Identification and Validation of SOXB1 Bound Developmental Enhancers
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Identification and Validation of SOXB1 Bound Developmental Enhancers Ella Paulina Thomson B.Sc. (Biomedical Science), Honours (Genetics) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Department of Molecular and Cellular Biology School of Biological Sciences University of Adelaide, Australia July 2019 ii Table Of Contents Abstract vi Statement of Authorship viii Acknowledgements ix Chapter One 1 Introduction 1 1 Enhancers 2 1.1 Transcription Factor Binding 3 1.2 Models of Enhancer Activity 7 1.2.1 The Enhanceosome Model 8 1.3 Enhancer-Promoter Interactions 11 1.4 Super enhancers 15 1.5 Trans interactions 17 1.6 E-RNAs 20 1.7 Enhancer States & Their Chromatin Signatures 21 1.8 Identifying Enhancers 26 1.9 Enhancers and Disease 29 1.10 Validating Enhancers 34 2 SOX Proteins 43 2.1 The SOX Family 44 2.2 SOXB1 Subgroup 47 2.4 Functional Redundancy 59 2.5 DNA Binding Partners 60 2.6 Identification of SOXB1-bound enhancers 61 2.7 Project Aims 62 Chapter Two 63 The Nestin neural enhancer is essential for normal levels of endogenous Nestin in neuroprogenitors but is not required for embryo development 63 iii Summary 64 Abstract 68 Introduction 69 Materials and Methods 72 Mouse Generation 72 Tissue Preparation 73 Immunohistochemistry 73 In situ Hybridization 74 qRT-PCR 75 Results 77 Generating a Nes Enhancer Deletion Mouse Model 77 Nes mRNA expression is reduced in enhancer deletion mice 78 Protein Expression within Enhancer Deleted Mice 79 Ectopic Nestin expression in vasculature of enhancer deleted embryos 81 Nes is not required for CNS development 82 Trans Interactions of the Nes neural Enhancer 84 Discussion 85 References 97 Chapter Three 99 Identification and in vivo validation of a Frizzled 3 neuroprogenitor enhancer bound by SOXB1 proteins 99 Summary 100 Abstract 103 Introduction 104 Materials and Methods 106 Generation of Enhancer Deletion and Knockout Mouse 106 Tissue Preparation 107 qRT-PCR analysis 107 In situ hybridisation 108 LacZ Reporter Mouse Generation 109 Immunohistochemistry 110 Results 111 Generating a putative Fzd3 enhancer deletion mouse model 111 Fzd3 mRNA Expression is altered in Enhancer Deleted Mice 112 iv The -573bp region drives floor plate reporter expression in transgenic embryos 113 Fzd3 Knockout 114 Compound Het Morphology 115 Discussion 116 References 128 Chapter 4 130 Identification of SOX3 bound putative enhancers within the postnatal mouse testes using ChIP-Seq 130 Statement 131 Introduction 132 Materials and Methods 134 Results 136 Genomic location of SOX3 bound regions 136 GO Terms associated with peak regions 138 Conservation of peak regions 140 Identification of SOX Motifs 142 Identification of putative co-regulator proteins 144 SOX3 bound regions present in both NPCs and Testes 146 Motif identification in peaks present in both NPCs and Testes 149 Peaks only present within Testes 151 Discussion 156 Chapter 5 165 General Discussion and Future Directions 165 Nestin 166 Genome Editing to study Enhancers 177 SOX3 function within postnatal testes 180 Concluding Remarks 186 Appendix One 187 Appendix Two 203 Bibliography 265 v This page intentionally left blank. vi Abstract Enhancers are regions of non-coding DNA bound by transcription factors that influence gene expression, and are essential for the precise regulation of embryonic development. Many enhancers have been identified through reporter assays and bioinformatic techniques, but these are unable to show the functional contribution of the enhancer to endogenous expression. The SOXB1 proteins, expressed within the neural progenitor and spermatogonial stem cell populations are important TFs, the absence of which leads to developmental defects in both humans and mice. NES is an intermediate filament protein, and is thought to be regulated via a SOXB1 bound enhancer, commonly used in transgenic mice models to direct expression to NPCs. Through CRISPR mediated deletion of the Nes enhancer we show it is active from 9.5dpc in the CNS, and is responsible for up to 70% of endogenous Nes expression. Further, we identify possible trans activity of the enhancer, a new field of mammalian enhancer research. SOX3 ChIP-Seq identified a region upstream of the Wnt-receptor gene, Fzd3 that appeared to be an enhancer. Subsequent deletion via CRISPR confirmed enhancer activity in the CNS. Combined with LacZ reporter mouse models we show the enhancer directs expression specifically to the floor plate, an important region for axon guidance for which FZD3 is required. SOX3s role within the postnatal testes is largely unknown. Through ChIP-Seq we identified putative enhancers bound by SOX3, and present evidence that it is important in the regulation of the complex chromatin reorganisation that occurs during spermatogenesis. Together, this data presents new insights into the role of SOXB1s in development as well as highlighting the importance of functional validation of putative enhancers which can be achieved through CRISPR. vii Acknowledgements Firstly, I would like to thank Paul Thomas for being a great supervisor. I have thoroughly enjoyed my time in the lab, and your help and guidance over the years has been wonderful. The members of the Thomas lab and MLS made coming into the lab everyday so enjoyable. Thanks to Murray Whitelaw for being a great co-supervisor. James, thank you for always being there to work through any problem. Sandie, thank you for all the microinjection sessions and animal work help. Dale, thank you for answering my many questions and all your help with the ChIP. Adi and Chee, thank you for all your work on the SOX projects. To everyone at morning tea and afternoon drinks – James, Dale, Sandie, Mel, Dan, Louise, Emily, Chan, Connor, Stef, Ruby, Chee and Adi - thanks for all the chats, scientific and not! Mel, Ruby and Louise – I couldn’t have asked for better friends to share this experience with, I hope you know how much your friendship means to me. Deb and Dad, thank you for your unconditional love and support always. Hayden, thank you for absolutely everything– you’re the best. Mum, I hope you would have been proud. ix This page intentionally left blank. x Chapter One Introduction 1 1 Enhancers In order to tightly control gene expression throughout development, regulatory elements are essential. Previously only thought of as ‘junk DNA, the non-coding genome was thought to be unimportant when compared to the 2% of DNA that encoded proteins (Ohno 1972). Within the last few decades it has become apparent that the non-coding genome is home to many regulatory elements, such as silencers, non-coding RNA, and enhancers (ENCODE Project Consortium 2012). Enhancers are cis-regulatory elements comprised of regions of DNA, usually 50- 1500 base pairs (bp) in length, to which specific transcription factors (TF) bind, allowing interaction with the target gene promoter, influencing transcription to allow for both spatial and temporal control of gene expression. 2 1.1 Transcription Factor Binding Enhancers are comprised of varying numbers of transcription factor binding sites (TFBS), which are recognised by an individual TF or TF family. Highly specific TF binding allows enhancers to precisely control gene expression; for example a gene only required in the developing brain will not be activated by a brain specific TF in a developing limb (Shlyueva et al. 2014). Figure 1, (Krijger & de Laat 2016) demonstrates the various ways in which each enhancer can control target genes. There can either be many enhancers for a single tissue, which would allow for it to be activated (or repressed) at different timepoints, or various enhancers which will be activated by tissue-specific TFs for expression within multiple body regions. The exact mechanism of how TFs are able to interact with DNA to regulate expression is a complex process which is still not completely understood. Firstly, for a TF to recognise and bind a DNA motif, it must contain a DNA binding domain. There are many different protein binding DNA domains found across the transcription factor families; these include homeodomains (HD), helix-turn-helix (HTH) domains and high mobility group box (HMG) domains. The focus of this thesis, the SOX family of TFs contain the HMG binding domain, and this will be discussed in more detail in 2.1. 3 Figure 1 (Krijger & de Laat 2016) Various types of enhancers exist within the genome. A. A single gene may contain a single or many distinct enhancers to control expression within one tissue type or expression region. Or, as shown in (B) a gene may have multiple enhancers which are individually able to direct expression to target tissue, for example one enhancer may direct expression to a limb, while another may direct expression to the brain. 4 The DNA binding domain of each TF will recognise a specific DNA motif, usually a 8-20 bp region to which the TF directly binds (Halford & Marko 2004). Whilst these motifs can be found repeated throughout the genome, it has been estimated by the ENCODE consortium that the vast majority of DNA that matches these specific motifs will never be bound by a TF (Yesudhas et al. 2017). The environment in which these DNA motifs are within is crucial, as will be discussed in section 1.7 where the epigenetic modifications and chromatin accessibility is vitally important. There are three main mechanisms proposed for how a TF finds its correct motif; sliding, hopping, and intersegmental transfer. The model lac repressor TF is an example of a sliding mechanism. Molecular dynamics simulations have shown the TF ‘sliding’ along the DNA searching for the specific binding sites within the major grooves of DNA (Hammar et al. 2012). The hopping mechanism is where the protein ‘hops’ between DNA motifs, governed by electrostatic interactions. It is thought that while this is slower that siding, the binding affinity between the protein and DNA is stronger (Yesudhas et al.