CtBP represses adult -like globin expression in haematopoiesis Jinfen Jasmine Yik A thesis submitted for the degree of Doctor of Philosophy (Biotechnology) School of Biotechnology and Biomolecular Sciences University of New South Wales October 2017 ORIGINALITY STATEMENT i Table of Contents Table of Contents ORIGINALITY STATEMENT ......................................................................................................... i Acknowledgements ................................................................................................................... v Publications arising from this candidature ................................................................................ vi Abstract .................................................................................................................................. vii List of abbreviations ............................................................................................................... viii 1 Chapter 1: Introduction .................................................................................................... 1 1.1 Mammalian gene regulation ..................................................................................... 1 1.2 Gene regulatory elements......................................................................................... 1 1.3 Transcription factors ................................................................................................. 2 1.4 Haematopoiesis ........................................................................................................ 2 1.5 Erythropoiesis ........................................................................................................... 3 1.6 Haemoglobin ............................................................................................................ 4 1.7 The - and -like globin loci ...................................................................................... 4 1.7.1 The α-like globin locus ....................................................................................... 5 1.7.2 The -like globin locus ....................................................................................... 6 1.8 Haemoglobin switching ............................................................................................. 6 1.9 Haemoglobinopathies ............................................................................................... 8 1.9.1 -Haemoglobinopathy ...................................................................................... 8 1.9.2 -Haemoglobinopathy ...................................................................................... 8 1.10 Understanding haemoglobin switching ..................................................................... 9 1.11 Current therapeutic approaches to haemoglobinopathies ........................................ 9 1.12 Transcriptional regulation of erythropoiesis .............................................................10 1.12.1 GATA1 ..............................................................................................................10 1.12.2 FOG1 ................................................................................................................11 1.12.3 Kruppel-Like Factors .........................................................................................11 1.12.4 SOX6 ................................................................................................................13 1.12.5 C-terminal binding proteins ..............................................................................14 1.13 Filling the gap in the knowledge: CtBP .....................................................................15 1.14 Gene editing: CRISPR/Cas9 .......................................................................................16 1.15 Hypothesis & Aims ...................................................................................................17 2 CHAPTER 2: Material and Methods ..................................................................................19 2.1 Materials .................................................................................................................19 2.1.1 Chemicals and Reagents ...................................................................................19 2.1.2 Enzymes ...........................................................................................................22 2.1.3 Antibodies ........................................................................................................22 2.1.4 Cytokines .........................................................................................................22 2.1.5 Plasmids ...........................................................................................................23 2.1.6 Oligonucleotides (Primers and ssODN) .............................................................23 iii 2.1.7 Bacterial strains and culture .............................................................................27 2.1.8 Commercial services and kits............................................................................27 2.2 Methods ..................................................................................................................27 2.2.1 General methods .............................................................................................27 2.2.2 Mammalian cell culture and Nucleofection ......................................................28 2.2.3 Genome editing: CRISPR/Cas9 ..........................................................................29 2.2.4 RNA extraction and cDNA synthesis..................................................................31 2.2.5 Quantitative real-time RT-PCR and Microarrays ...............................................31 2.2.6 Fluorescence-activated cell sorting and flow cytometry ...................................32 2.2.7 Microarray .......................................................................................................32 2.2.8 Statistical analysis ............................................................................................32 3 Chapter 3: The role of CtBP1, CtBP2 and SOX6 on -like globin regulation in HUDEP-2 cells 34 3.1 Introduction.............................................................................................................34 3.1.1 The human erythroid progenitor cell line: HUDEP-2 .........................................34 3.1.2 Existing knowledge on the roles of CtBP and SOX6 in -like globin regulation ...35 3.2 Determining endogenous expression of CtBP1, CtBP2 and SOX6 in HUDEP-2 ...........36 3.3 CRISPR/Cas9 gene engineering and validation of CtBP1-/-, CtBP2-/- and Sox6-/- HUDEP-2 clones ..................................................................................................................37 3.3.1 Western blot screening and validation of CtBP1-/- HUDEP-2 clones .................39 3.3.2 Western blot screening and validation of CtBP2-/- HUDEP-2 clones .................40 3.3.3 Western blot screening and validation of Sox6-/- HUDEP-2 clones....................41 3.4 Knocking out CtBP1 and CtBP2 in HUDEP-2 cells ......................................................43 3.5 Evaluation of -like globin expression in CtBP1-/-, CtBP2-/-, and Sox6-/- HUDEP-2 clones 43 3.6 CtBP1 ablation leads to downregulation of -globin expression in HUDEP-2 cells .....44 3.7 CtBP2 ablation leads to the down-regulation of -globin expression in HUDEP-2 cells 45 3.8 SOX6 ablation has no effect on -globin gene expression in HUDEP-2 cells ..............46 3.9 Discussion ................................................................................................................47 4 Chapter 4: CtBP represses adult - globin regulation in immature murine erythroid cells 51 4.1 Introduction.............................................................................................................51 4.1.1 Choosing MEL cells for the generation of mutant clones ..................................51 4.1.2 Evaluating the roles of CtBP1 and CtBP2 in -like globin expression in MEL cells 52 4.2 Generation and validation of CtBP1-/-, CtBP2-/-, CtBP1CtBP2-/-, Klf3-/-, Klf8-/-, Klf3Klf8-/-, and Sox6-/- mutations in CRISPR/Cas9 modified MEL cells .................................53 4.2.1 Screening and validation of CtBP1-/-, CtBP2-/-, and CtBP1CtBP2-/- MEL clones 55 4.2.2 Screening and validation of Klf3-/-, Klf8-/-, and Klf3Klf8-/- MEL clones .............61 4.2.3 Screening and validation of Sox6-/- MEL clones ................................................67 Table of Contents 4.3 Generation and validation of Sox6ΔDL and Klf3ΔDL mutations in MEL cells ..............69 4.3.1 Screening and validation of Sox6ΔDL MEL clones..............................................71 4.3.2 Screening and validation of Klf3ΔDL MEL clones ...............................................73 4.4 -like globin expression levels in CtBP1-/-, CtBP2-/-, CtBP1CtBP2-/-, Klf3-/-, Klf8-/-, Klf3Klf8-/-, Sox6-/-, Klf3∆DL, and Sox6∆DL MEL clones.........................................................75 4.4.1 -like globin expression levels in undifferentiated and induced differentiated CtBP1-/-, CtBP2-/- and CtBP1CtBP2-/- MEL clones ...........................................................76 4.4.2 Evaluating-like globin expression levels in undifferentiated and induced differentiated Klf3-/-, Klf3∆DL, Klf8-/-, and Klf3Klf8-/- MEL clones ...................................79 4.4.3 -like