A genetic screening identifies a component of the SWI/SNF complex, Arid1b as a senescence regulator Sadaf Khan A thesis submitted to Imperial College London for the degree of Doctor in Philosophy MRC Clinical Sciences Centre Imperial College London, School of Medicine July 2013 Statement of originality All experiments included in this thesis were performed by myself unless otherwise stated. Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives license. 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 license terms of this work. 2 Abstract Senescence is an important tumour suppressor mechanism, which prevents the proliferation of stressed or damaged cells. The use of RNA interference to identify genes with a role in senescence is an important tool in the discovery of novel cancer genes. In this work, a protocol was established for conducting bypass of senescence screenings, using shRNA libraries together with next-generation sequencing. Using this approach, the SWI/SNF subunit Arid1b was identified as a regulator of cellular lifespan in MEFs. SWI/SNF is a large multi-subunit complex that remodels chromatin. Mutations in SWI/SNF proteins are frequently associated with cancer, suggesting that SWI/SNF components are tumour suppressors. Here the role of ARID1B during senescence was investigated. Depletion of ARID1B extends the proliferative capacity of primary mouse and human fibroblasts. Furthermore, in cells expressing mutant RASG12V and PIK3CAH1047R, ARID1B is necessary for the maintenance of oncogene-induced senescence (OIS). Knockdown of ARID1B during OIS results in reduced expression of the CKIs p21Cip1 and p16INK4a. Many SWI/SNF proteins are post-transcriptionally induced during both replicative senescence and OIS, suggesting a broader role for the SWI/SNF complex in senescence. Ectopic expression of components of the SWI/SNF complex induced premature senescence associated with an increase in p21Cip1 and p16INK4a protein levels. SILAC analysis of global changes in protein levels identified enrichment of mitochondrial proteins and depletion of mitotic proteins upon ARID1B expression. Mitochondrial dysfunction and ROS production are important in ARID1B expressing cells as growth arrest was rescued using antioxidant N-acetyl-cysteine. Finally, analysis of cancer genome sequencing data has identified ARID1B as a mutational-driver gene in some cancers. In these tumours, ARID1B mutations are often associated with mutations in TP53 and PTEN. Altogether the present evidence suggests that regulation of senescence by different mechanisms contributes to explain the tumour suppressive properties of the SWI/SNF complex. 3 Acknowledgements All thanks is for God and Jesus. I am truly grateful for the opportunity to do what I love every day. Throughout my life I have been blessed with amazing teachers who are very important to me. Thank you to Ana, for always going the extra mile to help me. Thank you for always being there for me ♥. You are “the buzz lightyear to my woody”. Thank you to Juan Carlos and to Selina for the continual encouragement and help over the last few years, and for advising me with your experience every step of the way!! Much of the work wouldn’t have been possible without ‘the horrids’. Marta, thank you for being my inspiration, thank you for all that you did for me, I will never forget it. The day the two big slaves left the little slave was very Meireles, but Nik, YNWA! Thanks Migi for bringing me into your group of “kids” and for being a great friend. I would like to thank ALL the cool people in the CSC for making my time here so fun. Thank you my partner in distraction, Ines for teaching me ChIP sukhi para me :D. Where were you when Aguero scored 94th minute vs QPR? I was in Bram’s office destroying his ear drums :’), thanks for SO many things, not least the MS, and Vesela thanks for your 5% input ;). Thanks Tom for introducing me to Ronald ‘The Don’ Fisher and the Genomics lab for the NGS optimisation. Thanks also to Angeles, Nuria, Joao and Rita for fun times and everyone in CP. Thank you to Mike, for always checking up on me in the night, and Curtis for bringing my 20 boxes with a huge smile. You guys were “The Constant” when I was “unstuck in time”. To all the footie boys thanks for making Mondays worth it: thanks to my “favourite” Fico for filling a huge gap (fatass!) when Ana left. Jorge + Vlad your feedback on my work was absolutely priceless thank you! Fede, Ben, Olivier, Peter thank you for holding back the shots, you’re all true gentlemen . Francesco, I hope I finally convinced you fate exists, after you gave me ‘The Fall’ 4 days before I was that girl…thank you for giving me a break :oP! For Ken. Thank you for encouraging me to apply for studentships. If it wasn’t for your kind words and confidence in me, I never would have thought to take this path. Thank you for instilling in me your passion for science. Thank you to my friends who stood by me through all the late dinners, birthdays and engagement parties! Thank you to my big nurturing family for your unconditional love and prayers, especially to my Mummy. Thanks Chibbies for the room cleaning and Sally for ‘praying that I get a life one day’. Thank you for letting me follow my dreams. All praise for any good in this work belongs to God. Our helper when all doors are closed and our shelter when all solutions fail. Thank you for your guidance. 4 Table of Contents Statement of originality 2 Copyright declaration 2 Abstract 3 Acknowledgments 4 Table of contents 5 List of Figures and Tables 10 Abbreviations 13 Chapter 1. Introduction 20 1.1. Senescence 20 1.1.1. The senescence signature 21 1.1.2. Replicative senescence 24 1.1.3. Stress-induced senescence 25 1.1.4. Molecular mechanisms regulating senescence 28 1.1.5. Epigenetic regulation of senescence 31 1.1.5.1. Epigenetic regulation of the INK4b-ARF-INK4a locus 31 1.1.5.2. Chromosomal architecture during senescence 32 1.1.6. Senescence and ageing in vivo 34 1.2. Loss-of-function screenings using RNA interference (RNAi) 36 1.2.1. Systems for pooled shRNA screenings 38 1.2.2. Genetic screenings to identify novel senescence regulators 39 1.3. The SWI/SNF complex in senescence and cancer 44 1.3.1. The SWI/SNF complex of chromatin remodellers 44 1.3.1.1. A multi-subunit complex 45 1.3.1.2. ATP-dependent nucleosome remodelling 47 1.3.1.3. Regulation of transcription 50 5 1.3.1.4. Specialised domains and exchangeable isoforms 50 1.3.2. Role of the SWI/SNF complex in senescence and cancer 51 1.3.2.1. SWI/SNF subunits are frequently mutated in cancer 52 1.3.2.2. Relationship between the SWI/SNF complex and senescence 57 1.3.2.2.1. SNF5 57 1.3.2.2.2. BRG1/BRM 58 1.3.2.2.3. ARID1A/ARID1B 60 1.3.2.2.4. PBRM1/BRD7 61 1.3.2.2.5. Role of SWI/SNF in SAHF formation 62 Chapter 2. Materials and Methods 64 2.1. Libraries 64 2.1.1. HCC deletion library 64 2.1.2. pGIPZ genome-wide library 64 2.2. Cell lines and tissue culture methods 65 2.3. Retrovirus production and transduction of target cells 65 2.4. Lentivirus production and transduction of target cells 66 2.5. Generation of custom Solexa libraries 67 2.5.1. Genomic DNA isolation 67 2.5.2. Solexa PCR 67 2.5.3. DNA purification and quantification 68 2.6. Next-generation sequencing 68 2.6.1. Solexa library quality control 68 2.6.2. High-throughput sequencing 69 2.6.3. Bioinformatics 69 2.7. Growth assays 72 6 2.7. 1. Growth curves and colony formation assays 72 2.7.2. BrdU (5-Bromo-2’-deoxyuridine) incorporation assay 73 2.7.3. Senescence-associated β-galactosidase (SA-β-gal) assay 73 2.7.3.1. Cytochemical staining 73 2.7.3.2. Fluorescence visualisation 74 2.8. Generation of shRNA expressing retroviral vectors 74 2.8.1. shRNA constructs 74 2.8.2. Plasmid DNA purification 75 2.8.3. Agarose gel electrophoresis and DNA purification 75 2.8.4. DNA sequencing 75 2.9. RNA expression analysis 76 2.9.1. Total RNA purification 76 2.9.2. Gene expression profiling 76 2.9.2.1. RNA quality control 76 2.9.2.2. Microarray analysis 77 2.9.3. Quantitative RT-PCR (RT-qPCR) analysis 77 2.10. Protein expression analysis 78 2.10.1. Immunofluorescence 78 2.10.1.2. High throughput microscopy (HTM) 78 2.10.1.3. High content analysis (HCA) 78 2.10.1.4. Confocal microscopy 81 2.10.2. SILAC to identify changes in protein expression 81 2.10.2.1. Whole cell protein extraction 81 2.10.2.2. Protein quantification 82 2.10.2.3. Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 82 7 2.10.2.4. Co-immunoprecipitation of nuclear protein complexes 83 2.10.2.5. Identification of proteins by LC–MS/MS 83 Chapter 3. Screening for regulators of lifespan extension in MEFs using NGS 85 3.1.