Understanding the Architecture, Assembly, and Interactome of the Nucleosome Remodelling and Deacetylase (NuRD) Complex Mehdi Sharifi Tabar A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy School of Life and Environmental Sciences Faculty of Science University of Sydney Sydney, Australia June 2019 i DECLARATION The work described in this Thesis was performed between August 2015 and December 2018 in the School of Life and Environmental Sciences at the University of Sydney. The experiments were carried out by the author, unless stated otherwise. This work has not been submitted, in part or in full, for a higher degree at any other institution. Mehdi Sharifi Tabar June 2019 ii ABSTRACT This thesis presents data to enhance our understanding of the composition, assembly, and structure of the nucleosome remodeling and deacetylase (NuRD) complex, a transcriptional coregulator that is strongly linked to cancer. Understanding the structure and function of the gene regulating complexes will lead to a better understanding of mechanisms of gene regulation. This should ultimately both allow insight into disease processes that originate from gene regulatory network malfunction and might enable reprogramming of the gene expression for application in health, agriculture and industry. The Thesis is broken into four sections: the first Chapter is a general introduction to gene regulation, the second chapter is materials and methods. Chapter 3 is a manuscript that is written about the stoichiometry and composition of the complex in cancer cells. Chapters 4 and 5 are results chapters investigate the structure of the subcomplexes. Chapter 6 discuss the finding of this thesis. Chapter 1 represents a general introduction to gene regulation and the NuRD complex. The NuRD complex is a 1mDa multisubunit transcriptional coregulator protein complex, which is widely expressed and harbours both chromatin remodelling and histone deacetylase activities. Abnormalities in a number of its components are associated with various types of cancers and neurodegenerative diseases. Although the core components of the complex have been well established, the structural basis of the complex has poorly been studied. Chapter 2 describes the details of the materials and methods used in this research. Chapter 3 investigates NuRD complex stoichiometry and interactome using a label-free quantitative proteomics method and provides biochemical evidence for two new direct interactors of the complex. Chapter 4 and 5 investigate the recombinantly produced subcomplexes using biochemical and a mass spectrometry-based hybrid method for structural modelling of the NuRD complex. Chapter 6 discuss the findings and outlines the future directions. iii ACKNOWLEDGEMENT Three and a half years after starting on my PhD adventure I have finally reached the end. This journey would not have been possible without the support of my supervisor Joel Mackay and lab mates. First of all, I would like to sincerely thank Joel Mackay whose guidance has been invaluable in all aspects of the thesis production. His generosity with both time and knowledge is deeply appreciated. I am highly grateful for his patience and encouragement in the face of the many bewilderment of research. It was a real privilege and an honour for not only to undertake a PhD under his supervision but also of his extraordinary human qualities. Enormous thanks are owed to Dr. Jason Low for teaching and demonstrating me mass spectrometry based proteomics and also being an inexhaustible source of useful discussions. I have no doubt that without Joel and Jason I would not be able to smoothly undertake my PhD. Special thanks also goes to Dr. Mario Torrado Delray, for his abundant assistance with protein-protein interaction and pulldown experiments, and Dr. Ana Silva for providing advice and help at various stages of my PhD and in various forms. I also would like to thank the entire Mackay & Matthews lab members and everybody in Structural Biology group and also the Mass Spectrometry core facility at CPC. The lab environment has been a fantastic place to work, with everyone always willing to help each other out. Last, but definitely not least, thank you very much to my lovely and kind wife, Diba, for her tremendous help and for always putting a smile on my face and making this journey stress free. I also extend my gratitude to my wonderful family for their unconditional support throughout my PhD, and always. iv CONTENTS Chapter1. Introduction .................................................................................... 1 1.1 Gene regulation ............................................................................................. 1 1.2 Chromatin ...................................................................................................... 2 1.3 The histone code hypothesis and chromatin remodelling .................................. 2 1.4 Histone acetylation and deacetylation .............................................................. 3 1.5 Chromatin remodelling protein complexes ........................................................ 5 1.6 Our structural understanding of chromatin remodellers ..................................... 5 1.7 The NuRD complex ......................................................................................... 8 1.8 Recruitment of the NuRD to target sites .......................................................... 9 1.9 Molecular function of the NuRD ..................................................................... 10 1.10 NuRD in pluripotency .................................................................................. 11 1.11 NuRD in disease ......................................................................................... 12 1.12 NuRD subunits ........................................................................................... 14 1.12.1 MTA1-3 ................................................................................................... 15 1.12.2 HDAC1/2 ................................................................................................. 16 1.12.3 CHD3-5 ................................................................................................... 17 1.12.4 GATAD2A/B ............................................................................................. 19 1.12.5 MBD2/3 .................................................................................................. 20 1.12.6 RBBP4/7 ................................................................................................. 20 1.12.7 CDK2AP1/DOC-1 ...................................................................................... 22 1.13 Aims of this thesis ...................................................................................... 22 Chapter 2: Materials and Methods ................................................................ 24 2.1 Materials ...................................................................................................... 24 2.2 Methods ...................................................................................................... 26 v 2.2.1 Cloning ..................................................................................................... 26 2.2.1.1 Primer design ......................................................................................... 26 2.2.1.2 RNA isolation & cDNA synthesis ............................................................... 26 2.2.1.3 Polymerase chain reaction (PCR) ............................................................. 26 2.2.1.4 Gibson assembly ..................................................................................... 27 2.2.1.5 Colony PCR ............................................................................................ 27 2.2.2 Cell culture ................................................................................................ 28 2.2.2.1 Expi293 cells .......................................................................................... 28 2.2.2.2 NTERA2, PC3 and MCF7 cell culture ......................................................... 28 2.2.3 siRNA knock down experiment ................................................................... 28 2.2.4 Quantitative real-time PCR (qRT-PCR, qPCR) ............................................... 29 2.2.5 Protein production and FLAG-affinity purification ......................................... 29 2.2.5.1 Protein production in mammalian EXPI293 cells ........................................ 30 2.2.5.2 Protein production and purification in cell free system (Rabbit Reticulocyte Lysate) .............................................................................................................. 30 2.2.5.3 Preparing sucrose gradients (tilting method) ............................................ 31 2.2.5.4 Protein concentration and buffer exchange .............................................. 31 2.2.6 Native NuRD complex pull down ................................................................. 31 2.2.6.1 Preparation of bead-bound-GST-FOG ....................................................... 31 2.2.6.2 NuRD extraction from nucleus ................................................................. 32 2.2.6.3 NuRD pulldown experiment ..................................................................... 32 2.2.7 Protein analysis ......................................................................................... 32 2.2.7.1 SDS-PAGE .............................................................................................