COMPASS Mediates Transgenerational Epigenetic Inheritance in Caenorhabditis elegans By: Rosamund Clifford A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences School of Biology Submission Date February 2021 Acknowledgements There are a great many people who have contributed to the development of this thesis, who I would like to thank here. Professor Ian Hope, who took on the role of my primary supervisor midway through my PhD and guided me through to the end with constant support and advice during the bulk of the experiments and entire writing process. My first PhD supervisor, Dr Ron Chen, whose ideas and support in the first two years gave me a solid foundation on which to build this thesis. Professor Mark Dickman and the Dickman group at the University of Sheffield, in particular Dr Eleanor Hanson, Dr Joby Cole and Dr Caroline Evans, for their technical expertise in LC-MS/MS and support with performing the experiments described here. Professor Julie Ahringer and the Ahringer group at the University of Cambridge, for their support with the immunofluorescence microscopy experiments, which I learned to do under the supervision of Yan Dong on a visit to the Ahringer lab. Thanks also to the imaging team at the Gurdon Institute, particularly Dr Nicola Lawrence, for training me to use a confocal microscope, and Dr Richard Butler, for writing the code that enabled analysis of the images. At the University of Leeds, my thanks go to Dr Ruth Hughes at the Bio-imaging facility for her advice on image analysis, and Dr Brittany Graham for helping me settle into the Hope lab. The MRC DiMeN DTP for funding and training opportunities, in particular Dr Emily Goodall, the DTP manager, for facilitating my extra funding applications that enabled me to learn about commercial mass spectrometry applications on a summer placement, and to visit the Ahringer lab in Cambridge. My friends and family, for their moral support through the trials and tribulations of the PhD process. Dheemanth, for sharing an office with me while writing this thesis, putting up with frequent interruptions to read sections and cheering me on to the finish line. 2 Table of Contents Chapter 1. Introduction ......................................................................................................... 16 1.1 Epigenetics ......................................................................................................................................... 16 1.1.1 Interest and controversy ..................................................................................... 16 1.1.2 Definition ............................................................................................................. 16 1.1.3 Mechanisms ......................................................................................................... 16 1.2 The Histone Code .............................................................................................................................. 17 1.2.1 Overview .............................................................................................................. 17 1.2.2 The nucleosome core particle ............................................................................. 17 1.2.3 Nomenclature ...................................................................................................... 18 1.2.4 How histone modifications regulate gene expression ........................................ 19 1.2.5 Cross-talk ............................................................................................................. 20 1.2.6 Transgenerational epigenetic inheritance ........................................................... 21 1.2.7 Methods and limitations of study ....................................................................... 24 1.3 Mass spectrometry technologies ...................................................................................................... 24 1.3.1 Principle of mass spectrometry ........................................................................... 24 1.3.2 Ionisation methods .............................................................................................. 25 1.3.3 Mass analysers and detectors ............................................................................. 25 1.3.4 Tandem mass spectrometry ................................................................................ 26 1.3.5 The Q-Exactive mass spectrometer ..................................................................... 27 1.3.6 Data acquisition methods .................................................................................... 28 1.4 Histone Proteomics ........................................................................................................................... 28 1.4.1 Proteomic approaches ......................................................................................... 28 1.4.2 Preparation of histones for bottom-up MS ......................................................... 30 1.4.3 Reverse-phase high-pressure liquid chromatography ........................................ 33 1.4.4 Data analysis ........................................................................................................ 34 3 1.5 COMPASS ........................................................................................................................................... 36 1.5.1 The complex ........................................................................................................ 36 1.5.2 Set1/SET-2 ........................................................................................................... 38 1.5.3 Cfp1/CFP-1 ........................................................................................................... 42 1.6 Thesis aims ........................................................................................................................................ 45 Chapter 2. Materials and Methods ....................................................................................... 46 2.1 C. elegans maintenance..................................................................................................................... 48 2.1.1 Standard growth conditions ................................................................................ 48 2.1.2 Synchronization and/or decontamination of strains by bleaching ..................... 49 2.2 Single worm PCR genotyping ............................................................................................................. 49 2.3 Mass spectrometry ............................................................................................................................ 51 2.3.1 Histone extraction ............................................................................................... 51 2.3.2 Proprionylation of Histone Samples .................................................................... 57 2.3.3 Loading ................................................................................................................ 58 2.3.4 Data acquisition ................................................................................................... 59 2.3.5 Raw data analysis ................................................................................................ 59 2.4 RNAi ................................................................................................................................................... 64 2.4.1 Experimental design ............................................................................................ 64 2.4.2 Slide preparation ................................................................................................. 66 2.4.3 Scoring protocol................................................................................................... 66 2.4.4 Data analysis ........................................................................................................ 66 2.4.5 Statistical testing ................................................................................................. 66 2.5 Lifespan assays .................................................................................................................................. 67 2.5.1 Set up ................................................................................................................... 67 2.5.2 Maintenance ........................................................................................................ 67 2.5.3 Exclusions ............................................................................................................ 67 2.5.4 Data analysis ........................................................................................................ 68 4 2.6 Developmental progression assay ..................................................................................................... 69 2.7 Immunofluorescence ......................................................................................................................... 70 2.7.1 Worm preparation/selection ............................................................................... 70 2.7.2 Slide preparation ................................................................................................. 70 2.7.3 Antibody staining ................................................................................................. 71 2.7.4 Antibody validation by dot blot