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At the University of Edinburgh This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Investigating the role of RNA interference in the fission yeast Schizosaccharomyces japonicus Elliott Chapman Submitted for the degree of Doctor of Philosophy University of Edinburgh 2017 Table of Contents Abbreviations ............................................................. xiii Acknowledgements ..................................................... xvii Declaration .................................................................. xix Lay Summary ............................................................... xxi Abstract ..................................................................... xxiii Chapter 1 – Introduction ................................................. 1 1.1 – Epigenetics ............................................................................................. 3 1.2 - Chromatin ............................................................................................... 5 1.2.1 - Histone Post-Translational Modifications (PTMs) ......................... 8 1.2.2 - Histone Variants .............................................................................16 1.3 - The Centromere .................................................................................... 24 1.3.1 - DNA elements underlying the centromere .................................... 24 1.3.2 - The molecular components of the centromere ............................. 29 1.4 - Transposable Elements ........................................................................ 32 1.4.1 – DNA Transposons ......................................................................... 32 1.4.2 – Retrotransposons .......................................................................... 33 1.5 - RNA Interference ................................................................................. 37 1.5.1 - siRNAs ............................................................................................ 39 1.5.2 - miRNAs ........................................................................................... 41 1.5.3 - piRNAs ........................................................................................... 43 1.6 - Fission Yeast ......................................................................................... 45 i 1.6.1 - RNAi in S. pombe ........................................................................... 46 1.6.2 - Silencing of retrotransposons in S. pombe .................................... 50 1.6.3 – S. japonicus ................................................................................... 52 1.7 – Aims ..................................................................................................... 56 Chapter 2 – Materials and methods ............................. 59 2.1 – Cloning and Fragment construction ................................................... 61 2.1.1 – Preparation of chemically competent E. coli ................................ 61 2.1.2 – E. coli transformation ................................................................... 61 2.1.3 – E. coli Plasmid Purification .......................................................... 61 2.1.4 – Pfx PCR ......................................................................................... 62 2.1.5 – Agarose Gel Electrophoresis ......................................................... 62 2.1.6 – Split Marker Fusion PCR .............................................................. 63 2.1.7 – Tagging plasmid Construction and linearization ......................... 64 2.2 – Fission Yeast Growth .......................................................................... 65 2.2.1 – S. pombe/S. japonicus Media and Drugs ..................................... 65 2.2.2 – Cell culture and harvest ................................................................ 66 2.2.3 – Genetic crosses ............................................................................. 67 2.2.4 – Long term storage of fission yeast ................................................ 67 2.3 – Fission Yeast Transformation and Genotyping .................................. 68 2.3.1 – LiAcTE transformation of S. pombe ............................................. 68 2.3.2 – Electroporation of S. japonicus .................................................... 68 ii 2.3.4 – Colony PCR ................................................................................... 69 2.4 – Fission Yeast Nucleic Acid Methods ................................................... 69 2.4.1 – Genomic DNA extraction .............................................................. 69 2.4.2 – Total RNA extraction .................................................................... 70 2.4.3 – Small RNA extraction ................................................................... 70 2.4.4 – Reverse Transcription ................................................................... 71 2.4.5 – qPCR ............................................................................................. 72 2.4.6 – Sanger Sequencing ....................................................................... 72 2.4.7 – Southern Blot ................................................................................ 73 2.4.8 – Northern Blot ............................................................................... 74 2.4.9 – Retrotransposon integration sequencing .................................... 75 2.4.10 – RNA-Seq ..................................................................................... 76 2.4.11 – siRNA-Seq ....................................................................................77 2.4.12 – Genome Re-sequencing .............................................................. 78 2.5 – Fission Yeast Protein Methods ........................................................... 78 2.5.1 – Chromatin Immunoprecipitation (ChIP) ..................................... 78 2.5.2 – Protein extraction ......................................................................... 79 2.5.3 – Immunoprecipitation ................................................................... 80 2.5.4 – Western Blot ................................................................................. 80 2.6 – Fission Yeast Genetic Assays .............................................................. 82 2.6.1 – Spot Tests ...................................................................................... 82 iii 2.6.2 – TSA Assay ..................................................................................... 82 2.6.3 – Retrotransposition Assay ............................................................. 82 Chapter 3 – Investigating the role of RNAi and heterochromatin in S. japonicus ................................... 91 3.1 - Introduction.......................................................................................... 93 3.2 - Annotation and Sequence analysis of S. japonicus RNAi genes ......... 96 3.3 - Most core RNAi and heterochromatin factor deletions cannot be recovered in S. japonicus ............................................................................. 98 3.4 - A factor involved solely in heterochromatin establishment can be deleted in S. japonicus ............................................................................... 104 3.5 - RNAi factors can be genetically tagged with no impact on function 105 3.6 - Chp1-GFP co-localises with H3K9me2 at retrotransposons............. 109 3.7 - Discussion ........................................................................................... 113 Chapter 4 – Investigating the role of dcr1+ in S. japonicus .................................................................................... 119 4.1 - Introduction ........................................................................................ 121 4.2 – Deletion or disruption mutants of dcr1+ exhibit differing growth phenotypes ................................................................................................. 122 4.3 - Disruption of dcr1+ appears to impact functional centromere formation .................................................................................................... 124 4.4 - Disruption of dcr1+ dramatically changes the small RNA pool ........ 125 4.5 – Disruption of dcr1+also alters small RNA production from non- retrotransposon loci ................................................................................... 138 4.6 - Disruption of dcr1+ reduces H3K9me2 levels at specific retrotransposons ........................................................................................ 139 4.7 - Histone H3 is also lost from retroelements that lose H3K9me2 ....... 141 iv 4.8 - Overall nucleosome occupancy is reduced at elements that lose H3K9me2
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