Exploring the bZIP regulatory network in the sponge Amphimedon queenslandica: Insights into the ancestral animal regulatory genome Katia Jindrich B.Sc, M.Sc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Biological Sciences I Abstract What genomic innovations supported the emergence of multicellular animals, over 600 million years ago, remains one of the most fundamental questions in evolutionary biology. Increasing evidence suggests that the elaboration of the regulatory mechanisms controlling gene expression, rather than gene innovation, underlies this transition. Since transcriptional regulation is largely achieved through the binding of specific transcription factors to specific cis-regulatory DNA, understanding the early evolution of these master orchestrators is key to retracing the origin of animals (metazoans). Basic leucine zipper (bZIP) transcription factors constitute one of the most ancient and conserved families of transcriptional regulators. They play a pivotal role in multiple pathways that regulate cell decisions and behaviours in all kingdoms of life. Here, I explore the early evolution and putative roles of bZIPs in a representative of one of the oldest surviving animal phyla, the marine sponge Amphimedon queenslandica. Using recently sequenced genomes from across the Eukaryota, and in particular the Metazoa, I first reconstruct the evolution of bZIPs, and document that this transcription factor superfamily has undergone multiple independent kingdom-level expansions. I present here a thorough analysis of the whole superfamily of bZIP transcription factors across the main eukaryotic clades and the putative bZIP network that was in place in the ancestor of all extant animals. To then explore the putative role of bZIP transcription factors in the metazoan ancestor, I identify the bZIP complement in the demosponge Amphimedon queenslandica (phylum Porifera). As expected for regulatory molecules, a majority of the 17 A. queenslandica bZIPs display high temporal specificity, cell-type specific localisation and are dynamically expressed throughout development. Along with bZIPs high expression across developmental stages, these characteristics point towards bZIPs having a fundamental role in this sponge. Given the consistent central role of these transcription factors in the functioning of extant animals, I infer that were already integrated in developmental and cell specific regulatory networks in the ancestral regulatory genome and supported the emergence of multicellularity. The PAR and ATF4 orthologues particularly stand out: they are highly expressed throughout Amphimedon life cycle and peak in expression at key developmental transitions. A. queenslandica PAR-bZIP AqPARa is part of the sponge circadian network. In this thesis, I establish that AqPARa is both spatially and temporally co-expressed with A. queenslandica cryptochrome AqCRY2, and that expression of both genes is entrained by light. I also explore the involvement of circadian genes that are conserved in other phyla in A. queenslandica response to light. This work provides insights II on the animal ancestral circadian regulatory network, and the evolution of PAR-bZIPs implication in the regulation of environmentally cued behaviours. A. queenslandica ATF4 orthologue, AqATF4, is highly expressed in larval archeocytes. Using chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) of AqATF4 and five histone H3 post-translational modifications (H3K4me3, H3K4me1, H3K27ac, H3K36me3 and H3K27me3), I explore AqATF4 putative target genes and binding sites in A. queenslandica larva, and the complexity of A. queenslandica regulatory landscape. I conclude that many of the roles bZIPs play in bilaterians have a more ancient origin, and were present in the last common ancestor of all contemporary animals. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. IV Publications during candidature Peer-reviewed papers Jindrich, K. and Degnan, B.M. 2016. The diversification of the basic leucine zipper family in eukaryotes correlates with the evolution of multicellularity. BMC Evolutionary Biology 16: 28. Conference abstracts Jindrich K, Roper KE, Lemon S, Degnan S and Degnan BM. (2016). Functional diversity of bZIP transcription factors in the sponge Amphimedon queenslandica: insights into the ancestral animal regulatory genome (Oral presentation). The European Society of Evolutionary Developmental Biology, Sixth Meeting, Uppsala, Sweden. Jindrich K, Roper KE, Lemon S, Degnan BM and Degnan S. (2016). An ancient role for bZIP trancsrition factors? PAR-bZIPs and the sponge circadian clock (Poster presentation). Society for Molecular Biology and Evolution 2016, Gold Coast, Australia. Jindrich K, Roper KE, Lemon S, Reitzel AM, Degnan BM and Degnan S. (2015). Rhythmic gene expression in the demosponge Amphimedon queenslandica: how old is the animal circadian clock? (Oral presentation). 2015 EMBL Australia PhD Symposium, Melbourne, Australia V Publications included in this thesis Jindrich, K. and Degnan, B.M. 2016. The diversification of the basic leucine zipper family in eukaryotes correlates with the evolution of multicellularity. BMC Evolutionary Biology 16: 28. This work was incorporated as Chapter 2. Contributor Statement of contribution Katia Jindrich (Candidate) Designed study (80%) Conducted analysis (100%) Wrote and edited the paper (75%) Bernard Degnan Designed study (20%) Wrote and edited paper (25%) VI Contributions by others to the thesis Bernard M. Degnan contributed to the conception and design of this research, advised on methods and analyses, and provided critical comments on the thesis and all the associated publications. Milos Tanurdzic advised on methods and analyses in Chapter 3 and 5. Sandie Degnan contributed to the conception and design of Chapter 5 and provided critical comments on the associated manuscript. Sussan Lemon contributed with adult sample collection in Chapter 4. Kerry E. Roper contributed with qPCR analyses on adult samples and contributed to experiment design in Chapter 4. Selene L. Fernandez-Valverde produced several of the datasets analysed across this thesis (these contributions are specifically acknowledged in the relevant chapters). Federico Gaiti advised on methods and analyses in Chapter 5, and provided critical comments on the associated manuscript. Chromatin immunoprecipitation sequencing was conducted by Central Analytical Research Facility (CARF), Australia. Statement of parts of the thesis submitted to qualify for the award of another degree None. VII Acknowledgements I would like to start by thanking my remarkable and tireless supervisor, Bernie Degnan. There are quite a lot of things I would like to thank you for, so I’ll try and stay “focused and concise”, as you so relentlessly taught me. Thank you for your tireless enthusiasm, your guidance through this project and meeting my natural scepticism with a never-ending supply of optimism. I am deeply grateful for all the opportunities and independence you have given me to learn, and grow as a scientist. The passion you have for science and teaching is truly inspiring. And last but not least, thank you for the understanding and compassion you have never failed to show when I needed. All my thanks to my co-supervisor, Milos (I won’t write your Surname, since I’m bound to mess up the accents!). Thanks for making time for me, often on short notice, and trying to translate my nonsense into questions you could actually answer. And for your “You won’t know until you do it” attitude that helped me move forward more than once. I also wish to thank Sandie Degnan, not only for her contribution to this thesis, but for her infinite kindness and enthusiasm. You’ve always had a way of asking just the right question to make everything fall in place in my mind. On a more personal note, your genuine –and quite motherly, if I may say- interest in the details of our lives greatly contributes to that warm and welcoming atmosphere