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The Role of NF-Y in the Transcriptional Regulation of Human Topoisomerase II

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The Role of NF-Y in the Transcriptional Regulation of Human Topoisomerase II Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. The Role of NF-Y in the Transcriptional Regulation of Human Topoisomerase Ila A thesis presented to Massey University in partial fulfilment of the requirement for the degree of Master of Science in Biochemistry Patricia Ann Hintz 2001 ACKNOWLEDGEMENTS First of all, I would like to thank my very patient and wonderful supervisor Dr Kathryn Stowell for her support, enthusiasm, expertise, and for giving me the opportunity to be challenged and involved in a very interesting research project. I would also like to take this opportunity to thank the various and varied members of the Twilight Zone particularly Carole Flyger, Kirsty Allen and Angela Jones for their friendship, advice and support during the course of this study. I would also like to thank my parents, Geoffrey and Adrienne Hintz, and my sister Carolyn for their everlasting love, support and understanding. Finally, I would like to thank Dwayne, without you, none of what I have done or still might do would be possible. .1 ABSTRACT DNA topoisomerases are ubiquitous enzymes that catalyse reactions that alter the topological state of DNA during the various processes of DNA metabolism including transcription, recombination, replication and chromosome segregation. Human cells exhibit a Type II enzyme termed DNA topoisomerase Ila. This enzyme is expressed at higher levels in proliferating cells due to an increased demand for chromosome separation. This is advantageous with respect to some of the drugs used in chemotherapy. These drugs can specifically target cancer cells by only being effective at high levels of topoisomerase Ila gene expression. However, the use of such drugs has been limited by both toxicity and the development of resistance. This resistance has been associated with a decrease in topoisomerase Ila at both protein and mRNA levels. The topoisomerase Ila minimal promoter is 650 base pairs in length and includes promoter elements such as inverted CCAAT boxes (ICBs) and GC rich regions. It has been determined that the ICB elements are of the most interest in terms of regulation of the topoisomerase Ila gene expression. Several studies have shown that the transcription factor NF-Y binds to ICB 1-4 of the topoisomerase Ila promoter and regulates transcription through these elements. This stucly aimed to determine the importance of NF-Y in the transcriptional regulation of topoisomerase Ila and to investigate the molecular mechanisms by which NF-Y associates with the topoisomerase Ila promoter with a particular focus on the inverted CCAAT box elements. The binding of NF-Y to oligonucleotides containing selected consensus elements of the topoisomerase Ila promoter was analysed (in vitro) by electrophoretic mobility shift binding assays. The importance of NF-Y in the regulation of topoisomerase Ila expression was analysed by functional assays, usmg reporter gene constructs in transiently transfected HeLa cells. The binding studies indicated that the flanking sequences affect the affinity of the transcription factor NF-Y for ICBl and ICB2 and that a regulatory element flanking ICB2 may aid in NF-Y binding to that element. Functional assays showed that NF-Y appears to act a negative regulator of topoisomerase Ila with its effect being entirely -due to interaction with ICB2. 11 ABBREVIATIONS bp base pair BSA bovine serum albumin CAT chloroamphenicol acetyl transferase cDNA complementary DNA CDP CCAAT displacement protein C/EBP CCAAT enhancer binding protein CEM human leukemic ( cell line) CHO Chinese hamster ovary ( cell line) CO2 carbon dioxide cpm counts per minute DMSO dimethyl sulphoxide DNase I deoxyribonuclease I dNTPs deoxynucleotide triphosphates DTT dithiothreitol E. coli Escherichia coli EDTA ethylene diamine tetraacetic acid EMSA electrophoretic mobility shift assay FCS fetal calf serum G segment gate segment GCG Genetics Computer Group HADC histone deacetylase HAT histone acetyl-transferase HeLa human cervical carcinoma cell line HEPES N-[2-hydroxyethyl]piperazine-N' -[2-ethane sulfonic acid] HL-60 human promyelocytic leukaemia cells ICB inverted CCAAT box ICBP90 inverted CCAAT box binding protein Mr 90 kDa IgG isopropyl-~-D-thiogalactopyranoside kb kilobase LB Luria-Bertani MEM Eagle's minimal essential medium j\,fW molecular weight m NF-Y Nuclear factor-Y oligo oligonucleotide ONPG o-nitrophenol P-D-galacto-pyranoside PAGE polyacrylarnide gel electrophoresis PBS phosphate buffered saline PBSE phosphate buffered saline EDT A PCAF p3 00/CBP-associated factor PEA3 phenylmethylsulfonyl fluoride pGL2C pGL2Control pGL3B pGL3Basic PNK polynucleotide kinase poly( dI-dC) poly (dI-dC) poly (dl-dC) PSV-pgal pSV-P-galactosidase expression vector RT reverse transcriptase RT-PCR polymerase chain reaction coupled reverse transcriptase SDS sodium dodecylsulphate SDS-PAGE SDS-polyacrylarnide gel electrophoresis Spl Specificity protein 1 Sp3 Specificity protein 3 SV40 simian virus 40 T segment transport segment T80 80 cm2 flasks TAE tris acetate EDT A TBE tris boric acid EDT A TBST tris buffered saline triton X-100 TBP TATA-box binding protein TE~D N,N,N' ,N' -Tetramethylethylenediamine TK thymidine kinase Tris tris (hydroxymethyl)-arninomethane UV ultra violet light X-gal 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside YB-1 Y-box binding protein lV LIST OF FIGURES Page Figure 1.1 Schematic Diagram of the catalytic cycle of 4 topoisomerase Ila. Figure 1.2 The human topoisomerase Ila promoter 6 Figure 3.1 RNA isolated from HeLa cells 48 Figure 3.2 Gel photograph showing amplification ofNF-YA 51 probe Figure 3.3 Gel photograph of NF-YA probe quantitation 53 Figure 3.4 Gel photograph ofNF-Y PCR results 59 Figure 4.1 Schematic of EMSA 63 Figure 4.2 PAGE of end-labelled oligonucleotides 66 Figure 4.3 EMSA of HeLa extract with probes ICB2, ICB IF 67 and ICB2F Figure 4.4 EMSA ofHeLa extract with probes ICBl, ICB2, 68 ICB IF and ICB2F Figure 4.5 EMSA of the ICB2 probe with antibodies to NF-YA, 70 NF-YB and NF-YC Figure 4.6 EMSA ICB2, ICB IF and ICB2F probes with anti­ 72 NF-YA and -NF-YB Figure 4.7 EMSA with ICBl, ICB2, ICBlF and ICB2F probes 73 with antibodies to NF-YA and NF-YB V Page Figure 4.8 EMSA ofICBl probe with ICBlwt, ICBlmt, 76 ICB IF and ICB2F competitors Figure 4.9 EMSA ofICB2 probe with ICB2wt, ICB2mt, 79 ICB IF and ICB2F competitors Figure 4.10 EMSA of ICB IF probe with competitors ICB2wt, 81 ICB2mt, ICB IF and ICB2F Figure 4.11 EMSA ofICB2F probe with ICB2wt, ICB2mt, 84 ICB IF and ICB2F competitors Figure 5.1 Dosage dependence expression ofwt and ICB2- 92 Figure 5.2 Effect of promoter mutations on topo Ilalpha 93 expression Figure 5.3 Effect ofNF-YA dominant negative mutant on 95 pGL3B-6 l 7wt expression Figure 5.4 Effect ofNF-YA dominant negative vector on 96 pGL3B-617ICB2- expression Figure 6.1 Model for the regulation of topoisomerase Ila 111 Figure 6.2 Strategy for co-immunoprecipitation of proteins 113 from cultured cells Figure 6.3 Diagram of a GST-X fusion protein experiments 115 Figure 6.4 Schematic of a yeast two-hybrid experiment 116 Vl LIST OF TABLES Page Table 2.1 Mammalian cell lines and plasmids used in 30 this study Table 3.1 PCR thermocycling conditions used tor the 50 amplification of the NF-YA probe sequence Table 3.2 The monitoring progress of labelling with [ a-32P]dCTP 54 Table 3.3 PCR conditions for the amplification of NF-YA 58 NF-YB and NF-YC Table 4.1 The radioactivity of each probe ( 1 µL) in the first 64 labelling experiment determined by Cerenkov counting Table 4.2 The radioactivity of each probe ( 1 µL) in the second 64 labelling experiment determined by Cerenkov counting Table 5.1 Example of raw data and calculations involved 90 Table 6.1 DNA sequences of the ICB elements ICBl and ICB2 102 from the human topoisomerase Ila promoter Table 6.2 Transient transfection data from three separate sources 105 Vil TABLE OF CONTENTS Page ACKNOWLEDGEMENTS I ABSTRACT II ABBREVIATIONS III LIST OF FIGURES IV LIST OF TABLES VI TABLE OF CONTENTS VII Chapter One - Introduction I 1.1 DNA Topoisomerases 1 1.1.1 Prokaryotic DNA Topoisomerases 1 1.1.2 Yeast Topoisomerases 2 1.1.3 Human DNA Topoisomerases 2 J:2 Human Topoisomerase Ila 4 1.2.1 Human Topoisomerase Ila Promoter 5 1.2.2 Transcriptional Regulation of Human Topoisomerase Ila 7 1.2.3 Sp 1 and Sp3 Transcription Factors 9 1.2.4 Nuclear Factor-Y (NF-Y) 13 1.3 Topoisomerase Ila and Chemotherapy 20 1.3 .1 Topoisomerase Ila as a Drug Target 20 1.3 .2 Drug Resistance to Topoisomerase II Targeted Drugs 21 1.4 Aims of the Research 22 Chapter Two - Materials and Methods 23 2.1 Materials 23 2.2 Agarose Gel Electrophoresis 26 Vlll page 2.3 Quantitation ofDNA by Gel Electrophoresis 26 2.4 Quantitation of DNA by UV Spectroscopy 27 2.5 Restriction Digests 27 2.6 Annealing Single-stranded Oligonucleotides 27 2.7 Labelling Oligonucleotides for Electrophoretic Mobility Shift 28 Assays 2.8 Ethanol Precipitation 28 2.9 Isolation ofPlasmid DNA 29 2.10 Large Scale Preparation ofPlasmid DNA 29 2.11 Glycerol Stocks ofRecombinant Plasmids 30 2.12 DNA Sequencing 30 2.13 Isolation ofRNA from HeLa cells 31 2.14 Quantification and Analysis of RNA 31 2.15 Northern Blot Analysis 32 Preparation of Northern Blot 32 Preparation of Probe 32 Labelling a DNA Probe 33 Monitoring the Progress of the Reaction 34 Pre-hybridisation 34 Hybridisation 35 Washings 35 2.16 Reverse Transcriptase Polymerase Chain Reaction 36 DNase Treatment of RNA 36 First Strand Synthesis 36 2.17 Oligonucleotide Primers 40 2.18 Preparations for Tissue Culture 41 2.19 Starting HeLa Cell Cultures 41 2.20 Tissue Culture and Passage of Tissue Culture Cells 41 2.21 Transient Transfections 42 2.22 Harvesting Tissue Culture Cells (HeLa Cells) 43 2.23 Luciferase Assay 43 2.24 B-Galactosidase Assay 44 2.25 HeLa Cell Extract Pre/Xll'ation 44 lX page 2.
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