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Uncovering Transcriptional Cis- Regulatory Elements Active in Adult Zebrafish Exocrine Pancreas

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Uncovering Transcriptional Cis- Regulatory Elements Active in Adult Zebrafish Exocrine Pancreas Uncovering transcriptional cis- regulatory elements active in adult zebrafish exocrine pancreas Diogo José Coutinho Ribeiro Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências da Universidade do Porto e Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto 2018 Orientador Dr. José Bessa Principal Investigator, Instituto de Biologia Celular e Molecular / Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Coorientadores Dra. Renata Bordeira-Carriço Post-Doc Researcher, Instituto de Biologia Celular e Molecular / Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Prof. Dr. Pedro Rodrigues 1 Associate Professor, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Portugal Todas as correções determinadas Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 680156 - ZPR). Aos meus pais, Ao meu irmão, À memória da minha avó. FCUP/ICBAS Uncovering transcriptional cis-regulatory elements active in adult zebrafish exocrine pancreas Acknowledgments Ao meu Orientador Doutor José Carlos Ribeiro Bessa que me deu a oportunidade de participar neste projeto e, sem a liderança do mesmo, nada do que se segue no presente trabalho teria sido alcançável. Os meus agradecimentos recaem na sua incondicional disponibilidade e apoio em todos os aspetos e momentos, quer a nível profissional, quer pessoal. Por todas as sugestões, motivação, incentivo que se afiguraram fundamentais para o sucesso deste trabalho, bem assim por todo o seu conhecimento inigualável. Não é, de todo, possível imaginar um tão fantástico líder nesta fase crucial do meu percurso profissional. Para além de uma relação profissional de enorme relevo que foi estabelecida ao longo do presente ano, guardo igualmente com estima uma relação de amizade que nunca perderei. Por tudo isto, um inexprimível Obrigado. À minha coorientadora Doutora Renata Bordeira-Carriço que foi a minha companheira desde o primeiro dia ao último e que desempenhou um papel fundamental e extraordinário ao longo desta fase. Grande parte do que sei hoje se deve ao conhecimento que por si me foi transmitido e que levarei comigo no meu futuro caminho profissional. Agradeço, ademais, todas as palavras e momentos de amizade que guardarei com carinho. Ao Professor Pedro Rodrigues, um especial agradecimento pela disponibilidade, que constituiu uma peça fundamental para a realização deste trabalho. Aos restantes membros do grupo VDR e colaboradores, que constituíram uma peça basilar para a minha formação, um enorme Obrigado, por me permitirem a oportunidade de aprender com os melhores, pela partilha de conhecimento e entreajuda. Um grande abraço para todos com carinho e amizade ao João, Marta, Fábio, Ana G, Ana E, Leonor, Joana T, Joana M, Filipa, Mafalda e Isabel, a quem devo um enorme obrigado. Finalmente, àqueles a quem um obrigado nunca será suficiente: aos meus pais e ao meu irmão que foram, são e sempre serão o meu suporte na vida e que estiveram incondicionalmente ao meu lado. Aos meus amigos do coração que são uma família e a quem nunca terei oportunidade de lhes agradecer o suficiente. À minha namorada, cujas palavras de carinho, amor, paciência e apoio, que guardarei para a vida no meu coração, foram determinantes. À memória da minha avó que faleceu o ano passado vítima de cancro do pâncreas e de quem guardo as palavras de força, recordando-as diariamente e para o resto dos meus dias. Um perpétuo obrigado pela inspiração. 7 FCUP/ICBAS Uncovering transcriptional cis-regulatory elements active in adult zebrafish exocrine pancreas 8 FCUP/ICBAS Uncovering transcriptional cis-regulatory elements active in adult zebrafish exocrine pancreas Table of contents Abstract ............................................................................................................ 11 Resumo ............................................................................................................ 13 List of figures .................................................................................................... 15 List of tables ..................................................................................................... 17 List of abbreviations ......................................................................................... 18 Introduction....................................................................................................... 20 1. Pancreas ...................................................................................................... 20 1.1 Early pancreas development and morphology……………………………..20 1.2. Danio rerio as a model system to study pancreas development and function ......................................................................................................... 22 1.2.1. Zebrafish development .................................................................... 22 1.2.2. The zebrafish pancreas ................................................................... 24 1.3. Pancreatic diseases ............................................................................... 26 2. Gene regulation: general aspects ................................................................. 27 2.1. The role of cis-regulatory elements in the genome ................................ 28 2.2. Genome-wide techniques to predict cis-regulatory elements ................. 29 2.2.1. Chromatin epigenetic marks for enhancers and promoters ............. 31 2.2.2. Accessing open chromatin regions using ATAC-seq to find cis- regulatory elements ................................................................................... 34 2.2.3. Contemporary tools to identify chromatin interactions genome-wide 35 2.3. Genetic tools to study cis-regulatory elements in vivo ............................ 36 2.3.1. Enhancer assays to test for CREs activity ....................................... 37 2.3.2. Genome editing as a tool for loss of function assays ....................... 39 3. Objectives ..................................................................................................... 41 4. Material and Methods ................................................................................... 42 4.1. Zebrafish stocks, husbandry, breeding and embryo culture ................... 42 4.2. DNA extraction from zebrafish embryos ................................................. 42 4.3. Generation of the plasmids carrying the selected putative enhancer sequences ..................................................................................................... 43 4.3.1. PCR amplification of the selected sequences .................................. 43 4.3.2. Cloning of fragments into pCR®8/GW/TOPO vector ........................ 45 4.3.3. Recombination of the selected sequences into Z48 and ZED vectors ................................................................................................................... 46 4.3.4. Purification of the plasmids for injection ........................................... 48 9 FCUP/ICBAS Uncovering transcriptional cis-regulatory elements active in adult zebrafish exocrine pancreas 4.4. Generation of transgenic zebrafish ........................................................ 49 4.4.1. RNA synthesis ................................................................................. 49 4.4.2. Purification of the synthesized mRNA .............................................. 49 4.4.3. Injection of the plasmids and mRNA into zebrafish embryos ........... 50 4.5. Screening for enhancer activity .............................................................. 50 4.6. Mutagenesis using the CRISPR-Cas9 system ....................................... 51 4.6.1. Generation of Cas9 mRNA and sgRNAs ......................................... 51 4.6.2. Injection of sgRNAs into zebrafish embryos .................................... 52 4.7. Real time quantitative PCR .................................................................... 54 4.8. Statistical Analysis ................................................................................. 55 5. Results ......................................................................................................... 56 5.1. Analysis of enhancer activity of predicted cis-regulatory elements in pancreas ....................................................................................................... 56 5.2. Comparative analysis of the distal enhancers of ptf1a in human and zebrafish ...................................................................................................... 67 5.2.1. Cell-specific expression pattern of hPtf1aE and ptf1aE3 ................. 68 5.2.2. Deletion of ptf1aE3 in zebrafish ....................................................... 71 5.3. Analysis of the impact of hnf4a knockdown ........................................... 73 6. Discussion .................................................................................................... 76 7. Conclusions .................................................................................................. 82 References ....................................................................................................... 83 10 FCUP/ICBAS Uncovering transcriptional cis-regulatory elements active in adult zebrafish
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