Directing an artificial zinc finger protein to new targets by fusion to a non-DNA binding domain Wooi Fang (Catheryn) Lim A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science March 2016 Page | 0 THESIS/ DISSERTATION SHEET Page | i ORIGINALITY 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 material which have been accepted for the award of any other 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, with 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.’ WOOI FANG LIM Signed …………………………………………….............. 31-03-2016 Date …………………………………………….............. Page | i COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or 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 proprietary 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 Abstract International (this is applicable to doctoral theses only). 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Table of Contents ORIGINALITY STATEMENT ......................................................................................... i Table of Contents .............................................................................................................. ii Acknowledgement............................................................................................................. v Publications arising from this thesis ................................................................................ vi Abstract ........................................................................................................................... vii Abbreviations ................................................................................................................. viii Chapter 1 General Introduction ......................................................................................... 1 1.1 Transcriptional regulation .......................................................................... 1 1.1.1 DNA binding proteins ................................................................................... 3 1.1.2 Modularity of sequence-specific DNA binding proteins ................................. 4 1.1.3 C2H2 zinc finger containing DNA binding proteins ................................... 5 1.1.4 Target recognition by sequence-specific transcription factors ................. 9 1.1.5 KLF family of zinc finger transcription factors ....................................... 12 1.2 Artificial Zinc Finger DNA binding proteins .......................................... 17 1.2.1 Application of artificial zinc finger DNA binding proteins .......................... 18 1.2.2 Construction of artificial zinc finger DNA binding proteins .................. 21 1.2.3 Artificial zinc finger proteins targeting VEGF-A promoter ................... 23 1.2.4 Specificity of artificial zinc finger DNA binding proteins ....................... 25 1.3 Aims ............................................................................................................. 28 Chapter 2 Materials and Methods ................................................................................... 29 2.1 Materials .................................................................................................................. 29 2.1.1 Reagents and kits ........................................................................................ 29 2.1.2 Cell lines ....................................................................................................... 31 2.1.3 Oligonucleotides .......................................................................................... 31 2.1.4 Vectors ......................................................................................................... 31 2.2 Laboratory methods ............................................................................................... 32 2.2.1 General methods ......................................................................................... 32 Page | ii 2.2.2 Cell culture .................................................................................................. 32 2.2.3 Generation of retroviral and expression vectors ..................................... 32 2.2.4 Transient transfection for protein production ......................................... 33 2.2.5 Retroviral transduction to generate stable cell lines ............................... 34 2.2.6 Nuclear extracts .......................................................................................... 34 2.2.7 SDS-PAGE and Western blot .................................................................... 34 2.2.8 Electrophoretic mobility shift assay (EMSA) ........................................... 35 2.2.9 RNA extraction and cDNA synthesis ........................................................ 35 2.2.10 Real time PCR ............................................................................................ 36 2.2.11 Chromatin immunoprecipitation (ChIP)................................................. 36 2.2.12 DNA library preparation and next generation sequencing ................... 36 2.3 Bioinformatics methods ............................................................................. 37 2.3.1 Quality trimming ........................................................................................ 37 2.3.2 Alignment .................................................................................................... 37 2.3.3 Peak calling and IDR analysis ................................................................... 37 2.3.4 Quantification of ChIP tags ....................................................................... 38 2.3.5 Differential binding analysis ...................................................................... 38 2.3.6 Genomic annotation and visualization ........................................................... 39 2.3.7 De novo motif analysis ................................................................................ 39 2.3.8 Motif scanning ............................................................................................. 40 2.3.9 ENCODE data set and data accession ...................................................... 40 2.3.10 Common promoter binding events .......................................................... 40 2.3.11 Statistical test.............................................................................................. 41 Chapter 3 in vivo DNA binding specificity of a three zinc finger artificial DNA binding protein ............................................................................................................................. 42 3.1 Introduction ................................................................................................ 42 3.2 Experimental design and construct validation ........................................ 44 3.3 ChIP-Seq and Bioinformatics workflow .................................................. 47 3.4 AZF genomic occupancy ............................................................................ 51 3.4.1 AZF binds to a large number of sites within the genome ....................... 51 3.4.2 AZF occupancy is enriched at DNase hypersensitive sites ...................... 55 3.4.3 AZF binds predominantly to sites containing the target sequence ........ 57 Page | iii 3.5 Discussion .................................................................................................... 61 Chapter 4 Regions outside of DNA binding domain of the zinc finger transcription factor KLF3 are involved in in vivo DNA binding specificity........................................ 65 4.1 Introduction ................................................................................................ 65 4.2 Experimental design and construct validation ........................................ 67 4.3 ChIP-Seq and bioinformatics workflow