P53 and Sp1 Associated Rnas Act As Non-Coding Transcriptional Regulators at Homologous Loci
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p53 and Sp1 Associated RNAs Act as Non-coding Transcriptional Regulators at Homologous Loci Rachel Hughes A thesis in fulfilment of the requirements for the degree of Master of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science April 2016 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Hughes First name: Rachel Other name/s: Genevieve Abbreviation for degree as given in the University calendar: MPhil School: Biotechnology and Biomolecular Sciences Faculty: Science Title: p53 and Sp1 Associated RNAs Act as Non-Coding Transcriptional Regulators at Homologous Loci Abstract 350 words maximum: (PLEASE TYPE) RNA functionality has been proven to extend far beyond the outdated protein coding divide, as transcripts not bound for translation are instead found to act as endogenous messengers and moderators, utilising inherent sequence homology to interact with DNA or protein targets. A RIP-Seq of six major transcription factors including p53 and Sp1 uncovered multiple bound RNAs, some of which were interestingly protein coding. Of these, the HIST1 H1 D and SF3B5 mRNAs were knocked down in order to investigate the ability of their affiliated transcription factors to localise to the target genes. Both RNAs demonstrated an inherent ability to modulate transcription factor localisation to homology containing loci in cis by acting as protein guides in the case of SF3B5, and as decoys in HIST1 H1 D. Expression of other linker histone H1 proteins was also observed to be under H1ST1 H1 D RNA regulation, suggesting a network involving the p53 tumour suppressive cascade which induces senescence in damaged cells. Together, the data confirms an innate complexity of RNA that is only beginning to be unveiled, with major lies to tumour preventative pathways and therefore therapeutic possibilities. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or bo I or part of this thesis or dissertation. I also authorise University Mi se the 350 word abstract of my thesis in Dissertation doctoral theses only). The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. 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Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in this thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed Date Table of Contents Abstract i Acknowledgements ii Abbreviations iii List of Figures v List of Tables vi Chapter 1: Introduction 1.1 RNA 2 1.1.1 Protein Coding and Infrastructural RNA 2 1.1.2 Non-coding RNA 3 1.1.3 RNAs in Disease 8 1.2 The Search for Transcription Factor Associated RNAs 9 1.2.1 RIP-Seq 10 1.3 RNAs of Interest 10 1.3.1 HIST1H1D 10 1.3.2 SF3B5 12 1.4 Associated Transcription Factors 13 1.4.1 p53 14 1.4.2 Sp1 16 1.5 Aims 18 Chapter 2: Materials and Methods 19 2.1 Cell lines 19 2.2 Reagents 19 2.3 Buffers 20 2.4 Chemicals and Solutions 21 2.5 Media 21 2.6 Oligodeoxynucleotides 22 2.7 Primers 22 2.8 Antibodies 24 2.9 Biotinylated Antisense ODNs 24 2.10 Deep Sequencing 25 2.11 Cell Culture 25 2.12 Transfection 26 12.12.1 Seeding 26 2.12.2 Transfection Complexes 27 2.13 Knockdown Efficiency 28 2.13.1 RNA Isolation 28 2.13.2 Removing DNA Contaminants 28 2.13.3 Reverse Transcription 28 2.13.4 Quantitative Real Time PCR 28 2.14 Chromatin Immunoprecipitation for Transcription Factor Localisation 30 2.14.1 Cross-linking 30 2.14.2 Lysis and Sonication 31 2.14.3 Binding the Antibody 31 2.14.4 Immunoprecipitation 31 2.14.5 DNA Extraction 33 2.14.6 Quantitative PCR 33 2.14.7 Standard Curve 34 2.15 UCSC Genome Browser 34 2.16 Biotinylated asODN-RNA Pulldown 34 2.16.1 Immunoprecipitation 34 2.16.2 Validation of the Immunoprecipitated RNA 36 Chapter 3: Results 37 3.1 Deep Sequencing Alignment 37 3.2 Determining the Efficacy of asODN Mediated RNA Knockdown 42 3.3 Enrichment or Loss of Transcription Factor Localisation After RNA Knockdown 45 3.4 Enrichment or Loss at Homologous Loci 48 3.4.1 Determining Possible Homologous Targets 48 3.4.2 Enrichment or Loss of Transcription Factor Localisation at Homologous Loci 50 3.5 Expression at Homologous Loci 52 3.6 Biotinylated asODN-RNA Pulldown 54 Chapter 4: Discussion 55 4.1 RNA-Directed Transcriptional Activation and Repression 55 4.2 Trans-regulation at Homologous Loci 60 4.3 mRNA Acting as ncRNA 62 4.4 HIST1H1D in Cell Senescence 66 4.5 Looking Ahead 67 Chapter 5: Conclusion 69 References 70 Appendix 81 A. UCSC Genome Browser 81 B. Sequenced Transcripts From RIP-Seq Data 84 Abstract RNA functionality has been proven to extend far beyond the outdated protein coding divide, as transcripts not bound for translation are instead found to act as endogenous messengers and moderators, utilising inherent sequence homology to interact with DNA or protein targets. A RIP-Seq of six major transcription factors including p53 and Sp1 uncovered multiple bound RNAs, some of which were interestingly protein coding. Of these, the HIST1H1D and SF3B5 mRNAs were knocked down in order to investigate the ability of their affiliated transcription factors to localise to the target genes. Both RNAs demonstrated an inherent ability to modulate transcription factor localisation to homology containing loci in cis by acting as protein guides in the case of SF3B5, and as decoys in HIST1H1D. Expression of other linker histone H1 proteins was also observed to be under HIST1H1D RNA regulation, suggesting a network involving the p53 tumour suppressive cascade which induces senescence in damaged cells. Together, the data confirms an innate complexity of RNA that is only beginning to be unveiled, with major ties to tumour preventative pathways and therefore therapeutic possibilities. i Acknowledgements Preliminary immunoprecipitation and deep sequencing data was provided with permission by Kevin Morris, John Burdach and Merlin Crossley. Many thanks to Kevin Morris for the insightful guidance over the course of this project, as well as Rosie, Chris, Galina, Caio, Nick and Albert of the Morris lab who have been kind enough to answer questions along the way. Thanks also to Louise Lutze- Mann and Matthew Clemson for the additional support. This project is dedicated to Bill, Julie, Derek, Lauren and Zac who's continued encouragement has made the achievement possible. ii Abbreviations asODN - antisense oligodeoxynucleotide BLAT - basic local alignment tool cDNA - complementary deoxyribonucleic acid ChIP - chromatin immunoprecipitation Ct - cycle threshold DNA - deoxyribonucleic acid F1- forward primer 1 F2- forward primer 2 HIST1H1D - protein member D of the histone cluster 1 family H1 - histone cluster 1 lncRNA - long non-coding ribonucleic acid mRNA - messenger ribonucleic acid ncRNA - non-coding ribonucleic acid ODN - oligodeoxynucleotide PCR - polymerase chain