A Genetic Screening Identifies a Component of the SWI/SNF Complex, Arid1b As a Senescence Regulator

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. 2Abstract 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. 3Acknowledgements 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. 4Table of Contents Statement of originality Copyright declaration Abstract 223 Acknowledgments Table of contents List of Figures and Tables Abbreviations 4510 13 <ul style="display: flex;"><li style="flex:1">Chapter 1. Introduction </li><li style="flex:1">20 </li></ul><ul style="display: flex;"><li style="flex:1">1.1. Senescence </li><li style="flex:1">20 </li></ul>1.1.1. The senescence signature 1.1.2. Replicative senescence 1.1.3. Stress-induced senescence 21 24 25 28 31 31 32 34 36 38 39 44 44 45 47 50 1.1.4. Molecular mechanisms regulating senescence 1.1.5. Epigenetic regulation of senescence 1.1.5.1. Epigenetic regulation of the INK4b-ARF-INK4a locus 1.1.5.2. Chromosomal architecture during senescence 1.1.6. Senescence and ageing in vivo 1.2. Loss-of-function screenings using RNA interference (RNAi) 1.2.1. Systems for pooled shRNA screenings 1.2.2. Genetic screenings to identify novel senescence regulators 1.3. The SWI/SNF complex in senescence and cancer 1.3.1. The SWI/SNF complex of chromatin remodellers 1.3.1.1. A multi-subunit complex 1.3.1.2. ATP-dependent nucleosome remodelling 1.3.1.3. Regulation of transcription 5 1.3.1.4. Specialised domains and exchangeable isoforms 1.3.2. Role of the SWI/SNF complex in senescence and cancer 1.3.2.1. SWI/SNF subunits are frequently mutated in cancer 1.3.2.2. Relationship between the SWI/SNF complex and senescence 1.3.2.2.1. SNF5 50 51 52 57 57 58 60 61 62 1.3.2.2.2. BRG1/BRM 1.3.2.2.3. ARID1A/ARID1B 1.3.2.2.4. PBRM1/BRD7 1.3.2.2.5. Role of SWI/SNF in SAHF formation <ul style="display: flex;"><li style="flex:1">Chapter 2. Materials and Methods </li><li style="flex:1">64 </li></ul><ul style="display: flex;"><li style="flex:1">2.1. Libraries </li><li style="flex:1">64 </li></ul><ul style="display: flex;"><li style="flex:1">2.1.1. HCC deletion library </li><li style="flex:1">64 </li></ul>64 65 65 66 67 67 67 68 68 68 69 69 72 2.1.2. pGIPZ genome-wide library 2.2. Cell lines and tissue culture methods 2.3. Retrovirus production and transduction of target cells 2.4. Lentivirus production and transduction of target cells 2.5. Generation of custom Solexa libraries 2.5.1. Genomic DNA isolation 2.5.2. Solexa PCR 2.5.3. DNA purification and quantification 2.6. Next-generation sequencing 2.6.1. Solexa library quality control 2.6.2. High-throughput sequencing 2.6.3. Bioinformatics 2.7. Growth assays 6 2.7. 1. Growth curves and colony formation assays 2.7.2. BrdU (5-Bromo-2’-deoxyuridine) incorporation assay 2.7.3. Senescence-associated β-galactosidase (SA-β-gal) assay 2.7.3.1. Cytochemical staining 72 73 73 73 74 74 74 75 75 75 76 76 76 76 77 77 78 78 78 78 81 81 81 82 2.7.3.2. Fluorescence visualisation 2.8. Generation of shRNA expressing retroviral vectors 2.8.1. shRNA constructs 2.8.2. Plasmid DNA purification 2.8.3. Agarose gel electrophoresis and DNA purification 2.8.4. DNA sequencing 2.9. RNA expression analysis 2.9.1. Total RNA purification 2.9.2. Gene expression profiling 2.9.2.1. RNA quality control 2.9.2.2. Microarray analysis 2.9.3. Quantitative RT-PCR (RT-qPCR) analysis 2.10. Protein expression analysis 2.10.1. Immunofluorescence 2.10.1.2. High throughput microscopy (HTM) 2.10.1.3. High content analysis (HCA) 2.10.1.4. Confocal microscopy 2.10.2. SILAC to identify changes in protein expression 2.10.2.1. Whole cell protein extraction 2.10.2.2. Protein quantification 2.10.2.3. Sodium-dodecyl-sulphate polyacrylamide <ul style="display: flex;"><li style="flex:1">gel electrophoresis (SDS-PAGE) </li><li style="flex:1">82 </li></ul>7 2.10.2.4. Co-immunoprecipitation of nuclear protein complexes 2.10.2.5. Identification of proteins by LC–MS/MS 83 83 Chapter 3. Screening for regulators of lifespan extension in MEFs using NGS 3.1. Optimisation of the bypass of senescence screening 85 85 86 87 91 93 93 96 96 97 3.2. Proof of concept experiment verifies feasibility of the screening 3.3. NGS used to identify shRNAs from bypass of replicative senescence screen in MEFs 3.4. ARID1B is a candidate regulator of replicative senescence in MEFs 3.5. Depletion of Arid1b extends cellular lifespan in MEFs 3.6. ARID1B regulates cell proliferation and replicative senescence in IMR-90 3.7. Conclusions and perspectives 3.7.1. Screening for regulators of replicative senescence in MEFs 3.7.2 Advances in pooled shRNA screenings using NGS Chapter 4. ARID1B a component of the SWI/SNF complex regulates senescence100 <ul style="display: flex;"><li style="flex:1">4.1. Knockdown of ARID1B attenuates OIS </li><li style="flex:1">100 </li></ul>4.2. ARID1B regulates p21CIP1, p16INK4a and p53 expression during OIS 4.3. Gene expression profiling identifies multiple senescence effectors regulated by ARID1B 100 103 105 106 110 4.4. Expression of oncogenic PIK3CAH1047R induces senescence 4.5. Knockdown of ARID1B attenuates PIK3CAH1047R mediated senescence 4.6. ARID1B regulates p16INK4a and p21CIP1 expression during PIK3CA-induced senescence 4.7. High expression of SWI/SNF complex components during replicative senescence 4.8. Expression of SWI/SNF complex components increases during OIS 4.9. Post-translational stabilisation of the SWI/SNF complex during senescence 110 113 113 8 4.10. SILAC based quantitative proteomics identifies changes in the levels of <ul style="display: flex;"><li style="flex:1">ARID1B binding proteins during OIS </li><li style="flex:1">116 </li></ul>119 121 121 126 129 132 132 132 134 139 141 4.11. ARID1B is excluded from regions of repressed chromatin 4.12. Retroviral constructs overexpressing ARID1A and ARID1B 4.13. Ectopic expression of SWI/SNF subunits induces premature senescence 4.14. Premature senescence induced by ARID1B is mainly p53 dependent 4.15. Global changes in protein expression associated with ARID1B 4.16. ARID1B is deleted in cancer 4.17. Conclusions and perspectives 4.17.1. ARID1B depletion can bypass senescence through multiple mechanisms 4.17.2. SWI/SNF proteins are induced during senescence 4.17.3. Growth arrest induced by SWI/SNF is exerted through different mechanisms 4.17.4. ARID1B exhibits tumour-suppressive functions in cancer <ul style="display: flex;"><li style="flex:1">Chapter 5. Discussion & Future work </li><li style="flex:1">144 </li></ul>5.1. Genetic screens are useful tools for identifying novel genes and <ul style="display: flex;"><li style="flex:1">pathways involved in senescence </li><li style="flex:1">144 </li></ul>144 145 146 146 150 152 15X 154 5.1.1. Genome-wide screening for regulators of OIS in IMR-90 5.1.2. Library bias not detected from sequencing pGIPZ library pools 5.1.3. Set up of infection conditions for pGIPZ lentiviral screening 5.1.4. pGIPZ shp53-1 can be used as a control hairpin that bypasses OIS 5.2. ARID1B is an important senescence regulator 5.3. The SWI/SNF complex is implicated in cancer and ageing 5.4. The clinical utility of targeting the SWI/SNF complex in cancer detection and therapy 5.5. Concluding remark 9References Appendix 159 213 List of Figures and Tables Figure 1.1. Senescence is an intrinsic cellular response to multiple stresses. Figure 1.2. The senescence signature. 21 24 26 27 30 34 38 40 42 49 Table 1.1. Senescent cells are found in pre-malignant lesions in vivo. Figure 1.3. Senescence is a barrier to tumourigenesis in vivo. Figure 1.4. Key senescence pathways and effectors. Figure 1.5. Epigenetic regulation of senescence. Figure 1.6. Screenings using pooled shRNA libraries. Figure 1.7. Genetic screens for the bypass of replicative senescence. Figure 1.8. Workflow of shRNA identification using different methods. Figure 1.9. The SWI/SNF complex of nucleosome remodellers. Figure 1.10. The SWI/SNF complex interacts with proteins involved in many <ul style="display: flex;"><li style="flex:1">cancer-related pathways. </li><li style="flex:1">52 </li></ul>55 56 69 71 77 80 86 Figure 1.11. Mutations in SWI/SNF genes are predominantly deleterious. Table 1.2. SWI/SNF mutations occur frequently in human cancers. Figure 2.1. Bioanalyser profile of acceptable custom Solexa library for NGS. Figure 2.2. Solexa sequencing schematic. Figure 2.3. Bioanalyser profile of acceptable RNA profile for gene expression profiling. Figure 2.4. Workflow of establishing filters for High Content Analysis. Figure 3.1. An shRNA library targeting genes frequently deleted in human HCC. Figure 3.2. Proof of concept to establish the feasibility of an shRNA lifespan extension <ul style="display: flex;"><li style="flex:1">screen in MEFs. </li><li style="flex:1">87 </li></ul>Figure 3.3. Strategy for the genetic screening for bypass of replicative senescence 10 <ul style="display: flex;"><li style="flex:1">in MEFs. </li><li style="flex:1">90 </li></ul>Figure 3.4. Lifespan extension screen in MEFs identifies Arid1b as a candidate <ul style="display: flex;"><li style="flex:1">senescence regulator. </li><li style="flex:1">92 </li></ul>Figure 3.5. Depletion of Arid1b extends cellular lifespan in MEFs. Figure 3.6. Depletion of ARID1B extends cellular lifespan in IMR-90. Figure 4.1. Knockdown of ARID1B attenuates OIS. 94 95 101 <ul style="display: flex;"><li style="flex:1">102 </li><li style="flex:1">Figure 4.2. ARID1B regulates different senescence effectors. </li></ul>Figure 4.3. Validation of gene expression changes associated with ARID1B knockdown <ul style="display: flex;"><li style="flex:1">during OIS. </li><li style="flex:1">104 </li></ul>107 108 Figure 4.4.1. Expression of oncogenic PIK3CAH1047R induces senescence. Figure 4.4.2. Characterisation of the senescence response induced upon PIK3CAH1047R expression. Figure 4.5. Knockdown of ARID1B attenuates PIK3CAH1047R mediated senescence. Figure 4.6. ARID1B regulates p16INK4a and p21CIP1 during PIK3CA-induced senescence. Figure 4.7. Expression of SWI/SNF complex increases during replicative senescence. Figure 4.8. Expression of SWI/SNF complex increases during OIS. Figure 4.9. Increase in expression of SWI/SNF subunits is not transcriptionally regulated. Figure 4.10. SILAC based quantitative proteomics identifies SWI/SNF interactor exchange during OIS. 109 111 112 114 115 118 120 122 123 125 Figure 4.11. ARID1B is excluded from regions of repressed chromatin. Figure 4.12. Retroviral constructs overexpressing ARID1A and ARID1B. Figure 4.13.1. Overexpression of SWI/SNF subunits induces premature senescence. Figure 4.13.2. Molecular analysis of senescence induced by SWI/SNF subunits. Figure 4.14.1. Knockdown of important senescence regulators alleviates ARID1B <ul style="display: flex;"><li style="flex:1">mediated senescence. </li><li style="flex:1">127 </li></ul>Figure 4.14.2. Co-expression of Human papillomavirus (HPV) oncoproteins alleviates 11 ARID1B-mediated senescence. Figure 4.15.1. Global changes in protein levels upon ARID1B overexpression analysed by SILAC. 128 130 131 145 148 Figure 4.15.2. Production of oxidative stress may contribute to ARID1B mediated senescence. Figure 5.1. pGIPZ library set up and pool coverage determined by HTS. Figure 5.2. Optimising infection conditions with pGIPZ lentiviral vectors. Figure 5.3. Validation of pGIPZ controls for proof of principle experiment for bypass <ul style="display: flex;"><li style="flex:1">of OIS screening. </li><li style="flex:1">149 </li></ul>213 214 215 215 216 216 217 217 217 219 219 220 Table A1. Inferences from test NGS run at CSC. Table A2. Differential gene expression of ARID1B knockdown cells during OIS. Table A3. pGIPZ pool coverage determined by HTS. Table A4. Copy number deletions in ARID1B is significant across cancers. Table A5. cDNA constructs. Table A6. shRNA target sequences. Table A7. Solexa PCR primers. Table A8. Sequencing primers. Table A9. RT-qPCR primers. Table A10. Primers for cloning. Table A11. Antibodies. Figure A1. Validation of ARID1B antibody for immunoprecipitation 12 Abbreviations <ul style="display: flex;"><li style="flex:1">4-OHT </li><li style="flex:1">4-hydroxy tamoxifen </li></ul><ul style="display: flex;"><li style="flex:1">A</li><li style="flex:1">Adenine </li></ul><ul style="display: flex;"><li style="flex:1">Ac </li><li style="flex:1">Acetylated </li></ul>AKT ARF ARID ASF ATCC ATM ATP ATR v-akt murine thymoma viral oncogene homologue Alternative reading frame AT-rich interactive domain Anti-silencing function American Type Culture Collection Ataxia telangiectasia mutated Adenosine tri-phosphate Ataxia telangiectasia and Rad3 related <ul style="display: flex;"><li style="flex:1">BAF </li><li style="flex:1">BRG1/BRM associated factors </li></ul>BMP and activin membrane-bound inhibitor homologue B-cell CLL/lymphoma BAMBI BCL <ul style="display: flex;"><li style="flex:1">BH </li><li style="flex:1">Benjamini-Hochberg </li></ul>BMP bp Bone morphogenetic protein Base pair BRAF BRCA BRD BrdU BRG1 BRM BSA v-raf murine sarcoma viral oncogene homologue B1 Breast cancer susceptibility gene Bromodomain containing 5-bromo-2-deoxyuridine Brahma related gene 1 Brahma Bovine serum albumin <ul style="display: flex;"><li style="flex:1">C</li><li style="flex:1">Cytosine </li></ul>c-FOS C. elegans C(T) Cellular-FBJ osteosarcoma oncogene <a href="/goto?url=http://en.wikipedia.org/wiki/Caenorhabditis_elegans" target="_blank">Caenorhabditis elegans </a>Threshold cycle C/EBP C12FDG CASAVA cBAF CCAAT/enhancer binding protein Fluorescein-di-β-d-galactopyranoside Consensus assessment of sequence and variation Cardiac BAF 13 CBX7 CDC Chromobox protein homologue 7 Cell division cycle <ul style="display: flex;"><li style="flex:1">CDK </li><li style="flex:1">Cyclin dependent kinase </li></ul><ul style="display: flex;"><li style="flex:1">CKI </li><li style="flex:1">Cyclin dependent kinase inhibitor </li></ul>cyclin-dependent kinase inhibitor 1A cyclin-dependent kinase inhibitor 2A Complementary DNA CDKN1 CDKN2 cDNA CGH ChIP CML Comparative genomic hybridisation Chromatin immunoprecipitation Chronic myeloid leukaemia <ul style="display: flex;"><li style="flex:1">Clinical Sciences Centre </li><li style="flex:1">CSC </li></ul><ul style="display: flex;"><li style="flex:1">CSC </li><li style="flex:1">Cancer stem cell </li></ul><ul style="display: flex;"><li style="flex:1">CSF </li><li style="flex:1">Colony-stimulating factor </li></ul>CTGF CXCL CXCR Connective tissue growth factor CXC chemokine ligand CXC chemokine receptor DAPI DDR DMEM DMSO DNA 4,6-diamidino-2-phenylindole DNA damage response Dulbecco’s modified Eagle’s medium Dimethyl sulfoxide Deoxyribonucleic acid DNA methyltransferase Deoxyribonucleotide triphosphate Double strand DNMT dNTP ds <ul style="display: flex;"><li style="flex:1">DTT </li><li style="flex:1">Dithiothreitol </li></ul>E.coli EDTA EIF Escherichia coli Ethylenediaminetetraacetic acid Translation initiation factor ELAND EMBL ER Efficient large-scale alignment of nucleotide databases European Molecular Biology Laboratory Oestrogen receptor esBAF ETS Embryonic stem cell BAF E26 oncogene homologue <ul style="display: flex;"><li style="flex:1">EZH2 </li><li style="flex:1">Enhancer of zeste homologue 2 </li></ul>FBS FGF Foetal bovine serum Fibroblast growth factors 14 <ul style="display: flex;"><li style="flex:1">FOXO </li><li style="flex:1">Forkhead box, sub-group O </li></ul>Phosphorylated histone 2, variant X Gram γH2AX g<ul style="display: flex;"><li style="flex:1">G</li><li style="flex:1">Guanine </li></ul>GDF GFP GLB1 GM Growth differentiation factor Green fluorescent protein Galactosidase, beta 1 granulocyte-macrophage <ul style="display: flex;"><li style="flex:1">Gene ontology </li><li style="flex:1">GO </li></ul><ul style="display: flex;"><li style="flex:1">GR </li><li style="flex:1">Glucocorticoid receptor </li></ul>Gene Set Enrichment Analysis glycogen synthase kinase 3 β-glucuronidase GSEA GSK3 GUS-B <ul style="display: flex;"><li style="flex:1">H</li><li style="flex:1">Histone </li></ul><ul style="display: flex;"><li style="flex:1">HAT </li><li style="flex:1">Histone acetyl-transferase </li></ul><ul style="display: flex;"><li style="flex:1">High content analysis </li><li style="flex:1">HCA </li></ul>HCC HDAC HEK Hepatocellular carcinoma Histone deacetylase Human embryonic kidney 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hutchinson–Gilford progeria syndrome Hypoxia inducible factor 1 Histone repressor A HEPES HGPS HIF HIRA HMG HMT HP High mobility group Histone methyltransferase Heterochromatic protein Human pancreatic duct epithelial Human papilloma virus 16 Hour HPDE HPV16 hHRAS HSCs hTERT HTM HUGO HZI Harvey rat sarcoma gene Hematopoietic stem cells Human telomerase reverse transcriptase High throughput microscopy Human genome organisation Helmholtz Centre for Infection

A Genetic Screening Identifies a Component of the SWI/SNF Complex, Arid1b As a Senescence Regulator

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