Identification and functional characterisation of non-canonical DNA methylation readers in the mammalian brain Sufyaan Mohamed School of Molecular Sciences ARC Centre of Excellence in Plant Energy Biology Harry Perkins Institute of Medical Research 2020 Supervisors: Professor Ryan Lister (60%) Associate Professor Ozren Bogdanovic (20%) Dr Yuliya Karpievitch (10%) Professor Ian Small (5%) Associate Professor Monika Murcha (5%) i i. Thesis Declaration: I, Sufyaan Mohamed, certify that: The work in this thesis has been substantially accomplished during enrolment in this degree. This thesis does not contain material which has been accepted for the reward of any other degree or diploma in my name, in any university or other tertiary institution. No part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint award of this degree. This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text. The work(s) presented here are not in any way a violation or infringement of any copyright, trademark, patent, or other rights whatsoever of any person. Mass spectrometry samples were processed by Ino Karemaker. The ProteoMM code used for the analysis of the human and mouse datasets described in Chapter III was written by Dr. Yuliya Karpievitch This thesis contains published work and/or work prepared for publication, some of which has been co-authored. Signature: Date: 07/12/2020 ii ii. Abstract DNA methylation is a covalent modification found in all vertebrates and functions as an additional layer of information through which transcription may be controlled. Changes in transcription may be modulated by proteins that bind to DNA methylation termed methyl ‘readers’. DNA Methylation (mC) within mammals occurs predominantly in the CG dinucleotide context (mCG), but non-CG contexts (mCH, where H = A, C or T) exist within restricted cell types. In the brain, mCH accumulates to become the dominant form of DNA methylation in adult neurons, coinciding with synaptogenesis. Whilst many mCG reader proteins have been identified and characterised, the relatively recent discovery of mCA has meant that only one mCH reader, MECP2, has been discovered. This PhD focused on identifying many potential mCG and mCA reader candidate proteins in the human and mouse brain. A DNA pull-down utilising methylated probes and unmethylated controls were coupled with quantitative mass spectrometry (MS) to screen for potential mCG and mCA binders in human and mouse brain. Chapter 1 presents an introduction to DNA methylation in mammals, and the mechanisms by which this epigenetic mark is deposited, removed and read. Additionally, Chapter 1 discusses some well characterised mCG reader proteins, their effects on transcription and experimental challenges faced in characterising their binding. Chapter 2 contains relevant methods to experiments discussed in Chapters 3,4 and 5. A novel, multivariate Proteomics analysis tool, ProteoMM, was developed to analyse the DNA pull-down MS data. The rationale behind development of ProteoMM, its optimisation and efficacy are detailed in Chapter 3. The identification of novel mCG and mCA readers are discussed in Chapters 4 and 5 respectively. Results from the mCA screen established a list of candidate mCA readers. The top mCA reader, MBD2, was chosen for biochemical validation experiments to confirm a direct, specific interaction for mCA. Details regarding the recombinant expression, purification and DNA binding ability of this protein are also detailed in Chapter 5. This work constitutes the first comprehensive combined analysis of mCG readers in human and mouse brain. ProteoMM identified a significant overlap in the binding of proteins enriched in each species. Further, this study constitutes the first mCA reader screen, and confirms a direct, specific affinity of MBD2 for mCA providing a crucial repository of mCG and mCA binders future studies can build upon (Chapter 6). iii iii. Acknowledgements There are many names and faces of people that cross my mind when thinking about the completion of my PhD. First and foremost is my family, who have supported me through the ups and downs of my PhD and were there through the highs, lows and stressful situations. You were the backbone I needed, emotionally and financially, especially in the latter years of my PhD. Another individual I am eternally grateful to is my partner Nathan for his understanding, patience, support and confidence. I would like to thank my supervisor Prof. Ryan Lister for his feedback, support, and for inspiring a sense of drive and excellence within me, helping me become a better scientist. I would also like to thank my co-supervisors Prof. Ian Small, Assoc. Prof. Prof. Monika Murcha and Assoc. Prof. Ozren Bogdanovic for being good mentors and individuals I could go to for advice when needed. I am really grateful to Dr Yuliya Karpievitch for the numerous sessions spent in her office in which I learnt vital programming skills. I would also like to extend my gratification to Dr Ethan Ford, a master molecular biologist and great individual with sound advice in my early years, as well as Dr Marina Oliva for her advice, banter and comedic relief. Some other notable post-docs include Dr Daniel Poppe for his patience, and for sharing his knowledge and time in cell culture and microscopy, Christian Pflueger for his knowledge (in all matters) and Jahnvi Pflueger for her time in ensuring the laboratory functioned efficiently and for running the numerous NGS libraries I had prepared. Thanks to Tessa for helping revise parts of my thesis, and to the many other students in the lab. Extending out of the Lister lab, I would like to thank Prof. Charlie Bond for making time, having patience and allowing me to learn about recombinant protein expression in his lab. Special thanks to Dr Gavin Knott and Dr Amanda Blythe within this lab among the other, always friendly members for their invaluable advice and patience in teaching me how to operate the sensitive chromatography systems. I would also like to thank Dr Cathie Small, who has been a fond part of my memories since I was introduced to the PEB family as an undergraduate. Alongside, I would like to extend thanks to the admin team, most notably Deb for handling orders, errors and anything lab reagent related, and to Geetha for always being a friendly face, helpful and ever dependable in all matters related to admin. Thanks go out to the many individuals and PhD students that made my experience a more wholesome one. Some have, been and gone, but I am grateful to have known you all. Special mention to Katharina, Karina, Jakob, Jon, Dennis, Tim, Arnold and Max. Thank you for the memories and support, in so many instances, you were all instrumental to my wellbeing. This research was supported by an Australian Government Research Training Program (RTP) Scholarship. iv iv. Authorship declaration This thesis contains work that has been prepared for publication. Details of work: Identification of mCG and mCA readers in human and mouse brain Location in thesis: Chapters 3, 4 and 5 Contribution(s) to work: The processing of mass spectrometry samples was performed by Dr Ino Karemaker. The development of ProteoMM was performed by Dr Yuliya Karpievitch who was responsible for development of analysis code concerning the integration, imputation, and normalisation of these datasets. The totality of remaining experiments was performed by the student. These include isolation of protein extract from human and mouse brain, DNA pull-downs, Western blots, optimising and benchmarking ProteoMM, recombinant DNA cloning, recombinant protein expression and purification, electrophoretic mobility shift assays, and all other data analysis and plots within thesis. Student signature: Date:07/12/2020 I, Ryan Lister, certify that the student’s statements regarding their contribution to each of the works listed above are correct. Coordinating supervisor signature Date:07/12/2020 v Table of contents i. Thesis Declaration: ii ii. Abstract iii iii. Acknowledgements iv iv. Authorship declaration v Chapter I Introduction I-11 Epigenetics I-11 I.1.1 Definition of epigenetics I-11 Sculpting the epigenome landscape I-12 I.2.1 Layers of the epigenome I-13 Features of DNA methylation in mammals I-19 I.3.1 DNA methylation at CpG islands (CGIs) I-19 I.3.2 DNA methylation at enhancers and intergenic regions I-20 I.3.3 DNA methylation and alternative gene splicing I-22 I.3.4 CH methylation I-23 Writing, maintenance, and removal of DNA methylation in mammalian genomes I-24 I.4.1 Writers of DNA methylation I-24 I.4.2 Erasure of DNA methylation I-26 Readers of DNA methylation I-28 I.5.1 The MBD family I-28 I.5.2 Set and RING- associated (SRA) family I-35 I.5.3 Kaiso and the Broad complex, Tramtrack, Bric-á-brac or Poxvirus Zinc-finger (BTB/POZ) family I-36 I.5.4 Expansion of the mCG reader repertoire and the need for contextually relevant, multifaceted characterisation approaches I-38 I.5.5 A need for mCH reader characterisation I-40 Outline of thesis I-41 References I-42 Chapter II Materials and methods II-67 DNA pull-down coupled to Mass spectrometry II-67 II.1.1 Nuclei isolation and protein extraction from mammalian brain II-67 II.1.2 Preparation of biotinylated probes II-67 II.1.3