Identification of Common and Distinct Epigenetic Reprogramming Properties of Core-binding Factor Fusion Proteins (Justin) Ching Ting Loke A Thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Cancer and Genomic Sciences, College Of Medical and Dental Sciences, University of Birmingham November 2016 1 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract RUNX1, also known as CBFα, is a master regulator of haematopoiesis. In Acute Myeloid Leukaemia (AML) it is frequently disrupted by translocations to different epigenetic regulators, resulting in the expression of core-binding factor fusion proteins. We compared the chromatin landscape of t(8;21) and t(3;21) AML which express RUNX1-ETO and RUNX1-EVI1, respectively. We found that the diverse clinical outcomes of patients with these two forms of AML are reflected in fundamental differences in gene expression and chromatin landscape. Despite both fusion proteins sharing a RUNT DNA binding domain, we show that RUNX1-EVI-1 targets a more immature stem cell-related gene expression program of genes as compared to RUNX1-ETO. Despite the differences in the epigenomic landscape of t(3;21) and t(8;21) leukaemia, knockdown of either core-binding factor fusion protein activates a common myeloid differentiation program involving up regulation of C/EBPα. By blocking C/EBPα DNA binding through a dominant negative partner, we showed that this factor is required for the downstream effects of RUNX1-EVI-1 knockdown. Even in the continued presence of RUNX1-EVI-1, ectopic expression of C/EBPα is sufficient to initiate myeloid differentiation in t(3;21) cells. Overall, this suggests that deregulation of C/EBPα is a common pathway in the development of both t(8;21) and t(3;21) AML. 2 This work is dedicated to my wife, Rebecca, and our daughter, Bessie. 3 Acknowledgments I would like to thank my supervisor Prof Constanze Bonifer for this opportunity to work with her team. She has been a constant source of wisdom and infective enthusiasm. Dr Salam Assi performed the bioinformatics analysis which has brought this story alive; I would like to thank her for her perseverance with this project, and her insightfulness. I am also indebted to Dr Anetta Ptasinska and Dr Maria “Rosie” Imperato for teaching me many of the techniques used in this thesis. Furthermore, Dr Ptasinska generated the majority of the t(8;21) AML data used in this thesis. Dr Imperato was an important colleague in much of the lentiviral work and DNase I hypersensitivity site mapping experiments. I would also like to thank the rest of the Bonifer-Cockerill lab for their support. Prof Peter Cockerill and Dr Manoj Raghavan provided helpful advice. Prof Olaf Heidenreich designed the siRNA against RUNX1-EVI-1, on which much of this work relies, and he has also been a source of fruitful discussions. I would like to thank his lab, and especially Dr Natalia Martinez-Soria, for helping set up the RUNX1-EVI-1 knockdown. I would also like to thank Prof Ruud Delwel for advice on the RUNX1-EVI-1 ChIP-seq and for patient samples. I would like to thank ‘NHS Blood and Transplant’ for reagents, and colleagues at the Department of Haematology, Queen Elizabeth Hospital, Birmingham for their support. I would also like to thank both the high-throughput sequencing facility at Biosciences, University of Birmingham and also the facility run by Dr Andrew Beggs, for their efficiency. This work was funded through a Kay Kendall Leukaemia Fund junior clinical fellowship, of which I am grateful. Finally, I am indebted to my family for their support during these studies, and especially to my parents for guiding me to this stage. 4 Table of Contents Chapter 1. Introduction .................................................................................... 1 1.1 Haematopoiesis ........................................................................................ 1 1.1.1 The emergence of haematopoietic stem cells .................................... 1 1.1.2 Hierarchical relationship of blood cell development ........................... 2 1.1.3 Regulation of differentiation in haematopoietic cells .......................... 3 1.2 Transcriptional regulation in eukaryotic cells ............................................ 9 1.2.1 RNA polymerase and core transcriptional machinery......................... 9 1.2.2 Structure and modification of nucleosomes ...................................... 10 1.2.3 DNaseI hypersensitive sites ............................................................. 14 1.2.4 Regulation of transcription through DNA modifications .................... 17 1.3 . Deregulation of transcriptional regulation in acute myeloid leukaemia.. 18 1.3.1 Mutations found in acute myeloid leukaemia .................................... 19 1.3.2 Targeting transcriptional deregulation in acute myeloid leukaemia .. 24 1.4 Role of RUNX1 in normal haematopoiesis .............................................. 26 1.4.1 Role of Runx1 at the onset of haematopoiesis ................................. 27 1.4.2 Role of Runx1 in Adult Haematopoiesis ........................................... 27 1.4.3 RUNX1 as a transcription factor ....................................................... 29 1.5 Role of EVI-1 in normal haematopoiesis ................................................. 31 1.5.1 Role of EVI-1 in fetal haematopoiesis .............................................. 31 1.5.2 Role of EVI-1 in adult haematopoiesis ............................................. 32 1.5.3 Features of EVI-1 as a transcription factor ....................................... 33 5 1.6 RUNX1 fusion proteins in myeloid malignancies .................................... 36 1.7 Comparison of RUNX1-ETO and RUNX1-EVI-1 ..................................... 37 1.7.1 Clinical description of patients with core-binding factor fusion proteins .................................................................................................................. 39 1.7.2 RUNX1-ETO role in development of leukaemia ............................... 42 1.7.3 Comparison of the role of RUNX1-EVI-1 and RUNX1-ETO in the pathogenesis of leukaemia........................................................................ 47 1.8 Aims of the project .................................................................................. 52 Chapter 2. Methods and Materials ................................................................ 54 2.1 Cell line culture ....................................................................................... 54 2.2 Purification of blood samples from patients with AML ............................. 54 2.3 Purification of CD34+ mobilised peripheral blood stem cells .................. 55 2.4 Culturing CD34+ mobilised peripheral blood stem cells .......................... 58 2.5 siRNA mediated depletion of RUNX1-EVI-1 ........................................... 58 2.6 RNA extraction ........................................................................................ 58 2.7 RNA Seq library ...................................................................................... 59 2.8 cDNA synthesis ...................................................................................... 60 2.9 Real-time polymerase chain reaction ...................................................... 60 2.10 Dead cell removal and Annexin V/PI staining for flow cytometry .......... 60 2.11 DNaseI hypersensitivity site mapping and size selection ...................... 60 2.11.1 Library production of DNaseI material for high throughput sequencing ................................................................................................ 64 2.12 ChIP-qPCR and ChIP-seq library preparation ...................................... 66 2.12.1 Double cross-linking ....................................................................... 66 2.12.2 Chromatin immunoprecipitation (ChIP) .......................................... 66 2.12.3 Library production of ChIP material for high throughput sequencing .................................................................................................................. 67 2.13 Cloning of RUNX1-EVI-1 into pSiew and LeGO vectors ....................... 68 2.14 Retroviral production ............................................................................. 70 6 2.14.1 Transfection of HEK293T cells for lentiviral production .................. 71 2.14.2 Transfection of HEK293T cells for CEBPA-ER virus production .... 71 2.14.3 Virus concentration......................................................................... 72 2.14.4 Titration of viruses on HEK293T cells ............................................ 72 2.14.5 Lentiviral transduction of CD34+ PBSCs and SKH-1 ..................... 73 2.14.6 Retroviral transduction with Retronectin ......................................... 74 2.15 Titrating 17 β-estradiol treatment of CEBPA-ER SKH-1 cells ............... 74 2.16 Methylcellulose colony forming