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Molecular Analysis of the ZIC3 and TCF7L1 Interaction and Its Implication in Human Heterotaxy

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Molecular Analysis of the ZIC3 and TCF7L1 Interaction and Its Implication in Human Heterotaxy Molecular analysis of the ZIC3 and TCF7L1 interaction and its implication in human Heterotaxy Koula Esta Maria Diamand A thesis submitted for the degree of Doctor of Philosophy of the Australian National University August 2019 © Copyright by Koula Esta Maria Diamand, 2019 All Rights Reserved ii Except where otherwise acknowledged, the work presented in this thesis is my own. Koula Diamand iii Publications contributed to through the course of this thesis: Diamand K.E.M., Barratt K.S., Arkell R.M. (2018) Overview of Rodent Zic Genes. In: Aruga J. (eds) Zic family. Advances in Experimental Medicine and Biology, vol 1046. Springer, Singapore Barratt, K.S., Diamand, K. E. M. & Arkell, R.M (2018) Identification of reference genes suitable for RT-qPCR studies of murine gastrulation and patterning. Mammalian Genome. 29(9-10):656-662 Publication in preparation: Jehangir N. Ahmed, Koula E. M. Diamand and Ruth M. Arkell. Systematized reporter assays reveal ZIC protein regulatory abilities are subclass-specific and dependent upon transcription factor binding site context iv Acknowledgments If I could, my acknowledgements would be short and simple. Something along the lines of: ‘Thank you – you know who you are.’ Vague, to the point, and no one should feel left out. This PhD, however, has been anything but ‘short and simple’ and so I must express my gratitude to those who have helped me get through this milestone. First and foremost, I would like to offer my special thanks to my supervisor, Ruth Arkell, for the opportunity to conduct my PhD in your research group. I have learnt a great deal from you and you have helped me develop essential research, presentation and communication skills. Thank you for your supervision, encouragement and thesis writing advice. To all members of the Arkell laboratory, both past and present (Kristen, Alaa, Jerry, Hannah, Helen, Kathryn, Kelsey, Kyle and the adopted member, Lora), who all have at some point provided me with either support, reassurance, inspiration or motivation, particularly in these last few months. Thank you for all the coffee breaks, the ridiculous conversations, heated discussions trying to answer the real philosophical questions of the world (such as, at what point is a fruit salad not a fruit salad - a question with many differing opinions) and life advice -maybe one day I will open a bakery. I also want to give special thanks to Kristen, Kyle and Alaa for proof reading my thesis and Hannah for your infectious optimism. To Kristen, I give you a poem for you as you did for me, to summarise us as acquaintances, as co- workers and, yes, even friends. May this become a respected and honourable Arkell laboratory tradition that I will in no way encourage or enforce. I need you to know just in case, Please add this to your fun fact knowledge base, Uno a sprinkle is light, Not drizzle, don’t fight Now how about a game of fruit-face? I also wish to acknowledge the help, guidance and lab reagents provided by Nicole Thompson and the Fisher laboratory at JCSMR. Without your assistance I would not have been able to set up or conduct my yeast two-hybrid experiments. Thanks also must be given to the Australian Government for their support through the Australian Postgraduate Award. v Last but not the least, I would like to thank my family. To my parents, words cannot describe how thankful I am for your unconditional love and support. I could not have come this far in life without you both and I am grateful for everything you have done in making me who I am today. To my brothers, who never quite understood why I attempted a PhD, what I even do at university, and how I even managed to finish this thesis. Although you may not be aware, you remind me of all the positive opportunities life will present. Happy reading. vi Abstract Heterotaxy is a congenital disorder that occurs due to the defective establishment of the left-right (L- R) body axis during early embryo development. The disorder affects 1/10,000 live births, results in the abnormal alignment of internal organs with respect to the L-R axis, and is often accompanied by severe cardiac malformations. The most commonly mutated gene in human cases of Heterotaxy is the X- linked ZIC3, which was first associated with the condition in 1997. ZIC3 belongs to a family of multifunctional transcriptional regulator proteins (ZIC family) all characterised by a C2H2 type zinc finger domain (ZFD). This domain is implicated in the transactivation (DNA binding) and co-factor (protein binding) function of the proteins. However, despite both capabilities, it is still not known when during development the co-factor role of ZIC3 is necessary over the transcription factor role. Consequently, the exact cellular and molecular causes underlying ZIC3-associated Heterotaxy remain unknown. Mouse models of Zic3 loss-of-function reveal phenotypes that resemble human Heterotaxy and exhibit a defective node (the structure that confers the left-right axis during development). More recently, studies have shown murine Zic3 alleles lead to incompletely penetrant, partial (posterior) axis duplications and anterior truncation, phenotypes characteristic of elevated canonical Wnt signalling. The expression pattern of Zic3 during mammalian gastrulation overlaps with a known repressor of the canonical Wnt pathway, Tcf7l1 and it has also been shown that a related ZIC protein (ZIC2) can interact with TCF proteins to co-repress Wnt/β-catenin mediated transcription when overexpressed in cell lines. Overall, this leads to the novel hypothesis that ZIC3 controls the establishment of the L-R axis by interacting with TCF7L1 to repress Wnt signalling during mammalian gastrulation. In this thesis I examine the molecular role of ZIC3 in the Wnt pathway and investigate the functional consequences of ZIC3 Heterotaxy-associated protein variants. A physical interaction between ZIC3 and TCF7L1 was demonstrated by several molecular assays. Additionally, 17 Heterotaxy-associated human ZIC3 variants were functionally assessed in well- established cell-based assays. Four of the human protein variants exhibit non-pathogenic properties as they retain wildtype ZIC3 function in these assays. The remaining variants, which affect key residues within the ZFD or contain a premature termination codon, display impaired transcription factor and co-factor functions: they can no longer activate transcription at a ZIC responsive element and have reduced ability to inhibit β-catenin-mediated Wnt transcription. Loss of Wnt inhibition did not always correlate with loss of TCF7L1 interaction, suggesting ZIC3 and/or TCF7L1 must interact with additional co-repressors or repressive chromatin complexes to mediate transcriptional repression in the Wnt pathway. Additionally, in this thesis I identified several evolutionary conserved domains implicated in vii transactivation, including the newly annotated SANC and SACC domains, enhancing our understanding of the structural domains required for ZIC3 molecular function. Overall, this thesis supports a new model of ZIC3 molecular function: ZIC3 interacts with TCF7L1 to inhibit canonical Wnt signalling during mammalian gastrulation and correctly establish the L-R axis. Ultimately, ZIC3-associated Heterotaxy arises when ZIC3 is unable to function as a Wnt co-repressor. viii Table of Contents Acknowledgments ............................................................................................................ v Abstract ......................................................................................................................... vii Table of Contents ............................................................................................................ ix List of Figures ................................................................................................................ xiii List of Tables .................................................................................................................. xv Abbreviations .................................................................................................................xvi Chapter 1: Introduction .................................................................................................. 20 1.1 Heterotaxy ....................................................................................................................... 20 1.2 Development of the Left-Right axis ................................................................................... 22 1.3 ZIC3 and human Heterotaxy .............................................................................................. 25 1.4 ZIC3 is associated with Heterotaxy in the mouse ............................................................... 27 1.5 The ZIC family of transcription factors ............................................................................... 29 1.5.1 Genome organisation........................................................................................................... 29 1.5.2 Zic Pleiotropy ....................................................................................................................... 31 1.5.3 Expression during early embryo development .................................................................... 35 1.5.4 ZIC molecular functions ....................................................................................................... 38 1.6 The Wnt signalling pathway .............................................................................................. 47 1.7 Canonical
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