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The Schizosaccharomyces Pombe Mediator

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The Schizosaccharomyces Pombe Mediator FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN PhD thesis Michela Venturi The Schizosaccharomyces pombe Mediator. Roles in heterochromatinization and analysis of additional subunits Med9/Med11 This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen Academic advisor: Dr. Steen Holmberg Submitted: 28/03/13 Preface This PhD thesis represents the results of my PhD project carried out at the Transcription, Chromatin and DNA Repair Laboratory at University of Copenhagen, Department of Biology under the supervision of Dr. Steen Holmberg and as result of a collaboration with Dr. Claes M. Gustafsson’s Laboratory in Stockholm and Gothenburg, Sweden. This PhD project has been partly funded by “Regione Autonoma della Sardegna”, which provided funding for my living expenses and tuition fees. A paper written in collaboration with Michael Thorsen, Heidi Hansen, Steen Holmberg and Genevieve Thon is also included. 1 Table of Contents List of abbreviations....................................................................................................................................... 4 English abstract .............................................................................................................................................. 7 Danish abstract .............................................................................................................................................. 9 INTRODUCTION ......................................................................................................................................... 10 Transcription of protein-coding genes in eukaryotes ............................................................................. 10 Activators and co-activators of transcription ............................................................................................ 11 RNA polymerase II....................................................................................................................................... 11 Pre-Initiation Complex formation ............................................................................................................... 12 Initiation and promoter clearance .............................................................................................................. 13 Mediator ........................................................................................................................................................ 14 Mediator discovery and structure .............................................................................................................. 14 Mediator complex functions ....................................................................................................................... 16 Mediator sub-modules................................................................................................................................. 17 Head module ................................................................................................................................................ 17 The Middle module ...................................................................................................................................... 17 The CDK8 module ....................................................................................................................................... 18 The Tail module ........................................................................................................................................... 18 Novel Mediator subunits in S. pombe ....................................................................................................... 19 Med18 and Med20 ....................................................................................................................................... 19 scCSE2/scMED9 and scMED11 ............................................................................................................... 21 Chromatin ...................................................................................................................................................... 22 Location of heterochromatin in S. pombe ................................................................................................ 23 Heterochromatin assembly: initiation, spreading and maintenance .................................................... 25 Heterochromatin initiation ........................................................................................................................... 26 Effect of rik1+, raf1+, raf2+, pcu4+ and CLRC components transient deletion on heterochromatin establishment ............................................................................................................................................... 28 Effect of dcr1+ and RDRC components transient deletion on centromeric heterochromatin and H3K9Me2 ...................................................................................................................................................... 29 Heterochromatin spreading and maintenance ....................................................................................... 30 Aims of the project ....................................................................................................................................... 31 RESULTS ...................................................................................................................................................... 32 Identification of S. pombe Med11 (SPAC23A1.15c) .............................................................................. 32 Genetic analysis of SPAC23A1.15c ......................................................................................................... 32 Tagging of Med11a ...................................................................................................................................... 34 Genetic analysis of med11a+-3HA ............................................................................................................ 34 Western blot analysis of Med11a-HA ....................................................................................................... 35 2 Identification of S. pombe Med9 SPCC645.12c...................................................................................... 39 Genetic analysis of med9+ (SPAC645.12c) ............................................................................................. 41 Resistance of Δmed9 strains to stress ..................................................................................................... 42 Affinity tagging of Med9 .............................................................................................................................. 43 Western blot analysis of 13Myc-tagged Med9 ........................................................................................ 44 Tandem Affinity Purification (TAP) of Mediator ....................................................................................... 45 Tagging of Med9 with a FLAG epitope using Gateway® Recombination Cloning Technology ...... 50 Identification of med11b+ (SPAC644.10) in S. pombe ........................................................................... 54 Genetic analysis of med11b+ ..................................................................................................................... 56 Affinity tagging of Med11b .......................................................................................................................... 57 Contribution of Mediator to heterochromatin formation and maintenance .......................................... 60 Chromosome loss assay ............................................................................................................................ 60 Mini-chromosome loss assay in Δmed12 mutants ................................................................................. 62 Mini-chromosome loss assay in Δmed18 mutants ................................................................................. 62 Sensitivity of Δmed12 and Δmed18 mutants to Thiabendazole (TBZ) ................................................ 63 Sensitivity assay of Δmed12 and Δmed18 mutants to stress ............................................................... 64 Centromere transcript quantification for the Δmed18 mutant ............................................................... 64 Telomere length in Mediator mutants ....................................................................................................... 66 Mediator regulates non-coding RNA transcription at fission yeast centromeres ............................... 69 DISCUSSION ............................................................................................................................................... 80 Mediator subunits Med9, Med11a and Med11b and their role in heterochromatin ........................... 80 Chromosome loss in Mediator mutants .................................................................................................... 80 Drug sensitivity assay of Mediator mutants ............................................................................................. 81 Silencing of the centromeric regions in Mediator mutants..................................................................... 81 Mediator and telomere length ...................................................................................................................
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