Role of promyelocytic leukemia (PML) and small ubiquitin like modifier (SUMO) proteins in alternative lengthening of telomeres

THE ROLE OF PROMYELOCYTIC LEUKEMIA (PML) AND SMALL UBIQUITIN–LIKE MODIFIER (SUMO) PROTEINS IN THE ALTERNATIVE LENGTHENING OF TELOMERES YONG WEI YAN JACKLYN (B.SC (HONS), NATIONAL UNIVERSITY OF SINGAPORE) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE, NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to express my most heartfelt gratitude to both Assoc. Professor M. Prakash Hande and Dr Martin Lee for the opportunity to work under their supervision. They have been selfless in imparting their knowledge and wisdom unto me and I sincerely thank both of them for their guidance. I would also like to express my gratitude and appreciation for their encouragement for my participation in various international scientific conferences. The exposure and experience gained at these conferences were invaluable. I would like to thank my friends in both Genome Stability and Nuclear Receptor laboratory. I extend my heartfelt gratitude to Mdm Wang Yaju and Mr Khaw Aik Kia, who have taught me various experimental techniques and imparted their laboratory knowledge unto me. In particular, I would also like to thank Dr Grace Low, Miss Diana Hay, Ms Asha, Mr Khaw Aik Kia and Mr Resham Gurung for their company, support and encouragement for the past years. I have gained a lot of insight from our discussions which have extended beyond science. I would also like to thank the members from both laboratories who have provided help and feedback with regard to my project. I would also like to extend my appreciation towards all staff members and their respective laboratories in the Department of Physiology for generously sharing the research equipments and materials. Special thanks towards the administrative staff, especially Ms Asha Das for her help whenever needed. In addition, my heartfelt gratitude towards my examiners for undertaking this thesis examination. Finally, I would like to thank my family members, particularly my spouse. A big ‘Thank You’ to him for being so very understanding about my commitment towards my project and for his tolerance when weekends had to be spent in the laboratory. I would also like to express my gratitude towards him for his expert help in the formatting of this thesis. My gratitude also to my mother, who has been extremely encouraging for me undertaking graduate studies and for grooming me into who I am today. My utmost appreciation to all of my family members for just loving me for who I am.   i LIST OF SCIENTIFIC CONFERENCES Yong JWY, Lee MB, and Hande MP. The coiled-coil domain of the promyelocytic leukemia protein is required for the formation of Alternative Lengthening of Telomeres-associated nuclear bodies. Telomeres and Telomerase Meeting, Cold Spring Harbor Laboratories Meeting. April-May 2009. Long Island, New York, USA. Yong JWY, Lee MB, and Hande MP. The role of the promyelocytic leukemia protein in the Alternative Lengthening of Telomeres. 100th Annual Meeting of the American Association for Cancer Research. April 2009. Denver, Colorado, USA. Yong JWY, Hande MP, and Lee MB. SUMO-mediated regulation of p53 in cancer cells exhibiting Alternative Lengthening of Telomeres. Centennial Conference of the American Association for Cancer Research. November 2007. Singapore, Singapore.   ii TABLE OF CONTENTS ACKNOWLEDGEMENTS I LIST OF SCIENTIFIC CONFERENCES II TABLE OF CONTENTS III LIST OF TABLES XI LIST OF FIGURES XIII LIST OF ILLUSTRATIONS XVII ABBREVIATIONS XVIII SUMMARY XXIII 1CHAPTER INTRODUCTION 1.1 POST-TRANSLATIONAL MODIFICATIONS 1.1.1 SMALL UBIQUITIN-LIKE MODIFIER (SUMO) 1.1.2 SUMOYLATION 1.1.3 REGULATION OF SUMO CONJUGATION 1.1.4 BIOLOGICAL FUNCTIONS OF SUMOYLATION 1.2 PROMYELOCYTIC LEUKEMIA (PML) 1.2.1 RING FINGER MOTIF 10 1.2.2 B-BOXES 10 1.2.3 COILED-COIL DOMAIN 11 1.2.4 PML NUCLEAR BODIES 11   iii 1.3 CANCER 12 1.4 HALLMARKS OF CANCER 13 1.5 TELOMERES 15 1.5.1 STRUCTURE OF TELOMERES 15 1.5.2 FUNCTIONS OF TELOMERES 16 1.5.3 TELOMERE MAINTENANCE MECHANISMS 17 1.5.4 TELOMERASE 18 1.6 ALTERNATIVE LENGTHENING OF TELOMERES 20 1.6.1 HALLMARKS OF ALT 20 1.6.2 MECHANISMS OF ALT 26 1.6.3 ALT AS A POSSIBLE CONSEQUENCE OF TELOMERE DYSFUNCTION 30 1.6.4 ALT AND TELOMERASE 31 1.6.5 EXISTENCE OF ALT REPRESSOR GENES 32 1.6.6 GENES POTENTIALLY INVOLVED IN ALT 34 1.6.7 ALT IN HUMAN CANCER 35 1.6.8 ALT AND PROGNOSIS 36 1.6.9 ALT AND CANCER THERAPY 37 2CHAPTER OBJECTIVES 39 3CHAPTER MATERIALS AND METHODS 42 3.1 CELL CULTURES 42 3.1.1 CELL LINES AND CULTURE CONDITIONS 42 3.1.2 PASSAGING CELLS 43 3.1.3 STORING CELLS 43 3.2 DETERMINATION OF NUCLEI ACID CONCENTRATION 43   iv 3.3 PREPARATION OF CACL2 COMPETENT E.COLI CELLS 44 3.4 PLASMIDS AMPLIFICATION 45 3.4.1 TRANSFORMATION 45 3.4.2 QIAGEN MINIPREP KIT 45 3.4.3 QIAGEN HISPEED PLASMID KIT 46 3.5 PLASMID CONSTRUCTS 46 3.5.1 ADDITION OF HA TAG TO SENP1 46 3.5.2 PCR MUTAGENESIS TO GENERATE PML KR MUTANTS 48 3.5.3 SUB-CLONING OF HA-PML TO PCI NEO VECTOR 49 3.5.4 ADDITION OF FLAG TAG TO PML C/C- 50 3.6 RESTRICTION ENDONUCLEASE DIGESTION OF DNA 51 3.7 AGAROSE GEL ELECTROPHORESIS 52 3.8 PURIFICATION OF DNA FROM AGAROSE GEL 52 3.9 DNA LIGATION 52 3.10 DNA FAST PREP 53 3.11 AUTOMATED DNA SEQUENCING 53 3.12 TRANSIENT TRANSFECTION 54 3.13 DETERMINATION OF GENETICIN DOSAGE 55 3.14 GENERATION OF STABLY OVER-EXPRESSING CELL CLONES (STABLE TRANSFECTION)   55 3.15 CELL CYCLE SYNCHRONIZATION 56 3.16 PREPARATION OF WHOLE CELL EXTRACTS 56 3.17 DETERMINATION OF PROTEIN CONCENTRATION BY BRADFORD METHOD 57 3.18 WESTERN BLOT ANALYSIS 58 3.18.1 SEPARATION OF PROTEINS BY POLYACRYLAMIDE GEL ELECTROPHORESIS 58   v 3.18.2 PROTEIN TRANSFER 58 3.18.3 WESTERN BLOTTING 59 3.19 IMMUNOPRECIPITATION 61 3.20 IMMUNOFLUORESCENCE 61 3.21 CONFOCAL MICROSCOPY ANALYSIS 62 3.22 CRYSTAL VIOLET CELL VIABILITY ASSAY 62 3.23 CELL CYCLE ANALYSIS BY PROPIDIUM IODIDE STAINING 63 3.24 COLONY FORMATION ASSAY 64 3.25 TERMINAL RESTRICTION FRAGMENT ANALYSIS 64 3.25.1 PREPARATION OF DNA FROM CELLS 65 3.25.2 RESTRICTION ENDONUCLEASE DIGESTION OF DNA 65 3.25.3 DNA SEPARATION BY AGAROSE GEL ELECTROPHORESIS 66 3.25.4 SOUTHERN BLOTTING 66 3.26 METAPHASE CHROMOSOMES PREPARATION 68 3.27 FLUORESCENCE IN SITU HYBRIDIZATION 68 3.28 TELOMERASE ACTIVITY ASSAY (TRAP) 69 3.29 BIOINFORMATICS AND BIOSTATISTICS 70 4CHAPTER RESULTS 4.1 SUMOYLATION OF P53 71 71 4.1.1 DIFFERENT GLOBAL SUMO-1 AND SUMO-2 CONJUGATION PATTERNS IN ALT 71 AND NON-ALT CANCER CELL LINES 4.1.2 SUMO-P53 IS DETECTED IN JFCF-6/T.1R CELLS AND NOT IN MCF7 CELLS 74 4.1.3 PIASY IS THE MOST STABLY OVER-EXPRESSED MEMBER AMONG THE PIAS FAMILY   79 vi 4.1.4 OVER-EXPRESSION OF P53 IN JFCF-6/T.1R CELLS BARELY AFFECTS SUMOYLATED P53 LEVELS 79 4.1.5 STABILITY OF OVER-EXPRESSED P53 IN MCF7 CELLS IS AFFECTED BY SUMO 80 AND PIAS 4.1.6 PIAS AFFECTS THE STABILITY OF OVER-EXPRESSED P53 81 4.1.7 SUMO1 AND PIAS STABILIZES OVER-EXPRESSED P53 FURTHER 82 4.2 EFFECTS OF SUMO-P53 84 4.2.1 SUMO-P53 AND CELL VIABILITY 84 4.2.2 SUMO-P53 AND CELL CYCLE PROGRESSION 87 4.3 FACTORS THAT AFFECT SUMOYLATION 89 4.3.1 ARSENITE REDUCES GLOBAL SUMOYLATION AS WELL THAT OF P53 IN ALT 89 CELLS 4.3.2 PROPORTION OF SUMO-P53 IN JFCF-6/T.1R CELLS VARIES IN DIFFERENT PHASES OF THE CELL CYCLE 4.4 PML IN CANCER 92 101 4.4.1 LYSINE160 IS IMPORTANT FOR SUMOYLATION OF PML AND THE COILED-COIL 101 DOMAIN IS REQUIRED FOR SUMOYLATION 4.4.2 TRANSIENTLY TRANSFECTED PML KR MUTANTS CONTINUE TO FORM APBS 105 BUT NOT THE COILED-COIL DOMAIN DELETION MUTANT 4.4.3 TRANSIENT OVER-EXPRESSION OF PML AND PML C/C- ENHANCES THE VIABILITY OF ALT CELLS 112 4.4.4 TRANSIENT OVER-EXPRESSION OF PML AND PML C/C- INCREASES THE 115 POPULATION OF ALT CELLS IN G2/M PHASE OF THE CELL CYCLE 4.4.5 U2OS AND MCF7 CLONES OF STABLY OVER-EXPRESSED PML AND PML C/C- WERE GENERATED 120 4.4.6 STABLY OVER-EXPRESSED PML C/C- DOES NOT FORM APBS IN ALT CELLS 122   vii 4.4.7 WILD-TYPE PML AND PML C/C- ALT CLONES HAVE A SLOWER POPULATION 129 DOUBLING RATE 4.4.8 WILD-TYPE PML INHIBITS THE CLONOGENICITY OF U2OS CELLS 132 4.4.9 HIGHER PROPORTION OF CELLS IN SUB-G1 AND G2/M PHASE OF THE CELL 135 CYCLE IN U2OS PML CLONES 4.4.10 WILD-TYPE PML INCREASES TELOMERE LENGTH SLIGHTLY WHILE PML C/C143 REDUCES TELOMERE LENGTH IN U2OS CELLS 4.4.11 TELOMERE LENGTHENING AND ACCUMULATION OF MCF7 CLONES EXHIBITING ALT-LIKE TELOMERE PHENOTYPE 149 4.4.12 MCF7 PML STC10 CLONE HAS A MUCH LOWER TELOMERASE ACTIVITY 151 4.4.13 MCF7 CLONES DISPLAYING ALT-LIKE PHENOTYPES ARE MORE SENSITIVE TO 153 DOXORUBICIN 5CHAPTER DISCUSSION 156 5.1 SUMOYLATION OF P53 156 5.1.1 DIFFERENT GLOBAL SUMO-1 AND SUMO-2 CONJUGATION PATTERNS IN ALT 156 AND NON-ALT CELL LINES 5.1.2 SUMO-P53 IS DETECTED IN JFCF-6/T.1R AND NOT IN MCF7 CELLS 156 5.1.3 PIASY IS THE MOST STABLY OVER-EXPRESSED MEMBER AMONG THE PIAS 158 FAMILY 5.1.4 OVER-EXPRESSION OF P53 IN JFCF-6/T.1R CELLS BARELY AFFECTS SUMOYLATED P53 LEVELS 159 5.1.5 STABILITY OF OVER-EXPRESSED P53 IN MCF7 CELLS IS AFFECTED BY SUMO 159 AND PIAS 5.1.6 PIAS AFFECTS THE STABILITY OF OVER-EXPRESSED P53 160 5.1.7 SUMO1 AND PIAS STABILIZES OVER-EXPRESSED P53 FURTHER 160 5.2 EFFECTS OF SUMO-P53 161 5.2.1 SUMO-P53 AND CELL VIABILITY 161   viii 5.2.2 SUMO-P53 AND CELL CYCLE PROGRESSION 162 5.3 FACTORS THAT AFFECT SUMOYLATION 164 5.3.1 ARSENITE REDUCES GLOBAL SUMOYLATION AS WELL THAT OF P53 IN ALT 164 CELLS 5.3.2 PROPORTION OF SUMO-P53 IN JFCF-6/T.1R CELLS VARIES IN DIFFERENT 166 PHASES OF THE CELL CYCLE 5.4 PML IN CANCER 167 5.4.1 LYSINE160 IS IMPORTANT FOR SUMOYLATION OF PML AND THE COILED-COIL 167 DOMAIN IS REQUIRED FOR SUMOYLATION 5.4.2 TRANSIENTLY TRANSFECTED PML KR MUTANTS CONTINUE TO FORM APBS 168 BUT NOT THE COILED-COIL DOMAIN DELETION MUTANT 5.4.3 TRANSIENTLY OVER-EXPRESSION OF PML AND PML C/C- ENHANCES THE 170 VIABILITY OF ALT CELLS 5.4.4 TRANSIENT OVER-EXPRESSION OF PML AND PML C/C- INCREASES THE 170 POPULATION OF ALT CELLS IN G2/M PHASE OF THE CELL CYCLE 5.4.5 U2OS AND MCF7 CLONES OF STABLY OVER-EXPRESSED PML AND PML C/C- WERE GENERATED 171 5.4.6 STABLY OVER-EXPRESSED PML C/C- DOES NOT FORM APBS IN ALT CELLS 172   5.4.7 WILD-TYPE PML AND PML C/C- ALT CLONES HAVE A SLOWER POPULATION 173 DOUBLING RATE 5.4.8 WILD-TYPE PML INHIBITS THE CLONOGENICITY OF U2OS CELLS   174 5.4.9 HIGHER PROPORTION OF CELLS IN SUB-G1 AND G2/M PHASE OF THE CELL 175 CYCLE IN U2OS PML CLONES 5.4.10 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Cong (2009) Telomerase reverse transcriptase activates the expression of vascular endothelial growth factor independent of telomerase activity. Biochem Biophys Res Commun, 386, 739-43. Zhu, X. D., B. Kuster, M. Mann, J. H. Petrini & T. de Lange (2000) Cell-cycleregulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet, 25, 347-52.   210 [...]... groups, to proteins Post-translational modifications may also involve structural changes of a protein, including the formation of disulphide bridges between cysteine amino acids and proteolytic cleavage (of a peptide bond) Post-translational modifications of a protein may also involve the attachment of other proteins or peptides, such as ubiquitin and the small ubiquitin- like modifier (SUMO) Subsets of a... suppressor in APL and in other cancers; PML has also been implicated in the repression of gene expression and the promotion of both intrinsic and extrinsic apoptotic pathway (Jensen, Shiels and Freemont 2001, Ruggero, Wang and Pandolfi 2000) There are three cysteine-rich zinc-binding domains in PML, a RING-finger domain and two B-boxes (B1 and B2) Together with a predicted α-helical coiled-coil domain, these... localization signal in exon 6 is not present in all PML isoforms and this results in both nuclear and cytoplasmic isoforms of PML Thus while the RBCC motif is conserved in all PML isoforms, the isoforms differ in their C-terminal regions   9 1.2.1 RING finger motif The RING finger motif is a cysteine-rich zinc binding domain The conserved RING structural elements consists of two zinc atoms bound via... of proteins may be modified post-translationally according to the cellular conditions and microenvironment as part of the cellular response and regulatory processes 1.1.1 Small Ubiquitin- like Modifier (SUMO) The Small Ubiquitin- like Modifier (SUMO) protein is a 10-11 KDa polypeptide that has a strong structural homology to ubiquitin (Melchior 2000, Ulrich 2009, Johnson 2004) However, SUMO has distinct... (protein inhibitor of activated STAT) family is the most prominent among the SP-RING proteins In general, the E3 ligases appear to play a part in conferring substrate selectivity to the SUMO conjugation process 1.1.2.3.1 PIAS PIAS (protein inhibitor of activated STAT) proteins were named after their ability to interact and inhibit STAT proteins (Palvimo 2007) In mammals, four genes encode the PIAS proteins; ... family members and this suggests that it is an important determinant of the overall motif and its function (Reymond et al 2001) The B-boxes, B1 and B2, are two distinct cysteine-rich motifs adjacent to the RING domain Both bind zinc but differed in terms of the number and spacing of conserved Cys and His ligands (Borden et al 1996) While substitution of the conserved zinc ligands in B1 and/ or B2 disrupted... arrangement of Cys and His ligands Mutations of these ligands in PML disrupted its nuclear body formation and led to loss of its growth suppression and apoptosis abilities (Jensen et al 2001) The requirement of an intact RING finger for PML nuclear body formation could be due to specific proteinprotein interactions that are mediated by the RING motif In addition, the RING finger motif in PML specifically interacts... Besides being involved in the cleavage of the SUMO precursor to the mature form, SENPs are also involved in the deconjugation of SUMO from target proteins and in the processing of SUMO polymers The C-terminal hydrolase activity of SENP converts the SUMO precursor to its mature form The removal of SUMO from target proteins is accomplished through the isopeptidase activity of SENP and this occurs in a single... xxi TIN2 – TRF1-Interacting Partner TMM – Telomere Maintenance Mechanism TPG – Total Product Generated TPP1 – POT1 -and- TIN2 binding protein TRAP – Telomeric Repeat Amplification Protocol TRF – Terminal Restriction Fragment TRF1 – Telomeric Repeat Binding Factor 1 TRF2 – Telomeric Repeat Binding Factor 2 UV – Ultra-violet WRN – Werner Syndrome protein   xxii SUMMARY The Alternative Lengthening of Telomeres. .. fidelity of chromosome transmission (Takahashi et al 2006) and the SUMOylation of RAD52 to the regulation of recombination events at the ribosomal gene locus (Torres-Rosell et al 2007)   8 1.2 Promyelocytic Leukemia (PML) The promyelocytic leukemia (PML) protein is a growth and tumor suppressor that is inactivated in acute promyelocytic leukemia (APL) through the fusion of the PML gene with the retinoic . THE ROLE OF PROMYELOCYTIC LEUKEMIA (PML) AND SMALL UBIQUITIN LIKE MODIFIER (SUMO) PROTEINS IN THE ALTERNATIVE LENGTHENING OF TELOMERES YONG WEI YAN JACKLYN. MB, and Hande MP. The coiled-coil domain of the promyelocytic leukemia protein is required for the formation of Alternative Lengthening of Telomeres- associated nuclear bodies. Telomeres and. Telomerase Meeting, Cold Spring Harbor Laboratories Meeting. April-May 2009. Long Island, New York, USA. Yong JWY, Lee MB, and Hande MP. The role of the promyelocytic leukemia protein in the Alternative