Molecular regulation of BRD3 in forward programming of Megakaryocytes Maria Isabel Marques Rosa University of Cambridge Newnham College This dissertation is submitted for the degree of Doctor of Philosophy September 2018 Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit for the relevant Degree Committee. Isabel Rosa September 2018 “Quando de lá vimos, é que para lá havíamos de ir” “The moment when one finishes a journey, is when they are truly ready to start.” Ernesto Rosa, my Dad Table of contents Abstract ................................................................................................................................ 1 Introduction ................................................................................................................................. 2 1.1 Haematopoiesis................................................................................................................... 3 1.1.1. Embryonic haematopoiesis...................................................................................... 3 1.1.2. Adult haematopoiesis, an evolving model ................................................................ 5 1.2 Megakaryopoiesis................................................................................................................ 9 1.2.1. Megakaryopoiesis development .................................................................................... 9 1.2.2. Megakaryopoiesis regulation .......................................................................................12 1.2.3. Platelets......................................................................................................................16 1.2.4. Genome-wide association studies (GWAS) ....................................................................18 1.3 Bromodomains ...................................................................................................................22 1.3.1. Bromodomain modules ...............................................................................................22 1.3.2. Bromodomain proteins (BRDs) in transcription .............................................................24 1.3.3. BET proteins in transcription regulation and cell cycle progression .................................25 1.3.4. BET inhibition ..............................................................................................................26 1.3.5. BRD3-GATA-1 interaction.............................................................................................27 1.4 Model systems to study megakaryopoiesis...........................................................................28 1.4.1. Animal models ............................................................................................................28 1.4.2. Immortalised cell lines .................................................................................................28 1.4.3. Stem cell models .........................................................................................................29 1.4.4. in vitro models of megakaryopoiesis .............................................................................30 1.4.3. Genetic manipulation system- CRISPR/Cas9 ..................................................................32 1.5 Chromatin and gene transcription .......................................................................................34 1.5.1. Chromatin structure organisation.................................................................................34 1.5.2. Transcriptional regulation ............................................................................................35 1.6 The aims of my project........................................................................................................40 Material and Methods..................................................................................................................41 2.1. Cell culture ....................................................................................................................42 2.2. Molecular techniques .....................................................................................................46 2.3. Bioinformatics analysis ...................................................................................................56 Results ........................................................................................................................................61 3.1 Generation of an iPSC BRD3 KO model .................................................................................62 3.1.1 Introduction.................................................................................................................62 3.1.2 Results.........................................................................................................................64 3.1.3 Discussion ....................................................................................................................80 3.2 BRD3 regulation in Megakaryopoiesis ..................................................................................84 3.2.1 Introduction.................................................................................................................84 3.2.2 Results.........................................................................................................................85 3.2.3 Discussion ....................................................................................................................96 3.3 BET inhibition in Megakaryopoiesis.................................................................................... 100 3.3.1 Introduction............................................................................................................... 100 3.3.2 Results....................................................................................................................... 101 3.3.3 Discussion .................................................................................................................. 118 3.4 BET regulation in Megakaryopoiesis................................................................................... 121 3.4.1 Introduction............................................................................................................... 121 3.4.2 Results....................................................................................................................... 122 3.4.3. Discussion ................................................................................................................. 132 Conclusion and future work........................................................................................................ 134 References ................................................................................................................................ 138 Appendix ................................................................................................................................... 170 6.1. Plasmids.......................................................................................................................... 171 6.1.1 Plasmids used for generation of Knockouts .................................................................. 171 6.1.2 Plasmids used for generation viral particles containing TFs for FoP protocol .................. 174 6.1.3 Plasmids used for generation of Knockdowns............................................................... 177 6.2 Alignment of Sanger sequences to confirm sgRNAs insertions ............................................. 179 6.2. Differentially expressed genes in BRD3 KO ........................................................................ 181 6.2.1 BRD3 KO iPSC ............................................................................................................. 181 6.2.1 BRD3 KO MKs ............................................................................................................. 183 6.3. BET inhibition - appendix.................................................................................................. 186 Acknowledgments .............................................................................................................. 187 List of figures Introduction Figure 1.1.1 Sequential waves of embryonic haematopoiesis………………………………………………………………4 Figure 1.1.2 Diagram of canonical haematopoietic tree………………………………………………………………..…….7 Figure 1.2.1 Redefined model of human blood lineage formation……………………………………….…………….10 Figure 1.2.2 BRD3 locus zoom plot with SNP association for volume of platelets………………………………..20 Figure 1.2.3. BRD3 locus zoom plot with SNP association for distribution width of the volume of platelets………………………………………………………………………………………………....……………………………....………21