A bioinformatic approach was used to identify evolutionary conserved non-coding ATOH1 DNA sequences including those that are conserved between avian and mammals and those that are specific to both groups. Putative transcription factor binding sites predicted within these conserved elements were then investigated using EMSA analysis and reporter gene assays. These experiments suggest that the E2F1 transcription factor activates the chick ATOH1 gene and that this activation occurs predominantly via a direct interaction with a regulatory region that is conserved between avian species but absent from mammals. E2F transcription factors control cell cycle progression so the identification of this family as novel regulators of the chick ATOH1 expression links avian ATOH1 re-activation to cell proliferation. If confirmed, this would provide a possible mechanism to explain the different regenerative capabilities of mammalian and non-mammalian sensory cells and therefore contribute to the design of therapies for the regeneration of hair cells after damage. 2 Table of Contents 1 GENERAL INTRODUCTION 18 1.1 THE STUDY OF THE INNER EAR AND HEARING LOSS 18 1.2 GENERAL ANATOMY AND FUNCTION OF THE MAMMALIAN EAR 19 1.2.1 The outer ear 19 1.2.2 The middle ear 19 1.2.3 The inner ear 21 1.3 HAIR CELLS AND MECHANOELECTRICAL TRANSDUCTION 25 1.4 INNER EAR DEVELOPMENT 26 1.4.1 Early inner ear development 26 1.4.2 From the otic placode to the otocyst 28 1.4.3 Late morphogenesis and sensory development of the inner ear 31 1.4.4 Summary of inner ear development 32 1.5 DEVELOPMENT OF AUDITORY HAIR CELLS 33 1.5.1 Early cellular differentiation of the primordium cochlear epithelium 33 1.5.2 Specification of hair cells and supporting cells: Id proteins and Notch signalling 36 1.5.3 Hair cell differentiation 37 1.5.4 Maturation of the hair cells 41 1.6 ATOH1 41 1.6.1 Initial studies 42 1.6.2 Expression of Atoh1 in the developing inner ear 45 1.6.3 The role of Atoh1 in the inner ear 49 1.6.4 Atoh1 function in other tissues 50 1.7 TRANSCRIPTION FACTORS AND REGULATORY MECHANISMS OF GENE EXPRESSION 52 1.7.1 Transcriptional control of gene expression 54 1.7.2 Regulation of Atoh1 56 3 1.7.3 Transcription factors binding Atoh1 enhancers 59 1.7.4 Signalling pathways, ATOH1 expression and sensory cell fate in the inner ear 63 1.7.5 Can Atoh1 restore hearing? 64 1.8 NEW PERSPECTIVES AND TREATMENTS FOR HEARING LOSS 67 1.9 INVESTIGATION OF INNER EAR BIOLOGY 68 1.9.1 The challenge of studying the inner ear 68 1.9.2 The use of cell lines as a model to study gene regulation 69 1.10 PROJECT AIMS 70 2 MATERIAL AND METHODS 72 2.1 MATERIAL 72 2.1.1 General Equipment 72 2.1.2 Stock solutions 73 2.1.3 Safety 73 2.1.4 Primers 73 2.2 METHODS 74 2.2.1 DNA purification 74 2.2.2 Plasmid construction 78 2.2.3 Screening of bacterial recombinants 82 2.2.4 Sequencing of plasmid constructs 83 2.2.5 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 83 2.2.6 Cell culture 85 2.2.7 In vitro transcription and translation procedure 87 2.2.8 The electrophoretic mobility shift assay (EMSA) 87 2.2.9 Reporter Gene Assay 91 2.2.10 Quick Change Site-Directed Mutagenesis 99 2.2.11 Dissections of animal material 100 2.2.12 Tissue processing for cryo-sections 101 4 2.2.13 Immunohistochemistry 102 2.2.14 In ovo electroporation 104 2.2.15 In situ hybridization (ISH) 107 3 BIOINFORMATIC ANALYSIS 110 3.1 STRATEGY OF THE INVESTIGATION 110 3.2 IDENTIFICATION OF CONSERVED ELEMENTS BY COMPARATIVE GENOME ANALYSIS 111 3.2.1 Comparative alignment of the human and mouse Atoh1 locus 111 3.2.2 Comparative alignment of the chick and zebra finch Atoh1 locus 114 3.2.3 Multi-species alignment of the Atoh1 genomic sequence 117 3.3 IDENTIFICATION OF PUTATIVE TRANSCRIPTION BINDING SITES WITHIN ECRS 120 3.3.1 Predictions within Atoh1 Enhancer A and Enhancer B 120 3.3.2 Predictions within putative Enhancer C 125 3.3.3 Predictions within other evolutionary conserved regions 125 3.4 REFINEMENT OF PREDICTIONS 128 3.5 PRIORITIZATION OF YY1, NFKB, E2F AND ATOH1 131 3.5.1 YY1 131 3.5.2 NF-κB 132 3.5.3 E2F 133 3.5.4 ATOH1 133 3.6 BINDING PROFILES FOR YY1, NF-ΚB AND ATOH1 TRANSCRIPTION FACTORS 134 3.7 BINDING PROFILES FOR THE E2F TRANSCRIPTION FACTOR FAMILY 137 3.8 DISCUSSION 142 5 4 INVESTIGATION OF PUTATIVE CANDIDATE REGULATORS OF ATOH1 144 4.1 EMSA ANALYSIS OF CANDIDATE BINDING SITES 144 4.2 TRANSCRIPTIONAL ACTIVATION OF ATOH1 CONSERVED REGIONS BY CANDIDATE REGULATORS 149 4.2.1 Cloning of the mouse and chick Atoh1 conserved elements 149 4.2.2 Effect of YY1 on the Atoh1 conserved non-coding elements 150 4.2.3 Effect of NF-κB on the Atoh1 conserved non-coding elements 152 4.2.4 Effect of ATOH1 on the Atoh1 conserved non-coding elements 154 4.2.5 Investigation of the effect of E47, the ATOH1 co-factor 159 4.3 DISCUSSION 163 5 INVESTIGATION OF THE E2F TRANSCRIPTION FACTOR FAMILY AS A PUTATIVE CANDIDATE REGULATOR OF ATOH1 168 5.1 INTRODUCTION TO THE E2F FAMILY 168 5.1.1 Initial studies 168 5.1.2 E2F family members 169 5.1.3 E2F co-factors: The DP family 170 5.1.4 E2F functions 171 5.1.5 E2F and the inner ear 174 5.1.6 The E2F family and the study of Atoh1 regulation 176 5.2 REGULATION OF THE MAMMALIAN AND AVIAN ATOH1 CONSERVED REGIONS BY E2F1 177 5.3 EMSA ANALYSIS OF THE PREDICTED E2F BINDING SITES IN THE CHICK ATOH1 CONSERVED REGIONS. 181 5.3.1 Analysis of E2F1 expression in UB/OC2 cells 181 5.3.2 Binding of E2F in EMSA experiments 185 5.3.3 Characterization of binding affinities of the predicted E2F sites in the Atoh1 regulatory elements 189 6 5.3.4 Further analysis of Atoh1 probes 2 and 6 as putative E2F binding sites 193 5.4 SITE DIRECTED MUTAGENESIS OF SITES 2 AND 6 WITHIN THE AVIAN ATOH1 CONSERVED REGIONS 199 5.4.1 Design of mutagenesis sites 199 5.4.2 Site-directed mutagenesis of sites 2 and 6 201 5.5 RESPONSE OF THE ATOH1 CONSERVED REGIONS TO E2F1 WHEN PUTATIVE E2F SITES ARE MUTATED 203 5.6 TRANSCRIPTIONAL REGULATION OF THE ATOH1 CONSERVED REGIONS BY OTHER E2F TRANSCRIPTION FACTORS 205 5.7 CHARACTERIZATION OF E2F1 EXPRESSION IN THE MAMMALIAN AND AVIAN DEVELOPING INNER EAR EPITHELIUM 209 5.7.1 E2F1 expression in the developing chick inner ear epithelium 210 5.7.2 E2F1 expression in the developing mouse inner ear epithelium 216 5.8 SUMMARY AND DISCUSSION 221 6 IN VIVO INVESTIGATION OF THE ROLE OF PUTATIVE ENHANCER C AND E2F1 ON THE REGULATION OF CHICK ATOH1 228 6.1 INTRODUCTION TO THE TOL2 SYSTEM 228 6.2 IN VIVO CHARACTERIZATION OF THE CHICK ATOH1 CONSERVED REGIONS BY THE USE OF THE TOL2 SYSTEM. 230 6.2.1 Effect of the relative position of the Atoh1 conserved regions on the Tol2 reporter activity 238 6.3 IN VIVO EFFECT OF E2F1 IN THE CHICK INNER EAR AT EARLY STAGES 242 6.4 SUMMARY AND DISCUSSION 246 7 GENERAL DISCUSSION 250 8 CONCLUSIONS 259 APPENDIX 261 BIBLIOGRAPHY 266 7 List of Figures Figure 1.1. Anatomy of the mammalian ear. ...........................................................................20 Figure 1.2. General anatomy of the cochlea. ...........................................................................23 Figure 1.3. Anatomy of the mammalian vestibular system ....................................................24 Figure 1.4. Development of the inner ear .................................................................................27 Figure 1.5. Morphogenesis of the inner ear. ............................................................................29 Figure 1.6. Early cellular differentiation of the primordium cochlear epithelium. .............35 Figure 1.7. The Notch signalling pathway ...............................................................................37 Figure 1.8.