An Investigation Into The Function and Regulation Of ERG Exon 7b Samantha Jumbe A thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the degree of Doctor of Philosophy Department of Applied Science, Faculty of Health and Applied Sciences, University of the West of England June 2019 ABSTRACT The ETS family transcription factor ERG is a key oncoprotein in bone, blood, vascular and most notably prostate cancer where it is activated in at least 50% of cases. Of the several splice isoforms of ERG with variable biological activity those that include the cassette exon 7b are associated with aggressiveness and progression of disease in prostate cancer. Inclusion of exon 7b adds 24 amino- acids in frame to the central ‘alternative domain’ which also contains binding sites for other transcriptional regulators. Alignment of the amino acids of exon 7b showed it is evolutionary conserved in echinoderms emphasizing its functional importance. Splice switching oligonucleotides (SSO) targeting the splice sites for exon 7b were designed to induce exon 7b skipping. Successful SSO-induced skipping of exon 7b in the osteosarcoma MG63 cell line resulted in decreased cell migration, invasion and proliferation and increased apoptosis in vitro. ERG was shown to bind to the promoter of tissue non-specific alkaline phosphatase, a marker of cell differentiation, and SSO-induced skipping of exon 7b attenuated its expression. Moreover several splicing regulatory elements and proteins were identified using bioinformatics prediction methods and an RNA pull down of the 3´ splice site of exon 7b identified several potential splicing regulatory proteins for this exon. This study provides evidence that the inclusion of exon 7b, which encodes the full length transactivation domain enhances the oncogenic activity of ERG in vitro. Further refinement of in vivo experimentation is required but if successful SSOs that target oncogenes could potentially be developed as therapeutic agents. The study also highlights the requirement to understand splicing regulation of disease associated splicing events in ETS transcription factors and has provided pilot data for further study of ERG exon 7b splicing regulation. For those I love Samantha Jumbe- June 2019 1 ACKNOWLEDGEMENTS My sincere gratitude and appreciation go to my supervisors Dr Michael Ladomery, Dr Ian Wilson and Dr Bahareh Vahabi. Mike your tireless commitment and encouragement have been spurred me along and allowed me to develop my confidence and capabilities as a scientist. Thank you for your always open door, never ending time and patience and mostly for being there for me in the most difficult time. Ian for help with techniques and statistics and helping me remember that science is a creative discipline. For a listening ear and fresh perspectives - Bahareh thank you. I would like to acknowledge and thank my collaborators. Dr Sebastian Oltean (University of Exeter) and Dr Sean Porazinski (Ladomery Lab) for the xenograft work, Dr Lee Spraggon for advice on design and testing of SSOs and the RNAomics Platform team at the University of Sherbrooke, Canada for the high- throughput PCR analysis. I would like to thank the Malawi Government Scholarship Fund for funding this research and the University of the West of England for providing the research facilities to conduct the research in. Thank you to Kayla Friedman and Malcolm Morgan of the Centre for Sustainable Development, University of Cambridge, UK for producing the Microsoft Word thesis template used to produce this document. Thanks go to the superhero technical team of the CRIB labs with special thanks to David Corry and Dr Jeff Davey. All the fantastic members of Ladomery Lab and CRIB- thank you for the laughs, ideas and inspiration. 2 Samantha Jumbe- June 2019 So many friends who are like family – thank you for your prayers and so much more. My parents, sisters, brothers, nieces and nephew who are my most precious possession and have provided unsurpassable sustenance and wisdom. My niece Nina for always checking up on how my work on finding a cure for cancer was going (it’s still a work in progress). I could not be without all of your remarkable love and belief in me. A special mention for my late uncle Luke, and other men like him who fought prostate cancer. Your braveness was my inspiration to embark on this journey. “To Him who is able to do immeasurably more than all we ask or imagine, according to his power that is at work within us” Ephesians 3:20 (NIV) Samantha Jumbe- June 2019 3 ABBREVIATIONS AND ACRONYMS 1,25D calcitriol the active metabolite of vitamin D3 AMKL acute megakaryoblastic leukaemia AML acute myeloid leukaemia AP1 activator protein 1 AS alternative splicing ASO antisense oligonucleotide ASPCR alternative splicing using endpoint PCR coupled with microcapillary electrophoresis BCL-X B-cell lymphoma-extra-large BPS branch point site BRAF B-Raf Proto-Oncogene, Serine/Threonine Kinase BRCA1 breast cancer 1 CDK cyclin dependent kinase cDNA complementary DNA CELF CUGBP ELAV-like family member ChIP chromatin immunoprecipitation CLK cdc2-like kinase c-Myc MYC Proto-Oncogene, BHLH Transcription Factor 4 Samantha Jumbe- June 2019 CRISPR-Cas 9 Clustered Regularly Interspaced Short Palindromic Repeats CRISPR associated protein 9 CXCL12 C-X-C motif chemokine ligand 12 CXCR4 C-X-C motif chemokine receptor 4 DAPI 4′,6-diamidino-2-phenylindole DEF docking site for ERK DMD Duchenne muscular dystrophy DNA deoxyribonucleic acid DSCAM Downs syndrome cell adhesion molecule ELAVL1 ELAV Like RNA Binding Protein 1 EMT epithelial-mesenchymal transition ENCODE The Encyclopedia of DNA Elements ERG ETS-related gene or v-ets avian erythroblastosis virus E26 oncogene homolog ERK extracellular signal-regulated kinase ESE exonic splicing enhancer ESS exonic splicing silencer ETS E-26 transformation specific FAM98B Family With Sequence Similarity 98 Member B FHBP (3S)1-fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate, a synthetic analogue of LPA Samantha Jumbe- June 2019 5 FITC Fluorescein isothiocyanate G4 G-quadruplex GTEx Genotype-Tissue Expression HDR homology-directed repair hnRNP heterogeneous nuclear ribonucleoprotein ISE intronic splicing enhancer ISS intronic splicing silencer kDa kilodalton LPA lysophosphatidic acid MAPK mitogen-activated protein kinase mRNA messenger RNA MSI2 musashi RNA binding protein 2 ng nanogram NHEJ non-homologous end joining nM nanomolar NRG Neuroglian Nucleic acid bases A adenosine, T thymine, G guanine, C cytosine and U uracil PCa prostate cancer PCBP poly (rC) binding protein 6 Samantha Jumbe- June 2019 p-NP p-nitrophenol p-NPP p-nitrophenylphosphate PRAD prostate adenocarcinoma Pre-mRNA pre-messenger RNA PS phosphorothioate PSI percent spliced in PSM peptide spectrum match PTBP1 polypyrimidine tract-binding protein 1 qPCR quantitative polymerase chain reaction RBP RNA binding protein RNAPII RNA polymerase II RNA ribonucleic acid RNA-seq RNA sequencing RNPS1 RNA binding protein with serine rich domain 1 RPKM reads per kilobase transcript per million RT-PCR reverse transcriptase polymerase chain reaction SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SELEX systematic evolution of ligands by exponential enrichment SF splice factor SF3A1 splice factor 3 subunit 1 Samantha Jumbe- June 2019 7 SF3B4 splice factor 3b subunit 4 SFPQ Splicing factor proline and glutamine rich siRNA small interfering RNA Sm smith antigen SMA Spinal muscular atrophy smRNA small nuclear RNA snRNP small nuclear ribonucleoprotein SNW1 SNW domain containing 1 SR serine-arginine rich proteins SR-A1 steroid receptor RNA activator 1 SRE splicing regulatory element SRPK serine-arginine protein kinase SRSF serine-arginine splice factor SSO splice switching oligonucleotide SXL Sex-lethal T-ALL T-cell acute lymphoblastic leukaemia TARBP1 transactivation response RNA-binding protein 1 TCGA The Cancer Genome Atlas TCR T-cell receptor TNSALP tissue non-specific alkaline phosphatase 8 Samantha Jumbe- June 2019 UTR untranslated region VDR vitamin D receptor Wnt Wingless-related integration site μM micromolar Samantha Jumbe- June 2019 9 CONTENTS 1 Introduction ............................................................................................21 1.1 Alternative splicing ............................................................................22 1.1.1 Prevalence of alternative splicing ................................................. 24 1.1.2 Specificity and complexity of splicing ........................................... 25 1.1.3 Splicing regulatory elements and proteins involved in splicing regulation ................................................................................................ 27 1.1.4 The splicing reaction .................................................................... 32 1.1.5 Alternative splicing and cancer .................................................... 38 1.2 E26-transformation specific (ETS) family of transcription factors .....41 1.2.1 Transcription factors and gene expression .................................. 41 1.2.2 Structure and binding specificity of ETS transcription factors ...... 43 1.2.3 Expression and biological function of ETS transcription factors ... 48 1.3 The ERG transcription factor ............................................................50 1.3.1 Structure and isoforms ................................................................. 50 1.3.2 ERG and Cancer .........................................................................