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A University of Sussex Phd Thesis Available Online Via A University of Sussex PhD thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. 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 Please visit Sussex Research Online for more information and further details Exploring interactions between Epstein- Barr virus transcription factor Zta and The Human Genome By IJIEL BARAK NARANJO PEREZ FERNANDEZ A Thesis submitted for the degree of Doctor of Philosophy University Of Sussex School of Life Sciences September 2017 ii I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature:…………………………..…………………………..……………………… iii Acknowledgements I want to thank Professor Alison J Sinclair for her guidance, mentoring and above all continuous patience. During the time that I’ve been part of her lab I’ve appreciated her wisdom as an educator her foresight as a scientist and tremendous love as a parent. I wish that someday soon rather than later her teachings are reflected in my person and career; hopefully inspiring others like me. Thanks to Professor Michelle West for her help whenever needed or offered. Her sincere and honest feedback, something that I only learned to appreciate after my personal scientific insight was developed. I wish I had learned this sooner. Thanks to the people in the virology labs that make this place feel like home, Dr Kay Osborne has always been helpful and kind, Dr David Wood always lend his intelligence and “know how”, even when he didn't have to! Dr Michelle Brocard, Dr Andrea Gunnell, Dr Helen Webb and Dr Sharada Ramasubramanyan have been missed greatly since their departure. Dr Lina Chen and her flourishing spirit always makes any day brighter. Thank you to Yaqi Zhou, Anja Godfrey, Sarika Katnis, Hildegonda Veenstra, and Rajaei Al-Mohammad for their friendship and support. We should travel together more often. I know I take with me great friendships that will span far beyond my stay in the United Kingdom, so I hope it won’t be long before Dr Chris Traylen, Rob Simmons, Jake Evans and Dr Mike McClellan stop for a visit wherever I’m living. I want to thank the Mexican Council of Science and Technology for their economic support in the development of my scientific formation and by extension our nation’s progress. This of course is possible thanks to the Mexican taxpayer that still does what is correct, even when averse with the current administration. I want to thank my family for their support and direct involvement in my personal development. I thank my parents and the rest of the family. In particular I want to thank my sister Thais B. Naranjo P.-F. whose selflessness and generosity brings life to everyone that surrounds her. Special thanks to Anna K, you’ve made this so much easier I’m forever grateful to you. UNIVERSITY OF SUSSEX IJIEL BARAK NARANJO PEREZ FERNANDEZ PhD BIOCHEMISTRY “EXPLORING INTERACTIONS BETWEEN EPSTEIN-BARR VIRUS TRANSCRIPTION FACTOR Zta AND THE HUMAN GENOME” SUMMARY Epstein-Barr virus is a gamma herpesvirus that is present in human adult’s B- lymphocytes infecting 90% of the global population. EBV causes many types of lymphoma and carcinoma. The virus life cycle can be divided in two stages, latency and lytic cycle. Viral gene BZLF1 codes for the viral transcription and replication factor Zta (also known as BZLF1, ZEBRA, EB1, and Z) which is part of the signalling required to switch from latency to the lytic cycle. Zta is part of the bZIP family of proteins, it forms homodimers and can bind to specific sequences termed Zta Response Elements (ZREs). It binds to the EBV lytic origin of replication as well as to specific targeted promoters in the viral genome and regulates its expression. Recent research found and mapped interactions between the key viral transcription factor Zta and the B-cell genome, this showed interactions of Zta proximal (closer than 2Kb) and distal (farther than 2 Kb) to the transcription start site of several genes. In this work, I asked the questions: Can enhancer properties be found in the sequences where Zta binds to? Is Zta distally regulating expression by looping of DNA? This was approached first by identifying potential sequences that could be conferring enhancing activity, then inserting them into vectors and transfecting them into two different cell lines. In this way, through luciferase reporter assays, any enhancing capabilities of the sequences were tested when placed in a proximal and distal manner to promoters known to be regulated by Zta, as well as mutated promoters not regulated by Zta. This resulted in finding discreet enhancer activity in the sequences analysed, with some being specific to the cell type that was used in the experiment. To answer the second question, Chromosome conformation capture (3C) was used to test the possibility of a spatial rearrangement bringing together distal Zta binding regions and promotor regions of selected genes (looping). However, I did not find evidence of looping between Zta binding sites and the neighbouring promoters analysed, in the cell context employed. v Table of contents Chapter 1. Introduction .................................................................................... 1 1.1 Herpesvirales ......................................................................................... 1 1.2 Epstein-Barr Virus .................................................................................. 4 1.2.1 EBV genome structure .................................................................... 4 1.2.2 Viral Entry ........................................................................................ 6 1.2.3 EBV life cycle .................................................................................. 6 1.1.3.1 EBV latency cycle ..................................................................... 7 1.1.3.2 EBV associated pathologies and latency .................................. 9 1.1.3.2.1 EBV malignancies related to B cell infection ...................... 9 1.1.3.2.2 EBV malignancies in atypical cells ................................... 12 1.1.3 EBV lytic stage........................................................................... 14 Lytic Reactivation ............................................................................ 14 1.1.3 Zta ............................................................................................. 16 Structure and DNA binding ............................................................. 17 1.3 Computational methods for sequence analysis ................................... 19 1.4 Zta interacting with the human genome ............................................... 19 1.5 Enhancers ............................................................................................ 19 1.6 Chromatin looping events .................................................................... 23 1.7 Aims of project ..................................................................................... 24 Chapter 2. Materials and Methods ................................................................ 25 2.1 Materials .............................................................................................. 25 2.1.1 Vectors .......................................................................................... 25 2.1.2 Cell lines ........................................................................................ 25 2.1.3 Antibodies ..................................................................................... 26 2.1.4 Primers (oligonucleotides) ............................................................. 27 2.1.5 Solutions and buffers ..................................................................... 28 vi 2.1.6 Kits and reagents .......................................................................... 29 2.2 Methods ............................................................................................... 31 2.2.1 Molecular cloning by digestion-ligation .......................................... 31 2.2.2 Bacterial Transformation ............................................................... 32 2.2.3 Plasmid DNA extraction ................................................................ 33 2.2.4 Cell Culture ................................................................................... 33 2.2.5 Cell Transfection ........................................................................... 34 2.2.6 Protein gels and Western Blots ..................................................... 35 2.2.7 Luciferase reporter assays ............................................................ 36 2.2.7.1 Bicinchoninic Acid Assay (BCA) ............................................. 36 2.2.7.2 Normalization .......................................................................... 37 2.2.8 Polymerase Chain Reaction .......................................................... 37 2.2.9 Agarose Gel Electrophoresis ......................................................... 38 2.2.10 Chromatin conformation capture assay ....................................... 38 2.2.10.1 Formaldehyde crosslink and cell lysis ..................................
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