Investigation of a transcription factor complex and intrinsically disordered proteins Vania Santos Caldas 27, 524 words A thesis submitted in fulfilment of the requirements for the degree of Master of Philosophy Faculty of Science School of Life and Environmental Sciences The University of Sydney February 2018 i Statement of originaliy This is to certify that to the best of my knowledge, the content of this thesis is my own work. This thesis has not been submitted for any degree or other purposes. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. Vania Santos Caldas ii Acknowledgments I would like to thank everyone who helped me get through my Master’s degree. Firstly, thank you to my supervisor, Jacqui, for not only taking me on as a student but also always being encouraging and guiding me through this experience. And thank you of course, for all the thesis editing. You’ve helped me learn so much over the last few years. I would like to thank everyone in the Mackay/Matthews/Ataide lab, everyone was always so willing to help and provide advice. I feel very grateful to work in such a supportive environment, with people with such a variety of skills they are willing to share – advice on directions of my experiments, helping with using equipment, or even as simple as showing me where something was stored. Thank you especially to Flyp and Lorna for all the help in the lab. And a huge thank you to Neil for helping me with my cloning woes. Thank you to everyone to helped out with reading my thesis, Lorna, Flyp, Neil and Jason. I am very appreciative of all of your feedback. As well as all the excellent help with science provided from everyone, you also provided an excellent environment. I would not have enjoyed my experience in the lab as much as I did if it wasn’t for the people I got to meet and become friends with. Whether it be by chatting at our benches while doing minipreps, eating cake or commiserating over failed experiments, you all made each day better. I will truly miss working with you all each day. Thank you to all of my family and friends for their constant support. Thank you to my parents, for always encouraging and supporting me, and for putting up with my extreme stress levels. Thank you for taking over the cat parenting duties when I moved out. Thank you to Athina, for always being there to listen to me and support me over a coffee. Thank you to Taylor, for always encouraging me and making me smile. Knowing that I would go home and see you each day, made every day better. And finally, thank you to my partner James. You helped me through all the ups and downs (a lot of downs) of the past two years. I will forever be thankful for all your words of support and encouragement, and helping me stumble towards my future. I wouldn’t be where I am without you. I gratefully acknowledge the support of my scholarship, ‘Postgraduate Scholarship in Assembly of Transcriptional Complexes’. iii Abstract LIM domain binding protein 1 (Ldb1) is a chromatin looping factor that forms part of a transcriptional ‘pentameric complex’. Ldb1 contains two domains that are essential for looping: a self-association domain, and a LIM interaction domain (LID) that binds LIM proteins such as Lmo2, which in turn binds to DNA binding transcription factors, Tal1, E2a and Gata-1. It was proposed that the Gata-1 binding protein FOG1 could bind to the pentameric complex. GFP-tagged FOG1 was shown to bind the complex by multi angle laser light scattering, providing a mechanism by which the intrinsically weak Gata-1/FOG1 interaction is bolstered through binding to other units of the pentameric complex. Little is known about proteins that are distantly related to mammalian Ldb1. Two such proteins, Ldb1 from C. elegans and Adn1 from S. pombe were expressed in bacteria. They had generally poor solubility, but use of a maltose binding protein tag promoted solubility, and different expression systems may enable their further study. Ldb1 was reported to bind the E3 ubiquitin ligase protein, RLIM. No interaction could be detected between these proteins by yeast two-hybrid analysis using truncated or full length proteins. The interaction was detected in mammalian cells using FLAG pull-down experiments, but truncation mutants of these proteins could not be expressed. RLIM has high levels of predicted disorder, which may contribute to its degradation in both cell types. An assay was developed in which dimerization domains could stabilise disordered binding regions. Constructs containing GST, a coiled-coil domain of CtIP, or the leucine zipper of GCN4, were tethered to a test peptide and assayed for binding in yeast two-hybrid assays. The domains from CtIP and/or GCN4 allowed the interaction to be detected. Although the assay could not detect an interaction between RLIM and Ldb1, it shows promise for detecting interactions for disordered proteins, and can be adapted to different expression systems. iv Abbreviations 3-AT 3-amino-1,2,4-triazole 3C Chromosome conformation capture 3C-seq Chromosome conformation capture sequencing AD Activation domain Adn1 Adhesion defective protein 1 Amp Ampicillin AUC Analytical ultracentrifugation BD DNA binding domain bHLH Basic helix loop helix BSA Bovine serum albumin C. elegans Caenorhabditis elegans Cam Chloramphenicol cDNA Complementary DNA CeLdb1 Ldb1 from C. elegans CF/C-finger Carboxy-terminal zinc finger ChIP Chromatin immunoprecipitation CSM Complete supplement mixture CtIP DNA endoculease RBBP8 CV Column volume(s) D. melanogaster Drosophila melanogaster Da Dalton DD Dimerisation domain Deaf1 Deformed epidermal autoregulatory factor DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate DTT Dithiothreitol E. coli Escherichia coli E2a Transcription factor E2-alpha v E-box Enhancer box EDTA Ethylene ditetraacetic acid EMSA Electrophoretic mobility shift assay FOG1 Friend of GATA 1/ZFPM1 FRET Fluorescence/Förster resonance energy transfer Gata-1 GATA binding protein 1 (globin transcription factor 1) GCN4 General control protein GCN4 GFP Green fluorescent protein GSH Glutathione GST Glutathione-S-transferase HA Human influenza hemagglutinin HADDOCK High ambiguity driven protein-protein DOCKing HD Homeodomain HEK-293 Human embryonic kidney cells 293 HEPES 4-(2-hydroxylethyl)-1-piperazineethanesulfonic acid His-tag 6 × histidine tag HRP Horseradish peroxidase HRV-3C Human Rhinovirus 3C HSQC Heteronuclear single quantum correlation IDP Intrinsically disordered proteins IDR Intrinsically disordered regions IPTG Isopropyl β-D-thiogalactopyranoside ITC Isothermal titration calorimetry KA Association constant KD Dissociation constant kDa Kilodalton KO Knock-out LB Luria-Bertani broth LCCD Ldb1/CHIP conserved domain LCR Locus control region Ldb1 LIM domain binding protein 1 vi Ldb2 Lim domain binding protein 2 LDS Lithium dodecyl sulfate Lhx LIM homeobox protein LID LIM interaction domain LIM Lin1-Isl1-Mec3 (protein domain) LIM-HD LIM-homeodomain Lmo LIM only protein MALLS Multi angle laser light scattering MBP Maltose-binding protein MCS Multiple cloning site MEL Murine erythroleukemia MES 2-(N-morpholino)ethanesulfonic acid MQW MilliQ® water MW Molecular weight MWCO Molecular weight cut off N Stoichiometric ratio NF/N-finger Amino-terminal zinc finger Ni-NTA Nickel nitrile-triacetic acid NLS Nuclear localisation domain NMR Nuclear magnetic resonance spectroscopy OD600 Optical density at 600 nm PBD Protein data bank PBS Phosphate-buffered saline PCR Polymerase chain reaction PEG Polyethylene glycol PEI Polyethylenimine PMSF Phenylmethanesulfonyl fluoride PTM Post-translational modifications RING Really interesting new gene RIPA Radioimmunoprecipitation assay RLIM RING finger LIM-domain interacting protein vii RLIMBD RLIM basic domain RNA Ribonucleic acid S. pombe Schizosacchraomyces pombe SA Self-association SAXS Small-angle C-ray scattering SD Synthetic dextrose SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC Size exclusion chromatography SOC Super Optimal Broth SSBP Single stranded binding protein SSDP Single stranded DNA-binding protein TAE Tris/acetate/EDTA Tal1 T-cell acute lymphocytic leukemia protein 1 T-ALL T-cell acute lymphoblastic leukaemia` TBS Tris-buffered saline TCEP Tris(2-carboxyethyl)phosphine TE Tris/EDTA TF Transcription factor Tm Melting temperature Tris Tris(2-carboxymethyl)aminomethane USF-1 U-shaped finger 1 UV Ultraviolet WT Wild-type X-α-gal 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside YPD Yeast peptone dextrose viii Table of Contents Statement of originaliy ............................................................................................................. i Acknowledgments .................................................................................................................... ii Abstract ................................................................................................................................... iii Abbreviations .......................................................................................................................... iv Table of Contents ................................................................................................................. viii List of Figures .......................................................................................................................... xi List of Tables .........................................................................................................................