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This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Investigation into the role of LSH ATPase in chromatin remodelling Alina Gukova Doctor of Philosophy – University of Edinburgh – 2020 ABSTRACT Chromatin remodelling is a crucial nuclear process affecting replication, transcription and repair. Global reduction of DNA methylation is observed in Immunodeficiency-Centromeric Instability-Facial Anomaly (ICF) syndrome. Several proteins were found to be mutated in patients diagnosed with ICF, among them are LSH and CDCA7. LSH is a chromatin remodeller bearing homology to the members of Sf2 remodelling family. A point mutation in its ATPase lobe was identified in ICF. CDCA7 is a zinc finger protein that was recently found to be crucial for nucleosome remodelling activity of LSH. Several point mutations in its zinc finger domain were described in ICF patients. In vitro and in vivo studies have shown that LSH-/- phenotype demonstrates reduction of global DNA methylation, implying that chromatin remodelling LSH functions may be required for the efficient methyltransferase activity, linking this finding to ICF phenotype. Here, the LSH-mononucleosome interaction was explored in vitro using bioinformatics, biochemical, biophysical and structural techniques. LSH purification was further optimised, achieving near 100% purity, which is a useful improvement for any potential structural studies. LSH has been found to interact with the mononucleosome in vitro and no DNA linker was required for this interaction, indicating that LSH binds to the nuclesomal core through its ATPase domain. Qualitatively estimated Kd for this interaction was in nanomolar region, which did not translate into complex detection during size exclusion chromatography. CDCA7 was expressed in insect cell system and semi-purified, however, high nucleic acid presence in the final protein sample precluded any potential studies of CDCA7 interaction with chromatin. Homology and ab initio modelling for LSH and CDCA7, respectively, indicated that LSH is likely to bind the superhelical location 2 (SHL2), however, the exact location of CDCA7 and its interaction with LSH will have to be elucidated in further experimental work. i LAY SUMMARY Genetic information in living organisms is stored in a molecule called DNA. Being a long molecule, DNA required a special organisation within the cell nucleus. Specialised proteins called histones form a structural basis for DNA storage. Together, DNA and histones form structures called nucleosomes. Nucleus-wide DNA-histone mass is termed chromatin. Dynamic properties of chromatin in the nucleus are ensured by a specific class of proteins termed chromatin remodellers. LSH is a putative chromatin remodeller. A protein called CDCA7 is likely to be crucial in ensuring the remodelling activity of LSH. When mutated or truncated, LSH supposedly loses its remodelling activity and is implicated in a rare genetic disease called Immunodeficiency, centromeric instability, facial anomaly (ICF) syndrome. CDCA7, when mutated, is also implicated in ICF syndrome, indicating that they participate in a common process associated with chromatin remodelling. Here, work has been done to elucidate the mode of LSH-nuleosome interaction. Furthermore, initial work has been carried out to prepare CDCA7 for laboratory studies. ii DECLARATION The work presented in this thesis is the original work of the author. This thesis has been composed by the author and has not been submitted in whole or in part for any other degree. Alina Gukova February 2020 iii ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor Dr. Julia Richardson for supervising me over these years and providing useful comments on my thesis drafts and Dr. Irina Stancheva for suggesting the LSH project. Both made me gain crucial insights into scientific work and life in general, into how things should and should not be done. I would like to express my deep gratitude to my thesis committee: Dr. Philipp Voigt, Dr. Jeyaprakash (JP) Arulanandam and Prof. Bill Earnshaw. Philipp’s help was indispensable for carrying out the histone work and making nucleosome. JP provided many important insights into protein biochemistry and structural biology aspects of my project. Bill was the one who encouraged me to explore the CDCA7 topic. Apart from the scientific expertise, all of them provided their personal encouragement and motivation. Without my thesis committee, my work on this project would not be possible. I would like to thank Dr. Chris Jenness and Dr. Hiro Funabiki for their enthusiasm and help with the CDCA7 project, without them all my numerous questions about CDCA7 and the techniques associated with it would take me even longer to work through. The Edinburgh Protein Production Facility (EPPF) team played a cardinal role in my project, providing not only their scientific expertise in the art and skill of protein expression and purification, but also their human support. So, I’m thanking Dr. Liz Blackburn, Dr. Matt Nowicki and Dr. Martin Wear for being so amazing, professionally and personally. Also a big thank you to Dr. Meng Yuan and Dr. Iain McNae for their advice on homology modelling. I am sending my deepest gratitude to everyone on the 3rd floor of the Swann building, who made it almost pleasant to come to work every day (even including the weekends). Your positive attitude has really offset the negative aspects of a typical PhD existence. Those people include present and former members of Walkinshaw, Cook, Ly and Bramham labs. A special thank you to Dr. Simon Varzandeh, who worked on the LSH project before me and helped me a lot with it, to Dr. Jia Ning and to Dr. Ola Kasprowicz, who witnessed my struggles and supported me through the worst times in this PhD. I am also incredibly grateful to Karen Woodcock and Dr. Caroline Proctor for their emotional and practical support. I’m very grateful to my viva examiners Prof. Malcolm Walkinshaw and Dr. Helder Ferreira for their advice and useful insights into my project. iv Finally, I’d like to thank the University of Edinburgh and the School of Biological Sciences for providing financial support to me and my work and making it possible for me to carry out this work. v ABBREVIATIONS 5mC - 5 methyl-cytosine A - amper Å - ångström aa – amino acid ABC - ammonium bicarbonate ACN - acetonitrile ARPs - actin-related proteins ATP - adenosine triphosphate Bp – base pair BS3 - bis(sulfosuccinimidyl)suberate BSA – bovine serum albumin CC - coiled coil CDCA7 - Cell division cycle-associated protein 7 CDS - coding DNA sequence CHD - Chromodomain-Helicase DNA binding ChIP - chromatin immuno-precipitation C-NHEJ - non-homologous end joining CpG – cytosine-phosphate-guanine Cryo-EM – cryogenic electron microscopy CV - column volumes Da - Daltons DBD - DNA binding domain DBSs - double-strand breaks ddH2O – double-distilled water DMSO - dimethyl sulfoxide DNA - deoxyribonucleic acid DNMT1 - DNA Methyltransferase 1 DNMT3A - DNA Methyltransferase 3A DNMT3B - DNA Methyltransferase 3B dNTP - deoxynucleotide triphosphates dsDNA - double-stranded DNA DTT - Dithiothreitol dTTP – deoxythymidine triphosphate EDC - 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA - Ethylenediaminetetraacetic acid EMSA - electrophoretic mobility shift assay ESCs - embryonic stem cells ESI-MS - electrospray ionisation mass spectrometry FL -full length FRET - Fluorescent resonance energy transfer g - relative centrifugal force vi GFP - green fluorescent protein GraFix - gradient fixation GSH – reduced glutathione GST - Glutathione S-transferase HCL – hydrochloric acid HDAC1 and HDAC2 – histone deacetylase 1 and 2 HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMM - hidden Markov model HP1 - heterochromatin protein 1 HSA - helicase/SANT-associated domain HSCs - hematopoietic stem cells IAPs - intracisternal A-particles ICF - Immunodeficiency, centromeric instability, facial anomaly syndrome IEC - ion exchange chromatography IMAC - immobilised metal affinity chromatography IMAC FF - immobilised metal affinity chromatography fast flow IMAC HP - immobilised metal affinity chromatography high performance INO80 - INOsitol requiring 80 remodeller IPTG - isopropyl β-D-1-thiogalactopyranoside IR800 – infrared 800 nm dye ISWI - Imitation SWItch remodeller Kbp – kilo base pair KD - knockdown kDa - kiloDaltons KO – knockout LB - Lysogeny broth LCRs - Low complexity regions LIC - Ligation-independent cloning LINEs - long interspersed nuclear elements LSH - Lymphoid-specific helicase MALDI-ToF - matrix-assisted laser desorption/ionization-time-of-flight MAO - monoamine oxidase A Mbp – mega base pair MDa - megaDaltons MEFs - murine embryonic fibroblasts mESCs - the murine embryonic stem cells MNase - micrococcal
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