i Cell cycle regulation by post-translational and post-transcriptional mechanisms in an anaerobic extremist – The anoxic tolerant turtle, Trachemys scripta elegans Kyle Kevin Biggar B.Sc. Joint Honours St. Francis Xavier University, 2008 A Thesis Submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Carleton University Ottawa, Ontario, Canada © copyright 2013 Kyle K Biggar ii The undersigned hereby recommend to the Faculty of Graduate Studies and Research acceptance of this thesis Cell cycle regulation by post-translational and post-transcriptional mechanisms in an anaerobic extremist – The anoxic tolerant turtle, Trachemys scripta elegans submitted by Kyle Kevin Biggar, B.Sc. in partial fulfillment of the requirements for the degree of Doctor of Philosophy ____________________________________ Chair, Department of Biology ___________________________________ Thesis Supervisor ___________________________________ External Examiner Carleton University iii ABSTRACT As a model for vertebrate long-term survival in oxygen restricted environments, the red-eared slider turtle (T. s. elegans) can adapt at the biochemical level to deal with hibernation occurring in oxygen-free (anoxic) cold water (<10°C). In this thesis I hypothesized that the mechanisms which suppress ATP-expensive cell cycle activity, would contribute to establishing an hypometabolic state. To explore this possibility, this thesis studied the post-transcriptional and post-translational mechanisms of cell cycle arrest during anoxic stress in the freshwater turtle. Results indicated a general regulation of critical cell cycle components, in addition to the possible regulation by signaling cascades (Akt/GSK-3β and ATR/Chk2) that are known to regulate G /G phases of the cell cycle. Importantly, there is extensive 1 0 regulation of Cyclin D1 protein by (1) Akt/GSK-3β signaling, (2) post-translational modification, (3) an AU-rich region, and (4) microRNA-induced translational suppression. This study also identified a phase-specific cell cycle arrest mechanism involving the Rb/E2F DNA-binding complex in both anoxic liver and kidney tissues. A novel DNA-binding complex ELISA technique was able to identify that both kidney and liver establish an Rb/E2F1 mediated G arrest complex by 5 and 20 h anoxia, repectively. 1 By 20 h anoxia, kidney tissue established a reversible state of G , characterized by the 0 prescence of a p130/E2F4 DNA-bound complex. Overall, results from this thesis indicate that both kidney and liver enter into a G 1 arrest during anoxia. By contrast, the cell cycle in white skeletal muscle was found to be minimally regulated during anoxia and this finding is likely a reflection of its overall post-mitotic nature. Interestingly, kidney established a state of G arrest within 5 h anoxia 1 iv and subsequently transitioned to a sustainable G arrest by 20 h anoxia. However, it 0 appears that liver G arrest was not established until 20 h anoxia. Future studies will need 1 to explore the regulation of the cell cycle in liver after longer periods of anaerobiosis to determine whether hepatocytes are also able to transition into G arrest in a manner 0 similar to kidney tissue. v ACKNOWLEDGEMENTS First and foremost, I would like to thank my thesis supervisor, Dr. Kenneth Storey, for taking me into his lab when I was still an ecologist and turning me into a molecular biologist. I am grateful for his scientific advice, for giving me the opportunity to try new things and for shaping me into the scientist that I have become. He has been, and will continue to be, the major scientific role model throughout my career, and I am immensely thankful for my time under his tutelage. Of course, I must also thank Jan Storey. Her expansive knowledge and continuous hard work keeps the lab running. Unfortunately, I have been in the lab too long to list and thank all of my fellow lab members, past and present, but I would like to note that one of the most important qualities of the Storey lab is its incredible sense of community. It is a lab built on shared knowledge, and is all the more successful for it. A special thank you to both Mike Wu and Neal Dawson for our great scientific discussions – some of which, have turned into excellent scientific adventures. Most importantly, I would like to thank my family. I have always been grateful for their constant, unwavering support and involvement throughout my life. vi TABLE OF CONTENTS Title Page i Acceptance Sheet ii Abstract iii Acknowledgements v Table of Contents vi List of Abbreviations vii List of Figures viii List of Tables x List of Appendices xi Chapter 1 General introduction 1 Chapter 2 Characterization of cell cycle regulatory proteins 36 Chapter 3 Regulation of the G phase of the cell cycle 96 1 Chapter 4 Regulation of the Rb/E2F Transcription factor complex 134 Chapter 5 Evidence of microRNA regulation of cell cycle proteins 195 Chapter 6 General discussion 238 Appendices 260 vii LIST OF ABBREVIATIONS AA amino acid AEBSF 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride Akt protein kinase B AMP, ADP, ATP adenosine mono-, di-, or triphosphate APS ammonium persulphate ARE au-rich element ATM ataxia telangiectasia mutated ATR ATM-and Rad3 related BLAST basic Local Alignment Search Tool Bp base pairs BRM Brahma Cdc cell division cycle protein Cdk cyclin dependent kinase cDNA complementary deoxyribonucleic acid Chk checkpoint kinase CKI cdk inhibitor ddH O double distilled water 2 DEPC diethylpyrocarbonate dNTP deoxynucleotide triphosphate DTT dithiolthreitol E2F E2 promoter binding factor ECL enhanced chemiluminescence EDTA ethylenediamine tetraacetic acid ELISA enzyme-linked immunosorbent assay EMSA electromobility shift assay F6P fructose-6-phosphate G quiescence o G gap phase 1 1 G gap phase 2 2 GSK-3 glycogen synthase kinase-3 HAT histone acetyltransferase HDAC histone deacetylase HEPES N-(2-hydroxyethyl) piperazine-(2-ethanesulfonic acid) HIF-1 hypoxia-inducible factor-1 HMTase histone methyltransferase HRP horseradish peroxidase INK inhibitor of kinase viii kb kilobase kDa kilodalton Kip kinase inhibiting protein LINC Drosophila Rb/E2F and Myb complex M mitosis MAPK mitogen-activated protein kinase miRNA, miR microRNA mRNA messenger RNA MW molecular weight NCBI National Center for Biotechnology Information PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline PCNA proliferating cell nuclear antigen PCR p o l y m erase chain reaction PDK1 p hosphoinositide-dependent kinase PFK 6-phosphofructokinase PMSF phenylmethanesulfonyl fluoride PTM post-translational modification PVA poly-vinyl alcohol PVDF polyvinylidine fluoride Rb retinoblastoma protein RBP RNA-binding protein ROS reactive oxygen species RT room temperature (~21ºC) RT-PCR r e v erse-transcriptase polymerase chain reaction S DNA synthesis/replication SDS sodium dodecyl sulfate SOD super-oxide dismutase SSC saline-sodium citrate TAE tris-acetate-ethylenediamine tetraacetic acid buffer TBST tris-buffered saline containing Tween-20 TEMED N,N,N’,N’-tetra methylethylenediamine TMB 3,3’,5,5’-Tetramethylbenzidine Tris tris (hydroxymethyl) aminomethane UTR untranslated region ix LIST OF FIGURES Figure 1.1. North American distribution of anoxia tolerant turtles, (A) T. scripta elegans and (B) C. picta bellii. Figure 1.2. Lactate movement into a calcium carbonate rich shell during anoxia in the hypometabolic turtle. Figure 1.3. Transitions to and from a hypometabolic state in the anoxic turtle. Upon initial hypoxic sensing, cellular adjustments occur to reprioritize ATP metabolism and defend the cell from oxidative damage upon oxygen reperfusion. Figure 1.4. Generalized signaling pathways of ERK, SAPK/JNK, and p38 including their influences on cell functions. Figure 1.5. General mechanism of translational regulation by microRNA. MicroRNAs are targeted to the 3’ UTR of specific mRNA transcripts. Figure 2.1. Expression profiles of Cyclin/Cdk complexes throughout the cell cycle. Cyclic expression of these complexes allow for the completion of one phase before the initiation of the subsequent phase of the cell cycle. Figure 2.2. Regulation of Cyclin D1 degradation by Akt/PI3K signaling and regulation of GSK-3β activity. Figure 2.3. The Rb/E2F pathway. Sequential phosphorylation by kinase complexes Cyclin D1/Cdk4:Cdk6 and Cyclin E1/Cdk2, respectively, causes conformational changes to the Rb structure and release of E2F. The release of E2F is necessary for the expression of S-phases genes. Figure 2.4. Image files for immunoblotting and RT-PCR targets in control and anoxic (5 and 20 h) conditions from (A) liver, (B) kidney and, (C) white skeletal muscle tissue from T. scripta elegans. Figure 2.5. Effect of 5 and 20 h of anoxic submergence on relative protein and phosphorylation levels of Akt in T. s. elegans. Figure 2.6. Effect of 5 and 20 h of anoxic submergence on relative protein and phosphorylation levels of GSK-3b and phosphorylation of its downstream target, Cyclin D1 in T. s. elegans. Figure 2.7. Effect of 5 and 20 h of anoxic submergence on relative protein levels of Cyclins (types D1, E1, A1 and B1) in T. s. elegans. Figure 2.8. Effect of 5 and 20 h of anoxic submergence on relative nuclear protein levels of Cyclins (types D1, E1, A1 and B1) in T. s. elegans. Figure 2.9. Effect of 5 and 20 h of anoxic submergence on transcript levels of Cyclins (types D1, E1, A1 and B1) in T. s. elegans. Figure 2.10. Nucleotide and deduced amino acid sequence for T. s. elegans partial cyclin d1 sequence. Figure 2.11. Nucleotide and deduced amino acid sequence for T. s. elegans partial cyclin e1 sequence. Figure 2.12. Nucleotide and deduced amino acid sequence for T. s. elegans partial cyclin a1 sequence. Figure 2.13. Nucleotide and deduced amino acid sequence for T. s. elegans partial cyclin b1 sequence. Figure 2.14. Effect of 5 and 20 h of anoxic submergence on relative protein and phosphorylation levels of Cdks (types 4, 6, 2) and Cdc2 in T. s. elegans. Figure 2.15. Effect of 5 and 20 h of anoxic submergence on relative nuclear levels of Cdks (types 4, 6, 2) and Cdc2 in T. s. elegans. Figure 2.16. Effect of 5 and 20 h of anoxic submergence on transcript levels of Cdks (types 4, 6, 2) and Cdc2 in T. s. elegans. Figure 2.17. Nucleotide and deduced amino acid sequence for T. s. elegans partial cdk4 sequence. Figure 2.18. Nucleotide and deduced amino acid sequence for T. s. elegans partial cdk6 sequence. x Figure 2.19. Nucleotide and deduced amino acid sequence for T. s. elegans partial cdk2 sequence. Figure 2.20. Nucleotide and deduced amino acid sequence for T. s. elegans partial cdc2 sequence. Figure 3.1. Proposed hypoxia induced regulation of the cell cycle. Figure 3.2. The ATM/ATR pathway. Stress activation of either ATM and ATR results in downstream phosphorylation of checkpoint kinases (Chk1/2) and regulation of cell cycle effectors. Figure 3.3. Image files for immunoblotting and RT-PCR targets in control and anoxic (5 and 20 h) conditions from (A) liver, (B) kidney and, (C) white skeletal muscle tissue from T. scripta elegans. Figure 3.4. Effect of 5 and 20 h of anoxic submergence on relative protein expression of ATM and ATR in T. s. elegans. Figure 3.5. Effect of 5 and 20 h of anoxic submergence on relative protein and phosphorylation levels of Chk1 and Chk2 in T. s. elegans. Figure 3.6. Effect of 5 and 20 h of anoxic submergence on relative protein expression of Cdc25a and Cdc25c in T. s. elegans. Figure 3.7. Effect of 5 and 20 h of anoxic submergence on relative protein and phosphorylation levels of CKIs (types p27, p16 and p21) in T. s. elegans. Figure 3.8. Effect of 5 and 20 h of anoxic submergence on transcript levels of p27 in T. s. elegans. Figure 3.9. Nucleotide and deduced amino acid sequence for T. s. elegans partial p27 sequence. Figure 4.1. The Rb/E2F pathway. Sequential phosphorylation by kinase complexes Cyclin D1/Cdk4:Cdk6 and Cyclin E1/Cdk2, respectively, causes conformational changes to the Rb structure and release of E2F. The release of E2F is necessary for the expression of S-phases genes. Figure 4.2. Image files for immunoblotting and RT-PCR targets in control and anoxic (5 and 20 h) conditions from (A) liver, (B) kidney and, (C) white skeletal muscle tissue from T. scripta elegans. Figure 4.3. Effect of 5 and 20 h of anoxic submergence on relative protein expression of E2F1 and E2F4 in T. s. elegans. Figure 4.4. Effect of 5 and 20 h of anoxic submergence on relative nuclear protein expression of E2F1 and E2F4 in T. s. elegans. Figure 4.5. Effect of 5 and 20 h of anoxic submergence on relative protein and phosphorylation levels of Rb in T. s. elegans. Figure 4.6. Effect of 5 and 20 h of anoxic submergence on relative nuclear protein and phosphorylation levels of Rb in T. s. elegans. Figure 4.7. Effect of 5 and 20 h of anoxic submergence on relative protein expression of p130 in T. s. elegans. Figure 4.8. Effect of 5 and 20 h of anoxic submergence on the relative nuclear protein expression of p130 in T. s. elegans. Figure 4.9. Effect of 5 and 20 h of anoxic submergence on relative protein expression of epigentic modifying protein, Suv29H1 and HDAC4, in T. s. elegans. Figure 4.10. Effect of 5 and 20 h of anoxic submergence on the relative nuclear protein expression of epigentic modifying protein, Suv29H1 and HDAC4, in T. s. elegans. Figure 4.11. Electrophoretic mobility shift assay (EMSA) for the E2F family of transcription factors.
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