Candidate Enhancer and Transcription Dynamics in Early Xenopus tropicalis Development Rosa Gomes Faria University College London and The Francis Crick Institute MRC – National Institute for Medical Research A thesis submitted for the degree of Doctor of Philosophy University College London December 2017 Declaration I, Rosa Gomes Faria, 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. _________________________________ Rosa Gomes Faria 2 Abstract A previous study from the Gilchrist lab measured transcription in the Xenopus tropicalis embryo for the first 66 hours of development at a high temporal resolution. This data showed that early transcription in Xenopus tropicalis is a very dynamic process, indicating that the mechanisms regulating it should also present dynamic behaviours. The aim of this project was to understand the dynamics of enhancer usage and investigate whether this correlates with gene transcription, using p300 as an enhancer proxy and Xenopus tropicalis as a model. A 12.5-hour p300 ChIP-seq high-resolution time series, covering the end of the blastula stages, gastrulation and most of neurulation, was generated. ChIP-seq time series data analysis was optimised, including through the development of a normalisation method which allows for varying levels of background reads in different ChIP-seq samples. A dataset of 9,807 candidate enhancers and their respective usage dynamics was generated, a potentially useful tool for the Xenopus community. Furthermore, I showed that p300 binding dynamics at promoters and nearby candidate enhancers correlate well, reinforcing the enhancer-promoter loop model for transcription regulation. Additionally, I showed that p300 binding dynamics at promoters correlate with gene transcription, suggesting that the loop is maintained for the duration of transcription. I used both results to create a method to predict candidate enhancer-gene pairs and, with the addition of differential DNA motif analysis, to predict candidate target genes for some well-known transcription factors. The data generated in this project helped shed light on transcriptional regulation and led to the development of some useful tools both for Xenopus and transcription researchers. 3 For grandad Obrigada por existires 4 Acknowledgements I would like to thank several people without whom this thesis would not have been possible. Firstly I would like to thank Mike Gilchrist, for giving me the opportunity to do this PhD, for his guidance and advice. I would also like to thank Jim Smith for taking me into his lab in the final months of my PhD and, together with Peter Thorpe and Jean-Paul Vincent, for all the help and comments as part of my Thesis Committee. Thanks to Brook and Elena for all the frog and lab advice and help, to Ian and Ilya who had to suffer with my endless computational questions and the biggest thank you to Nick, for inspiring me and always being there to help me. You all made the Gilchrist lab a great group to work with. Thanks to the animal and the sequencing facility members for all the technical support and thanks to every past and current member of the Elgar and Smith lab for all the help, as well as availability for parties! A special thank you to Bathilde and Christina for listening to my constant whining and for all the fun in the 4NE corner of the Crick. And of course, to Rita, my fellow Portuguese, doing our PhDs at the same time was a gift and you were always my go-to person during these four years, for science advice and gossip (“o que é o vento?”). Thanks to all the fellow Portuguese people for our Monday lunches, making me feel closer to home. This PhD was done as part of the DevCom Marie Curie Initial Training Network. I would first like to thank the European Union for funding me, with the hope that many more people after me may get the same privilege. I would like to thank everyone that was part of it, it was the best opportunity of my life. A special thank you to Gert Jan Veenstra and José Luis Goméz-Skarmeta for all the advice and collaboration. Thanks to Simon van Heeringen for the long conversations and 5 email chains, your help and endless patience was essential for this PhD. Thanks to all the students for making all the courses and conferences so much fun. I would also like to thank all my friends and family, unfortunately I cannot mention everyone, but you were all essential and a bit of this thesis is also yours. Thanks to all the PG people, especially my three “Pauls”, you kept me sane in these past five months. Thanks to Joe and Meena for all the party nights but also serious and supportive conversations. Thanks to Carolina, Filipa and Rita for our travels and skype calls. Thanks to my two “persons”, Diane and Pedro, I miss you so much but know you are always there for me. Thanks to Rita (Táta) for the advice on how to make pretty images and for pushing me to the finish line. Thanks to Pádua for arranging my post-PhD holidays. Thanks to Jane, Nigel, Holly and Emily for taking me into their family and always making me feel at home. Thanks to Sofia, Leonor and Rita for filling my heart with pure love. Thanks to Inês and Ana for always supporting and believing in me, “Ró consé!”. Pai and Mãe, no words can ever thank you for all you have done for me, I hope I have made you proud. And to Tom, for being there every day, through the highs and lows that a PhD brings. Thanks for making me look forward to the future. 6 Table of Contents Abstract ............................................................................................................... 3 Acknowledgements ............................................................................................ 5 Table of Contents ................................................................................................ 7 Table of Figures ................................................................................................ 11 List of tables ...................................................................................................... 14 Abbreviations .................................................................................................... 15 Chapter 1. Introduction ................................................................................ 17 1.1 Transcription and its Regulation ......................................................... 17 1.1.1 Enhancers ......................................................................................... 20 1.1.1.1 Enhancers and disease ............................................................... 22 1.1.2 Insulators .......................................................................................... 23 1.1.3 Enhancer-promoter looping ............................................................... 23 1.1.4 Chromatin structure ........................................................................... 26 1.1.4.1 Accessible chromatin regions ...................................................... 26 1.1.4.2 Histone modifications ................................................................... 28 1.1.4.2.1 H3K4me ................................................................................... 28 1.1.4.2.2 H3K36me ................................................................................. 29 1.1.4.2.3 H3K27me ................................................................................. 29 1.1.4.2.4 H3K9me ................................................................................... 30 1.1.4.2.5 H3K20me ................................................................................. 31 1.1.4.2.6 Histone acetylation .................................................................. 31 1.1.5 Enhancer RNAs ................................................................................ 33 1.1.6 Transcriptional adaptor p300 ............................................................ 34 1.1.6.1 p300 and transcriptional regulation .............................................. 36 1.1.6.2 p300/CBP and disease ................................................................ 38 1.1.6.3 p300 and enhancer prediction ..................................................... 39 1.1.7 Sequence conservation ..................................................................... 42 1.1.8 Enhancer validation ........................................................................... 43 1.1.9 ChIP-seq ........................................................................................... 45 1.2 Xenopus tropicalis ................................................................................ 46 7 1.2.1 Dynamics of early transcription in Xenopus tropicalis ....................... 47 1.2.2 Enhancers and histone modifications in Xenopus tropicalis embryos49 1.2.3 p300 in Xenopus embryos ................................................................ 50 1.3 Thesis Aim ............................................................................................. 51 Chapter 2. Materials & Methods .................................................................. 52 2.1 Embryo in vitro fertilisation .................................................................. 52 2.1.1 Oocyte collection ............................................................................... 52 2.1.2 Sperm collection ............................................................................... 52 2.1.3 Fertilisation and embryo development .............................................