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Understanding the relationship between IRF-1 and the transcriptional repressor ZNF350 Lucy Mallin Doctor of Philosophy The University of Edinburgh October 2014 Declaration I hereby declare that I am the author of this thesis. The work presented here was performed by myself and any contributions by others has been clearly indicated and acknowledged. I can confirm that no part of this thesis has been submitted for any other degree or professional qualification. Lucy Mallin October 2014 i Acknowledgements I would like to dedicate this thesis to my Grandad, who always inspired me to stick in and try my hardest. His motto “always read the question” has helped throughout my academic career and I’m sure will continue to do so throughout my life. I would like to start off my thanking my supervisor Kathryn for her guidance and patience with me. I am very grateful to her for giving me the opportunity to study in her research group and for all the help and support provided along the way. Also a general big thank you to all the members of Kathryn and Ted’s groups for all their help in the lab as well as numerous entertaining tea break discussions. In particular, thanks to Vikram for helping me when I was starting out in the lab as well as Vivien, Yuh-Ping, Fiona, Fi and Kalai for help throughout my time there with experiments and insight. Also, thanks to my second supervisor Colin and members of the bioinformatics team including Graeme and GoGo, for helping me with all the data analysis. When I started out, I was a bit scared of computers but I gradually saw the light and developed a new love of bioinformatics. So much so that I have now undertaken a bioinformatics-based job so thank you for introducing me to the world of computational biology. Thank you to Richard, who I know was probably sick to death of stories of my cells dying, but has supported me endlessly and helped calm me down when things got a bit much. I can’t express how grateful I am that you allow me to drag you about the country for my jobs-I’ll pay you back some day. Thank you to my Mum and Dad for supporting me throughout my never-ending studies both financially and emotionally-I’m almost done I promise. Finally, to my awesome sister Sophie, thank you for the multitude of cup of tea visits and providing me with a nice distraction. They were really needed this past year and I’m really going to miss them and you over the next few years. ii Abstract Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumour suppressor, involved in many diverse cellular processes including immune responses and growth regulation. An interesting feature of IRF-1 is that it can both activate and repress gene expression, possibly by acting with co-activator or co-repressor proteins. In a previous phage display assay, a homologous peptide to the known repressor protein, zinc finger 350 (ZNF350), was found to bind to the C-terminus of IRF-1. ZNF350, also known as ZBRK1 (Zinc finger and BRCA1-interacting protein with KRAB domain-1), is a member of the Krüppel-associated box (KRAB)-containing zinc finger (KZF) proteins, which is a group of the widely distributed transcriptional repression proteins in mammals. ZNF350 has previously been shown to repress the expression of a number of genes including ANG1 and GADD45A, often in complex with other proteins. This study confirms the direct interaction between IRF-1 and ZNF350 and identifies key residues, including the LXXLL repression motif within the C-terminus of IRF-1, necessary for the binding interface. The two proteins have additionally been shown to interact within a cellular environment, shown by using techniques including immunoprecipitation and a proximity ligation assay. In addition, the ZNF350/IRF-1 complex formation appears to occur in the basal state of the cell, as opposed to in response to cellular stress such as viral infection or DNA damage. On the basis of ZNF350 being a negative regulator of transcription, a novel technique was developed to identify putative targets of both ZNF350 and IRF-1. This involved an initial bioinformatics screen using candidate IRF-1 binding site data obtained from CENTIPEDE, an algorithm that combines genome sequence information, with cell-specific experimental data to map bound TF binding sites. This allowed for the identification of novel target genes that contained the ZNF350 consensus binding site, GGGxxCAGxxxTTT, within close proximity to an IRF-1 consensus site, such as the immune response gene IL-12A. Lastly, a peptide phage display screen was combined with high-throughput sequencing to identify other potential binding partners of ZNF350 and perhaps help to understand the mechanism by which transcriptional repression is controlled by complex formation. iii Abbreviations aa Amino acid AMP Adenosine monophosphate ATP Adenosine triphosphate ATM Ataxia-telangiectasia Mutated Bax Bcl2-associated protein X bp Base pair BSA Bovine serum albumin CDK Cyclin dependent kinase ChIP Chromatin immunoprecipitation Co-IP Co-immunoprecipitation DBD DNA binding domain DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid DTT Dithiothreitol ECL Enhanced chemiluminesence E.coli Escherichia coli EDTA Ethylenediaminetetraacetic acid GST Glutathione S-transferase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid His Histidine HRP Horse radish peroxidase IFN Interferon iv IPTG Isopropyl-β-thio-galactoside IP Immunoprecipitation IR Ionising radiation kb Kilobase kDa Kilodalton mAb Monoclonal antibody MEF Mouse embryonic fibroblast mRNA Messenger RNA NGS Next generation sequencing OD Optical density pAb Polyclonal antibody PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction pfu Plaque-forming unit pH Potential hydrogen RLU Relative light unit RNA Ribonucleic acid RNase Ribonuclease rpm Revolutions per minute SDS Sodium dodecyl sulphate TAD Transactivation domain Tris 2-amino-2-hydroxymethyl-propane-1,3-diol wt Wild type Zn Zinc v Table of Contents Declaration i Acknowledgements ii Abstract iii Abbreviations iv Chapter 1: Introduction 1 1.1 Transcription 1 1.1.1 Transcriptional Activation 1 1.1.2 Transcriptional Repression 4 1.1.2.1 Repressor Proteins 4 1.1.2.2 Chromatin Remodelling 5 1.2 KRAB-containing zinc finger proteins 9 1.2.1 Structural Features 10 1.2.1.1 Zinc Fingers 10 1.2.1.2 KRAB domain 11 1.2.1.3 Other domains 11 1.2.2 Function and mechanism 13 1.3 ZNF350 13 1.3.1 Localisation 15 1.3.2 Mechanism of Action 17 1.3.2.1 Repression 17 1.3.2.1.1 GADD45A 17 1.3.2.1.2 ANG1 18 1.3.2.1.3 HMGA2 18 1.3.2.1.4 p21 19 1.3.2.1.5 HIV-1 LTR 19 1.3.2.1.6 MMP9 20 1.3.2.1.7 FGF2 20 1.3.2.1.8 KAP1 21 1.3.2.2 Activation 21 vi 1.3.2.2.1 SCA2 21 1.3.3 ZNF350 mRNA expression 22 1.3.4 Gene organization and structure 23 1.3.5 Regulation 23 1.3.5.1 Ubiquitin-Proteasome Pathway 23 1.3.5.2 Transcriptional Regulation 24 1.4 IRF-1 25 1.4.1 Protein Structure 27 1.4.1.1 DNA binding domain 27 1.4.1.2 Mf2 Domain 28 1.4.1.3 Nuclear localisation signal 28 1.4.1.4 Transactivation domain 30 1.4.1.5 Dimerisation domains 30 1.4.1.6 Enhancer domain 31 1.4.2 Gene Expression 32 1.4.2.1 JAK-STAT pathway 34 1.4.2.2 ATM pathway 34 1.4.3 Roles of IRF-1 35 1.4.3.1 Immune System 35 1.4.3.2 Autoimmunity 35 1.4.3.2.1 HIV-1 36 1.4.3.3 Human Cancer 37 1.4.3.4 Tumour suppressor 38 1.4.3.5 Invasion and metastasis 38 1.4.3.6 Cell cycle 39 1.4.3.7 Apoptosis 39 1.5 Objective of this thesis 42 Chapter 2: Materials and Methods 43 2.1 Reagents, Plasmids and centrifuges 43 2.2 General microbiological techniques 43 2.2.1 Maintaining bacterial cultures 43 2.2.2 Glycerol stocks 44 vii 2.2.3 Preparation of competent cells 44 2.2.4 Transforming bacterial cells 45 2.2.5 Plasmid DNA amplification, extraction and quantification 46 2.2.6 Agarose gel electrophoresis 46 2.2.7 DNA sequencing 47 2.2.8 Ethanol/EDTA precipitation 49 2.2.9 Amplification of gene by PCR 49 2.2.10 Amplification of chromatin isolated by immunoprecipitation 53 2.2.11 Site-directed mutagenesis 55 2.3 General biochemical techniques 57 2.3.1 SDS-PAGE 57 2.3.2 Staining of SDS-PAGE gels 58 2.3.2.1 Coomassie staining 58 2.3.2.2 Silver staining 59 2.3.3 Western Blotting 59 2.3.4 Stripping nitrocellulose blots 61 2.4 Cell culture 61 2.4.1 Cell lines 61 2.4.2 Sub-culturing of cells 62 2.4.3 Freezing and thawing cells 63 2.4.4 Transient transfection of DNA 63 2.4.5 Drug treatments 63 2.4.6 Cell irradiation 64 2.4.7 In vivo crosslinking 64 2.4.7.1 DSP (dithiobis[succinimidylpropionate]) 64 2.4.7.2 Formaldehyde 65 2.4.8 Cell harvesting and lysis 65 2.5 Protein expression and purification from E.coli 66 2.5.1 Expression of His-tagged ZNF350 66 2.5.2 Purification of His-tagged ZNF350 67 2.6 Assays 68 2.6.1 Peptide affinity chromatography (peptide