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Phosphorylation of KLF3 Affects Its DNA Binding Activity and Biological Function

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Phosphorylation of KLF3 Affects Its DNA Binding Activity and Biological Function Phosphorylation of KLF3 affects its DNA binding activity and biological function Vitri Aryani Dewi Supervisor: Prof. Merlin Crossley A thesis submitted for the fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry and Molecular Genetics) School of Biotechnology and Biomolecular Sciences The University of New South Wales August 2012 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: DEWI First name: VITRI Other name/s: ARYANI Abbreviation for degree as given in the University calendar: PhD School: BABS Faculty: SCIENCE Title: Phosphorylation of KLF3 affects its DNA binding activity and biological function Abstract 350 words maximum: (PLEASE TYPE) Krüppel-Like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor, which binds to CACCC-boxes and GC-rich regions in the promoters and enhancers of its target genes. Studies using knock-out mice have revealed functional roles for KLF3 in diverse tissues. Klf3-/- mice have a reduced life- span, leaner body composition, disturbed B-cell maturation and mild anaemia. This thesis explores the regulation of KLF3 function via post-translational modifications. We show that KLF3 exists as a phospho-protein in vivo and that post-translational modifications change in response to physiological stimuli. We have found that phosphorylation by Homeodomain Interacting Protein Kinase 2 (HIPK2) enhances KLF3’s DNA binding affinity. A mutant form of KLF3, in which serine 249 has been mutated to alanine has significantly reduced affinity for DNA, suggesting that phosphorylation at this site contributes to DNA binding capacity. Given that HIPK2 has been implicated in the cellular response to UV DNA damage, we investigated a potential role for KLF3 in this pathway. We found that cells lacking KLF3 have an altered response to UV stress, showing a complex phenotype indicative of a deregulated apoptotic pathway. Rescue of KLF3 null cells with wild-type KLF3, restored a normal response, while expression of a mutant version of KLF3, in which serine 249 has been mutated to alanine, showed only a partial rescue of the wild-type phenotype. Finally, we investigated the possibility of self-interaction within KLF3 using yeast two-hybrid assays and identified a putative self-association domain within the first 150 amino acids of KLF3. In conclusion, this study has identified post-translational modification of KLF3 as an important regulator of its activity. In particular, phosphorylation of serine 249 enhances KLF3’s DNA binding and plays a role in controlling its activity in biological pathways, such as the cellular response to UV stress and DNA damage. It also provides the first evidence of self-association within the KLF family. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. Date of completion of requirements for Award: ORGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of materials which have been accepted for the award of any degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, which whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged’ Signed Date ABSTRACT Krüppel-Like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor, which binds to CACCC-boxes and GC-rich regions in the promoters and enhancers of its target genes. Studies using knock-out mice have revealed functional roles for KLF3 in diverse tissues. Klf3-/- mice have a reduced life-span, leaner body composition, disturbed B-cell maturation and mild anaemia. This thesis explores the regulation of KLF3 function via post-translational modifications. We show that KLF3 exists as a phospho-protein in vivo and that post- translational modifications change in response to physiological stimuli. We have found that phosphorylation by Homeodomain-Interacting Protein Kinase 2 (HIPK2) enhances KLF3’s DNA binding affinity. A mutant form of KLF3, in which serine 249 has been mutated to alanine has significantly reduced affinity for DNA, suggesting that phosphorylation at this site contributes to DNA binding capacity. Given that HIPK2 has been implicated in the cellular response to UV DNA damage, we investigated a potential role for KLF3 in this pathway. We found that cells lacking KLF3 have an altered response to UV stress, showing a complex phenotype indicative of a deregulated apoptotic pathway. Rescue of KLF3 null cells with wild-type KLF3, restored a normal response, while expression of a mutant version of KLF3, in which serine 249 has been mutated to alanine, showed only a partial rescue of the wild-type phenotype. Finally, we investigated the possibility of self-interaction within KLF3 using yeast two-hybrid assays and identified a putative self-association domain within the first 150 amino acids of KLF3. In conclusion, this study has identified post-translational modification of KLF3 as an important regulator of its activity. In particular, phosphorylation of serine 249 enhances KLF3’s DNA binding and plays a role in controlling its activity in biological pathways, such as the cellular response to UV stress and DNA damage. It also provides the first evidence of self-association within the KLF family. TABLE OF CONTENTS ACKNOWLEDGEMENT ................................................................................................. i PUBLICATIONS ARISING FROM THIS THESIS................................................................ iii ABBREVIATIONS ........................................................................................................ iv CHAPTER 1 - INTRODUCTION ...................................................................................... 1 1.1. Regulation of gene expression ........................................................................ 2 1.1.1. The Krüppel-Like Factor (KLF) family of transcription factors ................ 3 1.1.1.1. Krüppel-Like Factor 3 (KLF3) .............................................................. 6 1.1.2. C-Terminal Binding Protein (CtBP) .......................................................... 8 1.1.3. Homeodomain-Interacting Protein Kinase (HIPK) .................................. 9 1.2. Post-translational modifications ................................................................... 12 1.2.1. Phosphorylation .................................................................................... 12 1.3. Project aims ................................................................................................... 14 CHAPTER 2 - MATERIALS AND METHODS ................................................................. 15 2.1. Materials ....................................................................................................... 16 2.1.1. Chemicals and Reagents ....................................................................... 16 2.1.2. Enzymes ................................................................................................ 21 2.1.3. Antibodies ............................................................................................. 21 2.1.4. Plasmids and oligonucleotides ............................................................. 22 2.1.4.1. Gift Plasmids .................................................................................... 22 2.1.4.2. Constructs ........................................................................................ 23 2.1.4.3. Oligonucleotides .............................................................................. 25 2.1.5. Bacterial strains and culture ................................................................. 26 2.1.6. Yeast strain and media ......................................................................... 26 2.1.7. Mammalian cell lines and culture media .............................................. 27 2.2. Methods ........................................................................................................ 28 2.2.1. General molecular biology techniques ................................................
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