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Copyright and Use of This Thesis This Thesis Must Be Used in Accordance with the Provisions of the Copyright Act 1968 COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Director of Copyright Services sydney.edu.au/copyright Biochemical and structural analysis of the Nucleosome Remodelling and Deacetylase (NuRD) complex Saad Salman M Alqarni A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Molecular Bioscience Faculty of Science University of Sydney 2013 ii Declaration The work described in this Thesis was performed between March 2009 and June 2013 in the School of Molecular Bioscience (formerly the School of Molecular and Microbial Biosciences) 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. Saad Salman M Alqarni June 2013 iii Abstract This PhD thesis is a biochemical and structural analysis of elements and interactions of the Nucleosome Remodelling and Deacetylase (NuRD) complex. Chapter 1 presents a general introduction to NuRD. The NuRD complex is a widely conserved transcriptional coregulator that harbours both nucleosome remodelling and histone deacetylase activities. Abnormalities in a number of its constituent proteins are associated with both cancer and ageing. Although we have a fairly complete ‘parts list’ for NuRD, our understanding of what the parts actually look like and, in particular, how they fit together lags a long way behind. In mammals, the NuRD complex consists of approximately 10 proteins that are consistently observed in purifications of the complex. These components are CHD3/4, HDAC1/2, p66α/p66β, MBD2/3, RbAp46/48 and MTA1-3. So far, there are three- dimensional structures of several subunits: namely RbAp46/48, HDAC2, and structures of a few isolated domains, including the PHD domains and one chromodomain of CHD4, the methyl-DNA binding domain of MBD2 and the SANT domain of MTA3. MTA1, MTA2 and MTA3 have been found in distinct NuRD complexes. MTA1 is now considered to be one of the most up-regulated proteins in human cancers. However, essentially nothing is known at the molecular level about how MTA1 either mediates its normal functions or acts in cancer. The N-terminal half of MTA-family proteins is well-conserved between family members and species and is predicted to harbour BAH, ELM2, SANT and GATA-type zinc finger (ZnF) domains. Chapter 2 presents efforts to obtain structural information on the N-terminal domains of MTA1 using bacterial and insect cell expression approaches. Although many inter-subunit interactions have been reported, the data are almost all limited to GST-pulldown and co-immunoprecipitation assays. The single structure that does exist is of a ~40-residue coiled-coil formed between MBD2 and p66α; otherwise we have no information on how this ~1-MDa complex is assembled. In order to begin to understand the makeup of the NuRD complex, we focused on inter- subunit interactions. RbAp48 and MTA1 are key components of the NuRD complex, and previous studies have reported that MTA1 interacts with the core complex RbAp46/48 and HDAC1/2. However, the molecular basis for this interaction is not iv known. Chapter 3 describes work carried out using pulldown assays to probe the interaction between the subunits RbAp46/48 and MTA1/2. Both MTA1 and MTA2 were found to bind to RbAp46/48 using the C-terminal end. Chapters 4 and 5 describe a more detailed analysis of this interaction. The interaction was confirmed by isothermal titration calorimetry (ITC) and the structure of MTA1-RbAp48 complex was determined using X-ray crystallography. The interaction is mediated by the C-terminal motif (KRAARR) of MTA1. It seems that the MTA1-RbAp48 interaction is conserved across species containing a NuRD-like complex. The structure reveals that RbAp48, which is a WD40 protein, interacts with MTA1 specifically via the histone H4 binding site, which is located to the side groove on RbAp48. These data provide a starting point for understanding both the assembly of the NuRD complex and its biochemical mechanism of action. Chapter 6 represents a final discussion that puts our results into context. v Acknowledgements In the Name of Allah, the Most Beneficent, the Most Merciful. The prophet Mohammed (Peace be upon him) said, He who would not be thankful to people, he who will not be thankful to ALLAH”. During this long and challenging journey, several people have helped me with their time, advice, experience, and patience. I would therefore like to offer my sincere thanks to all of them. First of all, I would like to express my deep thanks to my supervisor, Joel Mackay, for his guidance, encouragement and support throughout the study, and especially for his patience and guidance during the writing process. It has been a privilege to work with and learn from him over this period of intense effort. Thanks also to my associate supervisor, Jacqui Matthews, for her helpful advice, especially in matters of project planning and thesis completion. I would like to thank our collaborators from the Laue laboratory at the University of Cambridge, particularly Ernest Laue, who have been wonderful to work with. Special thanks go to Ana, who has helped me with all the work related to X-ray crystallography. Ana’s help has been invaluable on the structure-solving front. Thank you to Sandro for teaching me how to use Tecan and Mosquito. Thank you to Philippa for her help with the ITC experiments. Thanks to Chu Kong and Pep, whose guidance and friendship I will always appreciate, along with their helpful contribution to the work on insect cell expression. A big thank you to Nick, Michael, Philippa, and Cuong for their constructive advice and considerable efforts in proof reading this thesis; and to the occupants of Room 632― Cuong, Ivan and Mitch―who often acted as troubleshooters and helped me in other ways over the years. Thanks to past and current members of the Mackay, Matthews and Sunde labs (Alexei, Angela, Ann, Dorothy, Eija, Elise, Herman, Jane, Katschi, Krystal, Liza, Lorna, Margie, Marylène, Michael, Mugdha, Neil, Paul, Paula, Robyn, Roland, Sandra, Sarah, Soumya, Tom and Vanessa) for making it such a great environment to work in. All of them provided me with help and advice at various stages of my PhD. vi I also appreciate the financial support from King Saud bin Abdulaziz University for Health Sciences. thank you for all the support you have provided in your own special ways. I ,أمي ,أبي To am deeply indebted to my parents for their invaluable support and constant encouragement. All I can do is to say thanks for everything, and may Allah give you all the best in return. My heartfelt gratitude to my wife for all the love, endless support, timely advice, incredible patience and consideration she has shown to me throughout the course of this work. Without her support and encouragement, I could not have finished this work. She was the one who kept our family functioning, and I understand how difficult this was for her. Lastly, I am immensely thankful to my large family back home in Saudi Arabia. Thank you to my brothers and sisters for all their support. Thank you to all my friends here in Sydney and back in Saudi Arabia. vii Contents Declaration………………………………………………….………………………...ii Abstract ......................................................................................................................... iii Acknowledgment......................................................................................................... v Contents......................................................................................................................vii Abbreviations......................................................................................................... .xiv Publications ........................................................................................................... xvii Chapter 1. Introduction: DNA, Histones and Chromatin........................................... 1 1.1 DNA packaging into chromatin ......................................................................... 1 1.2 Gene regulation at the chromatin level .............................................................. 2 1.3 Histone modifications and chromatin remodelling ............................................ 3 1.3.1 Histone modifications ................................................................................. 3 1.3.2 Chromatin remodelling ..............................................................................
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