DETECTION AND ANALYSIS OF LIM DOMAIN-MEDIATED INTERACTIONS BETWEEN TRANSCRIPTION FACTORS Ivan Nisevic A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Life and Environmental Sciences University of Sydney Declaration i Declaration The experimental work described in this Thesis was performed between March 2008 and May 2013 in the School of Molecular Bioscience at the University of Sydney. I performed the experiments unless otherwise stated. This work has not been submitted, in part or in full, for the purpose of obtaining any other degree. Abstract ii Abstract LIM-homeodomain (LIM-HD) proteins are a class of transcription factors involved in tissue specification and cell determination during development and are important in adult gene regulation. Six families of LIM-HD proteins, with two close paralogues in each family, are commonly found in tetrapods. They bind DNA via HDs, whereas their interactions with other proteins are mediated mainly by a pair of closely spaced LIM-domains (LIMs) in each protein. These proteins take part in various transcriptional complexes with Ldb1 and other cofactors that contain LIM-interaction domains (LIDs). In this thesis, protein-protein interactions of LIM-HD proteins were analysed in order to better understand the molecular mechanisms of transcriptional complex formation. Based on previous research that showed LIM-LID mediated interactions between Lhx3 and Isl1, yeast two-hybrid mating arrays were used to investigate how widespread protein- protein interactions are amongst the 12 mammalian LIM-HD proteins. Due to high levels of background growth in experiments with full-length proteins in pGBT9 vectors, the mating arrays focused on LIM-domain mediated interactions with full-length LIM-HDs or known LIDs. The arrays revealed a relatively strong interaction between Lhx3 (or Lhx4) and Isl1 (or Isl2), and detected weaker interactions between Lmx1a or Lmx1b and the LIM-binding domain of Isl1. The contribution of separate LIM-domains to the overall interaction with Ldb1 for each of the proteins was analysed by the same method. In most cases one of the LIM domains in each protein was able to independently interact with the LID domain of Ldb1 by yeast two-hybrid analysis indicating a dominant binder: LIM1 in Isl1 and Isl2, or LIM2 in other proteins. The exceptions were paralogues Lhx1 and Lhx5, for which no separate domain showed interaction with Ldb1LID by this approach. All tandem LIM-domain constructs showed a much stronger interaction with Ldb1LID than any isolated LIM domain supporting the idea that both domains are required for high affinity binding to Ldb1. Bimolecular Fluorescence Complementation experiments in yeast were designed and conducted as an alternative approach to test interactions between full-length LIM-HD proteins in the hope that a non-transcription based assay would lead to no or less background signal compared to yeast two-hybrid analysis. A plasmid system was developed based on existing yeast Abstract iii two-hybrid vectors using split green fluorescent proteins in place of domains from the GAL4 transcription factor. The assay was able to detect interactions between different LIMs and their partners but unfortunately interactions between full-length proteins were still difficult to detect due to low fluorescence, self-complementation in the controls and localization effects. LIM domains from LIM-HD proteins cannot be used in standard bimolecular binding assays because they tend to be insoluble and/or aggregate in the absence of a binding partner. Stable, soluble intramolecular ‘tethered complexes’ can be generated in which LIMs are tethered to Ldb1LID via a flexible linker. Introduction of a specific protease site into the tether allows the formation of intermolecular cut complexes, which have previously been used in homologous competition ELISA experiments. In this thesis attempts were made to develop more robust biophysical binding assays that could be used to assess the binding affinities of different LIMs for Ldb1LID. Several different labelling approaches were used to generate proteins with fluorescent tags for use in fluorescence anisotropy assays. In one of these approaches expressed protein ligation was applied to generate proteins with an N-terminal fluorescein. Although this labelling strategy was of low efficiency for LIMs-Ldb1LID tethered constructs, some preliminary fluorescence anisotropy experiments were carried out, which indicated that this could be a useful strategy providing a more efficient labelling strategy can be found. GFP-tagged tethered complexes were easier to generate, but could not be used in anisotropy experiments because of the intrinsically high anisotropy of GFP proteins. However, preliminary experiments indicated that these proteins can be used in clear native gel shift competition assays to compare binding affinities of different tandem LIM domains to Ldb1LID. Data presented in this thesis provide valuable insight into protein-protein interactions of LIM-HD transcription factors and the advantages, as well as disadvantages, of applied experimental approaches. The results and their implications are discussed, raising questions that can be resolved in future studies. Acknowledgements iv Acknowledgements Most of all, I would like to thank my supervisor Prof. Jacqui Matthews for her support and help in writing this thesis. She has pulled me out of the quicksand many times, invested a lot of effort and showed incredible patience. I am indebted to her. To Prof. Joel Mackay, thank you for your inspirational talks and your passion in science and every sport we played. I would like to express my gratitude to Mugdha, Morgan, Liza, Sandra, Ann, Roland, Robyn, Eija, Pep, Philippa, Lorna, Arwen, Margie, Kate, Chris, Ingrid, Jason, Chu Wai, Cy, Paula, Sock, Cuong, Mitch, Saad, Marylene, Katschi, Soumya, Nick, David, Krystal, Sarah, Kaavya, Gonzalo, Ana, Angela, Paul and many other wonderful people who showed me how to get around the lab, gave me advice on my experimental work and made my PhD experience more fun. I would especially like to thank Philippa and Neil for proofreading my thesis. I am grateful to Dr Ben Crossett for his advice on MALDI-TOF, and Dr Brendan Wilkinson for his advice on native chemical ligation and help with reagent purification. Thanks are also due to Dr Tim Newsome for his microscopy-related advice. Thanks to Clare Woodley for everything and all the people from the SMB that I talked to at my progress interviews. Finally, I need to thank my wife Mirjana, my children and my close family that have stood by me and helped me throughout the years. I gratefully acknowledge the support and funding of an Australia Postgraduate Award. Table of Contents v Table of contents Declaration ................................................................................................................................................. i Abstract ..................................................................................................................................................... ii Acknowledgements .................................................................................................................................. iv Table of contents ....................................................................................................................................... v List of abbreviations ................................................................................................................................ xi Chapter 1 Introduction ....................................................................................................................... 1 1.1 Complex interactions of transcription factors ................................................................. 1 1.2 Multidomain nature of TFs ............................................................................................ 3 1.3 Homeodomains (HDs) ................................................................................................... 3 1.4 LIM-homeodomain (LIM-HD) proteins ......................................................................... 5 1.5 LIM-HD proteins and the TF-codes for neuron specification......................................... 8 1.6 LIM-domains ............................................................................................................... 11 1.7 Ldb1/2 cofactors stabilize LIM-HD/LMO proteins and can mediate interactions between relevant chromatin-bound complexes ...................................................................................... 13 1.8 Interactions between Isl1/2 and Lhx3/4 proteins .......................................................... 15 1.9 General characteristics of LIM domain interactions ..................................................... 16 1.9.1 Tethered LIM-LID ‘complexes’ ........................................................................................ 16 1.9.2 LIM domain interfaces and the β-zipper binding mode of linear partner motifs................ 17 1.10 Potential interactions between other LIM-HD proteins ................................................ 21 1.11 In vitro competition experiments can investigate binding affinities of competing LIMs:LID/LBD complexes ...................................................................................................... 22 1.12 Aims of the thesis ........................................................................................................