• Abstract and Figures
• Public Full-text

A genetic screen to identify novel regulators of Ikaros-mediated cell cycle arrest. By Lee Cooper A thesis submitted to Imperial College London for the degree of Doctor of Philosophy MRC Clinical Science Centre Imperial College School of Medicine September 2015 1 I, Lee Cooper, declare that the work presented in this thesis is my own, and that any work carried out by others has been acknowledged and appropriately referenced in the text. The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 2 The Fall of Icarus – Henri Matisse 1869-1954 3 Abstract Failure to regulate cellular proliferation is one of the hallmarks of cancer. The development of pre-B cells is demarcated by alternating stages of quiescence, in which immunoglobulin receptors are sequentially rearranged, and clonal expansion, in which signals from the assembled pre-B cell receptor lead to proliferation. Ikaros (encoded by Ikzf1 in mice) is a transcription factor that regulates gene expression in cycling B cell progenitors to enforce proliferative arrest. In humans IKZF1 mutations are prevalent in subsets of haematological malignancies and result in inappropriate proliferation, therefore the regulation of the cell cycle by Ikaros may be fundamental to its tumour suppressor function. The study of Ikaros-mediated cell cycle arrest in pre-B cells is complicated by the role of Ikaros in the regulation of genes involved in pre-B cell differentiation. I used 3T3 fibroblasts as a reductionist model to study the regulation of the cell cycle by Ikaros independently of pre-B cell receptor signalling. Using this model I performed an RNAi screen to discover novel regulators that cooperate with Ikaros to arrest the cell cycle. Amongst a number of candidates I identified the scaffolding protein SSeCKS (Akap12) as necessary for Ikaros-mediated proliferative arrest. Overexpression of Ikaros and SSeCKS synergistically arrested the cell cycle and silenced the expression of the proto-oncogene Myc. Utilising fibroblasts in which Ikaros and SSeCKS could be inducibly expressed I performed RNAseq to profile the global gene expression of cells that had undergone Ikaros and SSeCKS-mediated cell cycle arrest. Ikaros and SSeCKS together regulated the expression of hundreds of genes to coordinate proliferative arrest. The insights gained from these analyses may be applied to pre-B cells to deepen our understanding of the role of Ikaros in the regulation of proliferation in normal development and in the leukaemic state. 4 Acknowledgements First and foremost I would like to thank my supervisor Matthias for all the help and support over these years, and having faith in me when my own was lacking. I would like to thank Gopu for all the (occasionally begrudging) bioinformatics help. Without that analysis my project would not have progressed. I would also like to thank James for tirelessly sorting my cells and lending a sympathetic ear (and cup of tea) for when experiments didn’t work out quite the way I wanted to. I would like to thank my family for all the love and support they have given me. Without them I would not be here today. I would particularly like to thank my mum for fuelling my late night writing sessions with delicious gin and tonics. I would like to thank all the members of lymphocyte development for making the lab a productive, and deep down, enjoyable place to work. Thanks go to Isa and Ziwei for teaching me the techniques required for successful lab work. Special shout out to Anne, for stocking the ‘secret’ stash of sugary sweets that sustained me through many of my experiments. Many thanks to Grainne for her two rather large contributions at my birthday party. Thanks to Hakan for opening my eyes to the academic resource ‘urban dictionary’. Thanks to Leopard for being the object of my derision for four years. Thanks to Liz for all the cat pictures. Thanks to Preksha for her unintentionally hilarious jokes. Thanks to Allifia for giving me fantastic career guidance. Thanks to Sergi, who taught me that you are never too old to party. Thanks to Ludovica for coining the term ‘cucumberons’. Thanks to Stefan for the buffers I stole. Thanks to Feng for opening my eyes to the wonders of Tian Fu. Thanks to Lesly for making me cultured by taking me to the ballet. Thanks to Toby for never understanding a word I say. Thanks to Kotryna for so efficiently aliquoting the FCS. Thanks to Aljer, who is always a pleasant distraction from work at any hour of the day (mostly because he doesn’t seem to sleep). I’d like to thank Raj – we made it! Finally I would like to thank my cute kitty Tofu, who did his darndest to prevent me writing by sleeping on my laptop. 5 Table of contents Abstract…………………………………………………………………………………………………………………….4 Acknowledgements…………………………………………………………………………………………………..5 Figures and tables……..………………………………………………………………………………………….....9 Abbreviations………………………………………………………………………………………………………….12 Chapter 1 Introduction ............................................................................................... 14 1.1 Ikaros family members in haematopoiesis, cell cycle regulation and leukaemia .................................................................................................................................. 14 1.1.1 The function of Ikaros in early lymphocyte progenitors ............................ 14 1.1.2 The regulation of the cell cycle by Ikaros in B cell precursors .................... 19 1.1.3 Mechanisms of transcriptional regulation by Ikaros .................................. 25 1.1.4 Ikaros as a tumour suppressor .................................................................... 28 1.2 Regulation of the cell cycle ................................................................................ 31 1.2.1 The G1/S checkpoint ................................................................................... 33 1.2.2 Myc and the regulation of the cell cycle ..................................................... 36 1.2.3 Scaffolding proteins in the regulation of the cell cycle .............................. 40 1.3 RNA interference ............................................................................................... 42 1.3.1 Aims of the thesis ........................................................................................ 47 Chapter 2 Materials and Methods ............................................................................... 48 2.1 Materials ............................................................................................................ 48 2.1.1 Antibodies ................................................................................................... 48 2.1.2 Cell lines ...................................................................................................... 49 2.1.3 Primer and adaptor sequences ................................................................... 49 2.2 Methods ............................................................................................................. 52 2.2.1 Cell culture .................................................................................................. 52 2.2.2 Viral packaging and transduction ............................................................... 53 6 2.2.3 Cloning ........................................................................................................ 56 2.2.4 Cellular proliferation assays ........................................................................ 58 2.2.5 Positive selection shRNA screen ................................................................. 59 2.2.6 Immunofluorescence (IF) ............................................................................ 62 2.2.7 Western blot ............................................................................................... 63 2.2.8 Ikaros chromatin immunoprecipitation (ChIP) ........................................... 64 2.2.9 Protein pulldown ........................................................................................ 66 2.2.10 Real-time Quantitative PCR (RT-qPCR) ..................................................... 67 2.2.11 RNA-seq..................................................................................................... 68 Chapter 3 A model system to study the regulation of the cell cycle by Ikaros .......... 71 3.1 Introduction ....................................................................................................... 71 3.2 An inducible system of Ikaros activity ............................................................... 72 3.3 Ikaros regulates the cell cycle in pre-B cells and fibroblasts ............................. 74 3.4 Enforced Myc expression overrides Ikaros-induced cell cycle arrest ................ 82 3.5 Knockdown of Ikzf1 expression restored proliferation ..................................... 84 3.6 A proof of principle positive enrichment shRNA screen ................................... 87 3.7 Discussion ........................................................................................................... 89 3.7.1 Ikaros directs cells towards a quiescence-like state ..................................

Load more

  191

Copy Link