Regulation of DNA (cytosine 5) methyltransferase 1 in the cell cycle and its role(s) in doxorubicin mediated micronuclei formation

REGULATION OF DNA (CYTOSINE-5) METHYLTRANSFERASE IN THE CELL CYCLE AND ITS ROLE(S) IN DOXORUBICIN-MEDIATED MICRONUCLEI FORMATION TAN HWEE HONG NATIONAL UNIVERSITY OF SINGAPORE 2008 REGULATION OF DNA (CYTOSINE-5) METHYLTRANSFERASE IN THE CELL CYCLE AND ITS ROLE(S) IN DOXORUBICIN-MEDIATED MICRONUCLEI FORMATION TAN HWEE HONG (B.Sc, Murdoch University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY AND DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to thank my supervisor, Associate Professor Benjamin F.L.Li, for having faith in my ability to undertake graduate studies and for his guidance and encouragement throughout the years I would also like to thank Professor Alan Porter, Associate Professor Cai Mingjie and Dr Linda Chuang, members of my supervisory committee, for their advice and discussion I would like to extend my sincere appreciation to; Prof Alan Porter for his willingness to guide me through the last year of my PhD I am most grateful to him for giving me constructive suggestions for my project, and for reading and editing my thesis I really appreciate his constant encouragement A/Prof Manoor Prakash Hande for his kindness in helping me cross the final hurdle Dr Linda Chuang for teaching me many useful techniques and for sharing with me her invaluable knowledge on DNA methylation I am grateful for her guidance and her friendship all these years To Miss Yeo Wanlin and Dr Vinay Badal for their friendship and encouragement To Dr Oh Hue Kian and Miss Swa Li Foon for their collaborations Members of Prof Porter’s laboratory for sharing their knowledge in cell death Special thanks to my husband for his support and understanding throughout the years Finally, I would like to dedicate this thesis with great love to my parents and my husband, without whom none of this would have been possible i SUMMARY DNA methyltransferase (DNMT1) is the major methyltransferase involved in maintenance methylation of newly synthesized DNA The regulation of DNMT1 expression is critical in coordinating DNMT1 activity with biological processes and therefore must be tightly regulated in the cell cycle Interestingly, DNMT1 expression is inversely correlated with the cell cycle inhibitor p21WAF1 protein in mammalian cells, which is independent of the tumor suppresser p53 as shown here Using a combination of experimental protocols including cell synchronization studies, transient over-expression, siRNA-mediated depletion and luciferase reporter assays, the roles of the transcription factors E2F1 and Sp1, and the transcriptional coactivator p300 in the regulation of DNMT1 expression were investigated Transcription from the human DNMT1 promoter was shown to be dependent on E2F1 and Sp1 In addition, this study has identified p300 as a crucial transcriptional coactivator for E2F1 and Sp1 in regulating DNMT1 expression Most importantly, this report demonstrates for the first time that p21WAF1 negatively regulates DNMT1 at the transcriptional level The up-regulation of p21WAF1 by in-vitro over-expression or by treatment with Trichostatin A led to the corresponding down-regulation of DNMT1, which consistently coincided with the reduction of p300 Although p21WAF1 is known to inhibit the transcriptional activity of E2F1, my data show that p21WAF1 may potentially inhibit p300, either directly or indirectly Surprisingly, DNMT1 was down-regulated by TSA treatment in the absence of p300, which may be due to the selective depletion of Sp1 that only occurs when p300 is absent Nevertheless, my findings provide the outline of a mechanistic explanation for the inverse relationship between DNMT1 and p21WAF1 in mammalian cells This novel p21WAF1-E2F1/p300- ii DNMT1 pathway may play a pivotal role to ensure regulated DNMT1 expression and DNA methylation in mammalian cell division De-regulated DNMT1 expression is often associated with tumorigenesis, and there are reports suggesting that DNMT1 acts as a potential target for cancer therapy I attempted to compare the genotoxic effects of doxorubicin, a Topoisomerase II poison commonly used in chemotherapeutic treatments, on normal and cancer human cell lines, and the potential involvement of DNMT1 in this doxorubicin-induced cytotoxicity The physiological significance of the relationship between p300 and DNMT1 was further highlighted in the doxorubicin-mediated DNA damage response, which consistently depleted p300, and consequently DNMT1, in non-tumorigenic but not tumorigenic cells My data further show that doxorubicin selectively induces micronucleation preceded by senescence-like morphological changes in transformed or tumorigenic cells only, and this consistently correlates with a decrease in cell viability and an increase in cell death Importantly, I discovered for the first time a positive link between DNMT1 protein expression and micronuclei formation However, although I provide strong evidence that DNMT1 plays a significant role in doxorubicin-induced micronucleation, which may lead to cellular demise; it remains unclear to what extent DNMT1 contributes to the ultimate cell death Nevertheless, my findings strongly support DNMT1 as one molecular target for doxorubicininduced cytotoxicity in mammalian cancer cells I proposed that the expression levels of DNMT1 in tumor cells may potentially determine the effectiveness of doxorubicin in chemotherapy This novel observation enhances the understanding of drug response during doxorubicin administration in cancer therapy iii TABLE OF CONTENTS Acknowledgements i Summary ii Table of Contents iv List of Tables ix List of Diagrams ix List of Figures x Abbreviations xiv Chapter 1: 1.1 INTRODUCTION Historical Overview of DNA Methylation 1.1.1 1.1.2 DNA methylation in eukaryotic cells Mammalian DNA methylation 1.1.3 Demethylation of 5MeC 1.1.4 1.2 DNA methylation in prokaryotic cells Host defense versus DNA methylation Mammalian DNA Methyltransferases 1.2.1 DNMT1 DNMT1 variants 1.2.2 10 1.2.3 DNMT3a and DNMT3b 11 1.2.4 1.3 DNMT2 Cooperation among the DNMTs 12 Roles of DNA Methylation 1.3.1 Genomic imprinting 13 1.3.2 X-chromosome inactivation 15 1.3.3 Epigenetic regulation of genes 16 1.3.3a DNMT1 interacts with G9a histone methylase 17 1.3.3b DNMT1 interacts with Polycomb Group (PcG) proteins 17 1.3.3c DNMT1 interacts with UHRF1 18 Transcriptional suppression 19 1.3.4 iv 1.4 DNMT1 in the Cell Cycle 1.4.1 21 1.4.2 E2F1/RB pathway 23 1.4.3 p300/CBP co-activator 25 1.4.4 DNMT1 and DNA replication 27 1.4.5 DNMT1 and PCNA 27 1.4.6 1.5 Regulation of DNMT1 expression DNMT1 and p21WAF1 29 Regulation of p21WAF1 Expression 1.5.1 p53-dependent trancriptional regulation of p21WAF1 WAF1 31 1.5.2 31 1.5.3 1.6 p53-independent transcriptional regulation of p21 Translational regulation of p21WAF1 32 DNA Damage Response 1.6.1 33 1.6.2 Cell cycle checkpoints 34 1.6.3 DNMT1 and p53 36 1.6.4 DNMT1 in DNA damage and repair 37 1.6.5 1.7 ATM/ATR signaling pathways The nucleolus – a DNA damage response center 38 Micronuclei 1.7.1 40 1.7.2 DNMT1 and micronuclei 41 1.7.3 Possible DNA content in the micronuclei 42 1.7.4 1.8 Micronuclei - What are they? Doxorubicin 43 Research Objectives Chapter 2: 45 MATERIAL AND METHODS 2.1 Mammalian cell cultures 47 2.2 Antibodies 47 2.3 Bacterial strain 48 2.4 Drugs and Chemicals 48 2.5 Drug treatments 48 2.6 Harvesting of mammalian cells 48 2.7 Flow cytometry analysis 49 2.8 Cell viability and cell proliferation assays 49 v 2.9 Lactate Dehydrogenase (LDH) cell death assay 49 2.10 Purification of total RNA 50 2.11 Reverse transcription and PCR 50 2.12 Purification of genomic DNA 51 2.13 DNA agarose gel electrophoresis 52 2.14 Methylation-sensitive McrBc restriction digestion 52 2.15 Cell lysis 52 2.16 Western blot analysis 53 2.17 DNA manipulations 54 2.18 Mammalian expression plasmids 54 2.19 Cloning of full length DNMT1 into pXJ40-Flag vector 56 2.20 Cloning of DNMT1 promoter construct into pGL3-Basic vector 56 2.21 Preparation of competent cells 57 2.22 Transfection 58 2.23 Luciferase reporter assay 58 2.24 siRNA experiments 59 2.25 Cell staining 60 2.26 Cell count for micronuclei frequency 60 2.27 Comet assay 61 Chapter 3: 3.1 RESULTS AND DISCUSSION Regulation of DNMT1 expression in the Cell Cycle 3.1.1 Inverse relationship between DNMT1 and p21WAF1 in the 63 cell cycle 3.1.1.1 3.1.1.2 DNMT1 expression in the cell cycle 63 WAF1 in DNA 67 p21WAF1 68 Transient over-expression of DNMT1 does not inhibit 71 Inverse relationship between DNMT1 and p21 damage 3.1.1.3 Inverse relationship between DNMT1 and expression is independent of p53 3.1.1.4 p21WAF1 3.1.1.5 siRNA-mediated depletion of DNMT1 does not induce 72 p21WAF1 vi 3.1.1.6 Transient over-expression of p21WAF1 inhibits DNMT1 73 3.1.1.7 TSA-mediated induction of p21WAF1 results in inhibition of 76 DNMT1 3.1.1.8 TSA-mediated induction of p21WAF1 is independent of p53 85 3.1.2 Transcriptional regulation of human DNMT1 promoter 89 3.1.2.1 siRNA-mediated depletion of E2F1 results in down- 89 regulation of DNMT1 3.1.2.2 siRNA-mediated depletion of p300 results in down- 92 regulation of DNMT1 3.1.2.3 Transcriptional regulation of DNMT1 promoter by E2F1, 97 Sp1 and p300 3.1.3 3.2 Discussion 101 The Role(s) of DNMT1 in Doxorubicin-mediated micronuclei formation 3.2.1 Doxorubicin mediates selective depletion of DNMT1 in non- 108 transformed cell lines 3.2.2 Doxorubicin induces micronuclei formation in transformed 112 cell lines 3.2.3 Doxorubicin-mediated micronuclei formation is a general 118 phenomenon in transformed cell lines 3.2.4 Doxorubicin retards proliferation in both transformed and 120 non-transformed cell lines 3.2.5 Doxorubicin induces micronuclei formation in transformed 124 cell lines in a dose and time-course dependent manners 3.2.6 Induction of micronuclei correlates with decrease in cell 125 viability and increase in cell death in the transformed cell lines 3.2.7 Doxorubicin-mediated micronuclei structures are sites of 129 DNA damage 3.2.8 Depletion of DNMT1 by 5AzadC treatment attenuates Dox- 132 mediated micronuclei formation vii 3.2.9 Knockout of DNMT1 attenuates Dox-mediated micronuclei 136 formation 3.2.10 Knockdown of DNMT1 by siRNA-mediated transfection 141 attenuates Dox-mediated micronuclei formation 3.2.11 Chapter 4: Discussion CONCLUSIONS, 144 IMPLICATIONS AND FUTURE DIRECTIONS Conclusions 151 Implications and Future Directions 154 Chapter 5: REFERENCES 164 Chapter 6: APPENDIX List of Publications 190 viii Klisovic RB, Stock W, Cataland S, Klisovic MI, Liu S, Blum W et al (2008) A phase I biological study of MG98, an oligodeoxynucleotide antisense to DNA methyltransferase 1, in patients with high-risk myelodysplasia and acute myeloid leukemia Clin Cancer Res 14: 2444-9 Korba BE, Hays JB (1982) Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA V Recombinogenic plasmids from arl mutants of Escherichia coli are unusually sensitive to nuclease S1 and partially deficient in cytosine methylation at C-C-(A/T)-G-G sequences J Mol Biol 157: 213-35 Kottke TJ, Blajeski AL, Meng XW, Svingen PA, Ruchaud S, Mesner PW, Jr et al (2002) Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells J Biol Chem 277: 804-15 Koutsodontis G, Moustakas A, Kardassis D (2002) The role of Sp1 family members, the proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WAF-1/Cip1 gene promoter Biochemistry 41: 12771-84 Koutsodontis G, Tentes I, Papakosta P, Moustakas A, Kardassis D (2001) Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein J Biol Chem 276: 29116-25 Kovesdi I, Reichel R, Nevins JR (1987) Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control Proc Natl Acad Sci U S A 84: 21804 Krek W, Xu G, Livingston DM (1995) Cyclin A-kinase regulation of E2F-1 DNA binding function underlies suppression of an S phase checkpoint Cell 83: 1149-58 Krishna TS, Kong XP, Gary S, Burgers PM, Kuriyan J (1994) Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA Cell 79: 1233-43 Kurihara Y, Kawamura Y, Uchijima Y, Amamo T, Kobayashi H, Asano T et al (2008) Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase Dev Biol 313: 335-46 Kurki S, Peltonen K, Latonen L, Kiviharju TM, Ojala PM, Meek D et al (2004) Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation Cancer Cell 5: 465-75 Kurz EU, Douglas P, Lees-Miller SP (2004) Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species J Biol Chem 279: 53272-81 Kuzmichev A, Jenuwein T, Tempst P, Reinberg D (2004) Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3 Mol Cell 14: 183-93 176 Lambert PF, Kashanchi F, Radonovich MF, Shiekhattar R, Brady JN (1998) Phosphorylation of p53 serine 15 increases interaction with CBP J Biol Chem 273: 33048-53 Lavin MF (2007) ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks Oncogene 26: 7749-58 Lavin MF, Kozlov S (2007) ATM activation and DNA damage response Cell Cycle 6: 931-42 Le Gac G, Esteve PO, Ferec C, Pradhan S (2006) DNA damage-induced downregulation of human Cdc25C and Cdc2 is mediated by cooperation between p53 and maintenance DNA (cytosine-5) methyltransferase J Biol Chem 281: 24161-70 Lee BH, Yegnasubramanian S, Lin X, Nelson WG (2005) Procainamide is a specific inhibitor of DNA methyltransferase J Biol Chem 280: 40749-56 Lee CW, Sorensen TS, Shikama N, La Thangue NB (1998) Functional interplay between p53 and E2F through co-activator p300 Oncogene 16: 2695-710 Lee H, Zeng SX, Lu H (2006) UV Induces p21 rapid turnover independently of ubiquitin and Skp2 J Biol Chem 281: 26876-83 Lee JH, Paull TT (2007) Activation and regulation of ATM kinase activity in response to DNA double-strand breaks Oncogene 26: 7741-8 Lee SD, Alani E (2006) Analysis of interactions between mismatch repair initiation factors and the replication processivity factor PCNA J Mol Biol 355: 175-84 Leonhardt H, Page AW, Weier HU, Bestor TH (1992) A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei Cell 71: 865-73 Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development Nat Rev Genet 3: 662-73 Li E, Beard C, Jaenisch R (1993) Role for DNA methylation in genomic imprinting Nature 366: 362-5 Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality Cell 69: 915-26 Li L, He S, Sun JM, Davie JR (2004) Gene regulation by Sp1 and Sp3 Biochem Cell Biol 82: 460-71 Li X, Amazit L, Long W, Lonard DM, Monaco JJ, O'Malley BW (2007) Ubiquitinand ATP-independent proteolytic turnover of p21 by the REGgamma-proteasome pathway Mol Cell 26: 831-42 177 Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E et al (2002) Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements Mol Cell Biol 22: 480-91 Lin MJ, Lee TL, Hsu DW, Shen CK (2000) One-codon alternative splicing of the CpG MTase Dnmt1 transcript in mouse somatic cells FEBS Lett 469: 101-4 Lin WC, Lin FT, Nevins JR (2001) Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation Genes Dev 15: 1833-44 Lindholm C, Norppa H, Hayashi M, Sorsa M (1991) Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures Mutat Res 260: 369-75 Liu G, Xia T, Chen X (2003) The activation domains, the proline-rich domain, and the C-terminal basic domain in p53 are necessary for acetylation of histones on the proximal p21 promoter and interaction with p300/CREB-binding protein J Biol Chem 278: 17557-65 Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi Science 305: 1437-41 Liu L, Scolnick DM, Trievel RC, Zhang HB, Marmorstein R, Halazonetis TD et al (1999) p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage Mol Cell Biol 19: 1202-9 Liu X, Wang L, Zhao K, Thompson PR, Hwang Y, Marmorstein R et al (2008) The structural basis of protein acetylation by the p300/CBP transcriptional coactivator Nature 451: 846-50 Low GK, Fok ED, Ting AP, Hande MP (2008) Oxidative damage induced genotoxic effects in human fibroblasts from Xeroderma Pigmentosum group A patients Int J Biochem Cell Biol 40: 2583-95 Lutz WK, Tiedge O, Lutz RW, Stopper H (2005) Different types of combination effects for the induction of micronuclei in mouse lymphoma cells by binary mixtures of the genotoxic agents MMS, MNU, and genistein Toxicol Sci 86: 318-23 Ma S, Tang J, Feng J, Xu Y, Yu X, Deng Q et al (2008) Induction of p21 by p65 in p53 null cells treated with Doxorubicin Biochim Biophys Acta 1783: 935-40 MacLachlan TK, Takimoto R, El-Deiry WS (2002) BRCA1 directs a selective p53dependent transcriptional response towards growth arrest and DNA repair targets Mol Cell Biol 22: 4280-92 Majumder S, Ghoshal K, Datta J, Smith DS, Bai S, Jacob ST (2006) Role of DNA methyltransferases in regulation of human ribosomal RNA gene transcription J Biol Chem 281: 22062-72 178 Manser E, Huang HY, Loo TH, Chen XQ, Dong JM, Leung T et al (1997) Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes Mol Cell Biol 17: 1129-43 Mao DY, Watson JD, Yan PS, Barsyte-Lovejoy D, Khosravi F, Wong WW et al (2003) Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression Curr Biol 13: 882-6 Marciniak RA, Lombard DB, Johnson FB, Guarente L (1998) Nucleolar localization of the Werner syndrome protein in human cells Proc Natl Acad Sci U S A 95: 688792 Markey MP, Bergseid J, Bosco EE, Stengel K, Xu H, Mayhew CN et al (2007) Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function Oncogene 26: 6307-18 Marti A, Wirbelauer C, Scheffner M, Krek W (1999) Interaction between ubiquitinprotein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation Nat Cell Biol 1: 14-9 Masih PJ, Kunnev D, Melendy T (2008) Mismatch Repair proteins are recruited to replicating DNA through interaction with Proliferating Cell Nuclear Antigen (PCNA) Nucleic Acids Res 36: 67-75 Mathieu O, Bender J (2004) RNA-directed DNA methylation J Cell Sci 117: 4881-8 Matsumura I, Tanaka H, Kanakura Y (2003) E2F1 and c-Myc in cell growth and death Cell Cycle 2: 333-8 Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ (2000) Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro Proc Natl Acad Sci U S A 97: 10389-94 McCabe MT, Davis JN, Day ML (2005) Regulation of DNA methyltransferase by the pRb/E2F1 pathway Cancer Res 65: 3624-32 Meder VS, Boeglin M, de Murcia G, Schreiber V (2005) PARP-1 and PARP-2 interact with nucleophosmin/B23 and accumulate in transcriptionally active nucleoli J Cell Sci 118: 211-22 Mellor J (2006) Dynamic nucleosomes and gene transcription Trends Genet 22: 3209 Milutinovic S, Brown SE, Zhuang Q, Szyf M (2004) DNA methyltransferase knock down induces gene expression by a mechanism independent of DNA methylation and histone deacetylation J Biol Chem 279: 27915-27 Milutinovic S, Knox JD, Szyf M (2000) DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21(WAF1/CIP1/sdi1) J Biol Chem 275: 6353-9 179 Milutinovic S, Zhuang Q, Niveleau A, Szyf M (2003) Epigenomic stress response Knockdown of DNA methyltransferase triggers an intra-S-phase arrest of DNA replication and induction of stress response genes J Biol Chem 278: 14985-95 Miranda TB, Jones PA (2007) DNA methylation: the nuts and bolts of repression J Cell Physiol 213: 384-90 Mishra MV, Bisht KS, Sun L, Muldoon-Jacobs K, Awwad R, Kaushal A et al (2008) DNMT1 as a molecular target in a multimodality-resistant phenotype in tumor cells Mol Cancer Res 6: 243-9 Mocquet V, Laine JP, Riedl T, Yajin Z, Lee MY, Egly JM (2008) Sequential recruitment of the repair factors during NER: the role of XPG in initiating the resynthesis step EMBO J 27: 155-67 Morgan HD, Santos F, Green K, Dean W, Reik W (2005) Epigenetic reprogramming in mammals Hum Mol Genet 14 Spec No 1: R47-58 Mortusewicz O, Schermelleh L, Walter J, Cardoso MC, Leonhardt H (2005) Recruitment of DNA methyltransferase I to DNA repair sites Proc Natl Acad Sci U S A 102: 8905-9 Mullan PB, Quinn JE, Harkin DP (2006) The role of BRCA1 in transcriptional regulation and cell cycle control Oncogene 25: 5854-63 Muller H, Moroni MC, Vigo E, Petersen BO, Bartek J, Helin K (1997) Induction of S-phase entry by E2F transcription factors depends on their nuclear localization Mol Cell Biol 17: 5508-20 Muniyappa H, Das KC (2008) Activation of c-Jun N-terminal kinase (JNK) by widely used specific p38 MAPK inhibitors SB202190 and SB203580: a MLK-3MKK7-dependent mechanism Cell Signal 20: 675-83 Myant K, Stancheva I (2008) LSH cooperates with DNA methyltransferases to repress transcription Mol Cell Biol 28: 215-26 Nan X, Campoy FJ, Bird A (1997) MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin Cell 88: 471-81 Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN et al (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex Nature 393: 386-9 Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, Erdjument-Bromage H et al (1999) MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex Nat Genet 23: 58-61 Ng K, Pullirsch D, Leeb M, Wutz A (2007) Xist and the order of silencing EMBO Rep 8: 34-9 180 Norppa H, Falck GC (2003) What human micronuclei contain? Mutagenesis 18: 221-33 Obata T, Yaffe MB, Leparc GG, Piro ET, Maegawa H, Kashiwagi A et al (2000) Peptide and protein library screening defines optimal substrate motifs for AKT/PKB J Biol Chem 275: 36108-15 Ogden GB, Pratt MJ, Schaechter M (1988) The replicative origin of the E coli chromosome binds to cell membranes only when hemimethylated Cell 54: 127-35 Oka M, Meacham AM, Hamazaki T, Rodic N, Chang LJ, Terada N (2005) De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'-deoxycytidine Oncogene 24: 3091-9 Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development Cell 99: 247-57 Olson MO, Hingorani K, Szebeni A (2002) Conventional and nonconventional roles of the nucleolus Int Rev Cytol 219: 199-266 Ooi SK, Qiu C, Bernstein E, Li K, Jia D, Yang Z et al (2007) DNMT3L connects unmethylated lysine of histone H3 to de novo methylation of DNA Nature 448: 714-7 Oridate N, Lotan R (2005) Suppression of DNA methyltransferase levels in head and neck squamous carcinoma cells using small interfering RNA results in growth inhibition and increase in Cdk inhibitor p21 Int J Oncol 26: 757-61 Orsiere T, Sari-Minodier I, Iarmarcovai G, Botta A (2006) Genotoxic risk assessment of pathology and anatomy laboratory workers exposed to formaldehyde by use of personal air sampling and analysis of DNA damage in peripheral lymphocytes Mutat Res 605: 30-41 Owens DM, Keyse SM (2007) Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases Oncogene 26: 3203-13 Pabla N, Huang S, Mi QS, Daniel R, Dong Z (2008) ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis J Biol Chem 283: 6572-83 Palii SS, Van Emburgh BO, Sankpal UT, Brown KD, Robertson KD (2008) DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases and 3B Mol Cell Biol 28: 752-71 Pao GM, Janknecht R, Ruffner H, Hunter T, Verma IM (2000) CBP/p300 interact with and function as transcriptional coactivators of BRCA1 Proc Natl Acad Sci U S A 97: 1020-5 181 Papait R, Pistore C, Grazini U, Babbio F, Cogliati S, Pecoraro D et al (2008) The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin Mol Biol Cell Patra SK, Patra A, Rizzi F, Ghosh TC, Bettuzzi S (2008) Demethylation of (Cytosine-5-C-methyl) DNA and regulation of transcription in the epigenetic pathways of cancer development Cancer Metastasis Rev Peng DF, Kanai Y, Sawada M, Ushijima S, Hiraoka N, Kosuge T et al (2005) Increased DNA methyltransferase (DNMT1) protein expression in precancerous conditions and ductal carcinomas of the pancreas Cancer Sci 96: 403-8 Peterson EJ, Bogler O, Taylor SM (2003) p53-mediated repression of DNA methyltransferase expression by specific DNA binding Cancer Res 63: 6579-82 Poizat C, Puri PL, Bai Y, Kedes L (2005) Phosphorylation-dependent degradation of p300 by doxorubicin-activated p38 mitogen-activated protein kinase in cardiac cells Mol Cell Biol 25: 2673-87 Polley S, Guha S, Roy NS, Kar S, Sakaguchi K, Chuman Y et al (2008) Differential recognition of phosphorylated transactivation domains of p53 by different p300 domains J Mol Biol 376: 8-12 Potter AJ, Rabinovitch PS (2005) The cell cycle phases of DNA damage and repair initiated by topoisomerase II-targeting chemotherapeutic drugs Mutat Res 572: 2744 Pradhan S, Kim GD (2002) The retinoblastoma gene product interacts with maintenance human DNA (cytosine-5) methyltransferase and modulates its activity EMBO J 21: 779-88 Prendergast GC, Ziff EB (1991) Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region Science 251: 186-9 Ramchandani S, Bigey P, Szyf M (1998) Genomic structure of the human DNA methyltransferase gene Biol Chem 379: 535-40 Ramsahoye BH, Biniszkiewicz D, Lyko F, Clark V, Bird AP, Jaenisch R (2000) Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a Proc Natl Acad Sci U S A 97: 5237-42 Razin A, Urieli S, Pollack Y, Gruenbaum Y, Glaser G (1980) Studies on the biological role of dna methylation; IV Mode of methylation of DNA in E coli cells Nucleic Acids Res 8: 1783-92 Renaud S, Loukinov D, Abdullaev Z, Guilleret I, Bosman FT, Lobanenkov V et al (2007) Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene Nucleic Acids Res 35: 1245-56 182 Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE et al (2002) DNMT1 and DNMT3b cooperate to silence genes in human cancer cells Nature 416: 552-6 Rhee I, Jair KW, Yen RW, Lengauer C, Herman JG, Kinzler KW et al (2000) CpG methylation is maintained in human cancer cells lacking DNMT1 Nature 404: 10037 Riccio A, Aaltonen LA, Godwin AK, Loukola A, Percesepe A, Salovaara R et al (1999) The DNA repair gene MBD4 (MED1) is mutated in human carcinomas with microsatellite instability Nat Genet 23: 266-8 Robert MF, Morin S, Beaulieu N, Gauthier F, Chute IC, Barsalou A et al (2003) DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells Nat Genet 33: 61-5 Robertson AB, Pattishall SR, Gibbons EA, Matson SW (2006) MutL-catalyzed ATP hydrolysis is required at a post-UvrD loading step in methyl-directed mismatch repair J Biol Chem 281: 19949-59 Robertson KD (2001) DNA methylation, methyltransferases, and cancer Oncogene 20: 3139-55 Robertson KD (2002) DNA methylation and chromatin - unraveling the tangled web Oncogene 21: 5361-79 Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP (2000) DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters Nat Genet 25: 338-42 Rodriguez J, Frigola J, Vendrell E, Risques RA, Fraga MF, Morales C et al (2006) Chromosomal instability correlates with genome-wide DNA demethylation in human primary colorectal cancers Cancer Res 66: 8462-9468 Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139 J Biol Chem 273: 5858-68 Roman-Gomez J, Castillejo JA, Jimenez A, Gonzalez MG, Moreno F, Rodriguez Mdel C et al (2002) 5' CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia Blood 99: 2291-6 Roninson IB, Broude EV, Chang BD (2001) If not apoptosis, then what? Treatmentinduced senescence and mitotic catastrophe in tumor cells Drug Resist Updat 4: 30313 183 Rossig L, Badorff C, Holzmann Y, Zeiher AM, Dimmeler S (2002) Glycogen synthase kinase-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation J Biol Chem 277: 9684-9 Rouleau J, Tanigawa G, Szyf M (1992) The mouse DNA methyltransferase 5'-region A unique housekeeping gene promoter J Biol Chem 267: 7368-77 Rountree MR, Bachman KE, Baylin SB (2000) DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci Nat Genet 25: 269-77 Rubbi CP, Milner J (2003) Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses EMBO J 22: 6068-77 Sado T, Okano M, Li E, Sasaki H (2004) De novo DNA methylation is dispensable for the initiation and propagation of X chromosome inactivation Development 131: 975-82 Sage J, Mulligan GJ, Attardi LD, Miller A, Chen S, Williams B et al (2000) Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization Genes Dev 14: 3037-50 Sajedi E, Gaston-Massuet C, Andoniadou CL, Signore M, Hurd PJ, Dattani M et al (2008) DNMT1 interacts with the developmental transcriptional repressor HESX1 Biochim Biophys Acta 1783: 131-43 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A laboratory manual Cold Spring Harbor, USA Santini V, Kantarjian HM, Issa JP (2001) Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications Ann Intern Med 134: 573-86 Santoni-Rugiu E, Falck J, Mailand N, Bartek J, Lukas J (2000) Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway Mol Cell Biol 20: 3497-509 Sasaki H, Ishihara K, Kato R (2000) Mechanisms of Igf2/H19 imprinting: DNA methylation, chromatin and long-distance gene regulation J Biochem 127: 711-5 Sharif J, Muto M, Takebayashi S, Suetake I, Iwamatsu A, Endo TA et al (2007) The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA Nature 450: 908-12 Shay JW, Roninson IB (2004) Hallmarks of senescence in carcinogenesis and cancer therapy Oncogene 23: 2919-33 Shin JY, Kim HS, Park J, Park JB, Lee JY (2000) Mechanism for inactivation of the KIP family cyclin-dependent kinase inhibitor genes in gastric cancer cells Cancer Res 60: 262-5 184 Sliwinska MA, Mosieniak G, Wolanin K, Babik A, Piwocka K, Magalska A et al (2008) Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells Mech Ageing Dev Smallwood A, Esteve PO, Pradhan S, Carey M (2007) Functional cooperation between HP1 and DNMT1 mediates gene silencing Genes Dev 21: 1169-78 Smit AF (1999) Interspersed repeats and other mementos of transposable elements in mammalian genomes Curr Opin Genet Dev 9: 657-63 Smit AF, Riggs AD (1996) Tiggers and DNA transposon fossils in the human genome Proc Natl Acad Sci U S A 93: 1443-8 Snowden AW, Anderson LA, Webster GA, Perkins ND (2000) A novel transcriptional repression domain mediates p21(WAF1/CIP1) induction of p300 transactivation Mol Cell Biol 20: 2676-86 Somasundaram K, Zhang H, Zeng YX, Houvras Y, Peng Y, Wu GS et al (1997) Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1 Nature 389: 187-90 Sowa Y, Orita T, Hiranabe-Minamikawa S, Nakano K, Mizuno T, Nomura H et al (1999a) Histone deacetylase inhibitor activates the p21/WAF1/Cip1 gene promoter through the Sp1 sites Ann N Y Acad Sci 886: 195-9 Sowa Y, Orita T, Minamikawa-Hiranabe S, Mizuno T, Nomura H, Sakai T (1999b) Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor Cancer Res 59: 4266-70 Spada F, Haemmer A, Kuch D, Rothbauer U, Schermelleh L, Kremmer E et al (2007) DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells J Cell Biol 176: 565-71 Stewart FJ, Raleigh EA (1998) Dependence of McrBC cleavage on distance between recognition elements Biol Chem 379: 611-6 Stopper H, Eckert I, Wagener P, Schulz WA (1997) Formation of micronuclei and inhibition of topoisomerase II in the comet assay in mammalian cells with altered DNA methylation Recent Results Cancer Res 143: 183-93 Stopper H, Korber C, Schiffmann D, Caspary WJ (1993) Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells Mutat Res 300: 165-77 Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F (2006) Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines Cancer Res 66: 2794-800 Suske G (1999) The Sp-family of transcription factors Gene 238: 291-300 185 Swift LP, Rephaeli A, Nudelman A, Phillips DR, Cutts SM (2006) DoxorubicinDNA adducts induce a non-topoisomerase II-mediated form of cell death Cancer Res 66: 4863-71 Szyf M, Gruenbaum Y, Urieli-Shoval S, Razin A (1982) Studies on the biological role of DNA methylation: V The pattern of E.coli DNA methylation Nucleic Acids Res 10: 7247-59 Tate PH, Bird AP (1993) Effects of DNA methylation on DNA-binding proteins and gene expression Curr Opin Genet Dev 3: 226-31 Ting AH, Jair KW, Suzuki H, Yen RW, Baylin SB, Schuebel KE (2004) CpG island hypermethylation is maintained in human colorectal cancer cells after RNAi-mediated depletion of DNMT1 Nat Genet 36: 582-4 Trimarchi JM, Lees JA (2002) Sibling rivalry in the E2F family Nat Rev Mol Cell Biol 3: 11-20 Trouche D, Cook A, Kouzarides T (1996) The CBP co-activator stimulates E2F1/DP1 activity Nucleic Acids Res 24: 4139-45 Unoki M, Nishidate T, Nakamura Y (2004) ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain Oncogene 23: 7601-10 Unsal-Kacmaz K, Makhov AM, Griffith JD, Sancar A (2002) Preferential binding of ATR protein to UV-damaged DNA Proc Natl Acad Sci U S A 99: 6673-8 Unterberger A, Andrews SD, Weaver IC, Szyf M (2006) DNA methyltransferase knockdown activates a replication stress checkpoint Mol Cell Biol 26: 7575-86 Usdin K, Woodford KJ (1995) CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro Nucleic Acids Res 23: 4202-9 Uziel T, Lerenthal Y, Moyal L, Andegeko Y, Mittelman L, Shiloh Y (2003) Requirement of the MRN complex for ATM activation by DNA damage EMBO J 22: 5612-21 Vasques LR, Stabellini R, Xue F, Tian XC, Soukoyan M, Pereira LV (2005) XIST repression in the absence of DNMT1 and DNMT3B DNA Res 12: 373-8 Vertino PM, Sekowski JA, Coll JM, Applegren N, Han S, Hickey RJ et al (2002) DNMT1 is a component of a multiprotein DNA replication complex Cell Cycle 1: 416-23 Vidal A, Koff A (2000) Cell-cycle inhibitors: three families united by a common cause Gene 247: 1-15 186 Vigneron A, Cherier J, Barre B, Gamelin E, Coqueret O (2006) The cell cycle inhibitor p21waf1 binds to the myc and cdc25A promoters upon DNA damage and induces transcriptional repression J Biol Chem 281: 34742-50 Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C et al (2006) The Polycomb group protein EZH2 directly controls DNA methylation Nature 439: 8714 Wakeman TP, Kim WJ, Callens S, Chiu A, Brown KD, Xu B (2004) The ATMSMC1 pathway is essential for activation of the chromium[VI]-induced S-phase checkpoint Mutat Res 554: 241-51 Walsh CP, Bestor TH (1999) Cytosine methylation and mammalian development Genes Dev 13: 26-34 Wang H, Zhao Y, Li L, McNutt MA, Wu L, Lu S et al (2008) An ATM- and Rad3related (ATR) signaling pathway and a phosphorylation-acetylation cascade are involved in activation of p53/p21Waf1/Cip1 in response to 5-aza-2'-deoxycytidine treatment J Biol Chem 283: 2564-74 Wang KY, James Shen CK (2004) DNA methyltransferase Dnmt1 and mismatch repair Oncogene 23: 7898-902 Wigler M, Levy D, Perucho M (1981) The somatic replication of DNA methylation Cell 24: 33-40 Wilson GG, Murray NE (1991) Restriction and modification systems Annu Rev Genet 25: 585-627 Wischermann K, Popp S, Moshir S, Scharfetter-Kochanek K, Wlaschek M, de Gruijl F et al (2008) UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes Oncogene Wolffe AP, Jones PL, Wade PA (1999) DNA demethylation Proc Natl Acad Sci U S A 96: 5894-6 Woo LL, Futami K, Shimamoto A, Furuichi Y, Frank KM (2006) The RothmundThomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress Exp Cell Res 312: 3443-57 Woodcock DM, Crowther PJ, Diver WP (1987) The majority of methylated deoxycytidines in human DNA are not in the CpG dinucleotide Biochem Biophys Res Commun 145: 888-94 Wu S, Cetinkaya C, Munoz-Alonso MJ, von der Lehr N, Bahram F, Beuger V et al (2003) Myc represses differentiation-induced p21CIP1 expression via Miz-1dependent interaction with the p21 core promoter Oncogene 22: 351-60 187 Wu X, Ranganathan V, Weisman DS, Heine WF, Ciccone DN, O'Neill TB et al (2000) ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response Nature 405: 477-82 Xiao H, Hasegawa T, Isobe K (1999) Both Sp1 and Sp3 are responsible for p21waf1 promoter activity induced by histone deacetylase inhibitor in NIH3T3 cells J Cell Biochem 73: 291-302 Xiao H, Hasegawa T, Isobe K (2000) p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor J Biol Chem 275: 1371-6 Xiao H, Hasegawa T, Miyaishi O, Ohkusu K, Isobe K (1997) Sodium butyrate induces NIH3T3 cells to senescence-like state and enhances promoter activity of p21WAF/CIP1 in p53-independent manner Biochem Biophys Res Commun 237: 45760 Xie S, Wang Z, Okano M, Nogami M, Li Y, He WW et al (1999) Cloning, expression and chromosome locations of the human DNMT3 gene family Gene 236: 87-95 Xiong Y, Dowdy SC, Podratz KC, Jin F, Attewell JR, Eberhardt NL et al (2005) Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells Cancer Res 65: 2684-9 Yen RW, Vertino PM, Nelkin BD, Yu JJ, el-Deiry W, Cumaraswamy A et al (1992) Isolation and characterization of the cDNA encoding human DNA methyltransferase Nucleic Acids Res 20: 2287-91 Yoder JA, Bestor TH (1998) A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast Hum Mol Genet 7: 279-84 Yoder JA, Walsh CP, Bestor TH (1997) Cytosine methylation and the ecology of intragenomic parasites Trends Genet 13: 335-40 Yoder JA, Yen RW, Vertino PM, Bestor TH, Baylin SB (1996) New 5' regions of the murine and human genes for DNA (cytosine-5)-methyltransferase J Biol Chem 271: 31092-7 Yokochi T, Robertson KD (2004) Doxorubicin inhibits DNMT1, resulting in conditional apoptosis Mol Pharmacol 66: 1415-20 You JS, Kang JK, Lee EK, Lee JC, Lee SH, Jeon YJ et al (2008) Histone deacetylase inhibitor apicidin downregulates DNA methyltransferase expression and induces repressive histone modifications via recruitment of corepressor complex to promoter region in human cervix cancer cells Oncogene 27: 1376-86 Young JI, Smith JR (2001) DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal J Biol Chem 276: 19610-6 188 Yuan ZM, Huang Y, Ishiko T, Nakada S, Utsugisawa T, Shioya H et al (1999) Role for p300 in stabilization of p53 in the response to DNA damage J Biol Chem 274: 1883-6 Zhang H, Somasundaram K, Peng Y, Tian H, Bi D, Weber BL et al (1998) BRCA1 physically associates with p53 and stimulates its transcriptional activity Oncogene 16: 1713-21 Zhao H, Piwnica-Worms H (2001) ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1 Mol Cell Biol 21: 4129-39 Zheng QH, Ma LW, Zhu WG, Zhang ZY, Tong TJ (2006) p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation J Cell Biochem 98: 1230-48 Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective Nature 408: 433-9 Zhou Q, Agoston AT, Atadja P, Nelson WG, Davidson NE (2008) Inhibition of histone deacetylases promotes ubiquitin-dependent proteasomal degradation of DNA methyltransferase in human breast cancer cells Mol Cancer Res 6: 873-83 Zhu B, Zheng Y, Angliker H, Schwarz S, Thiry S, Siegmann M et al (2000) 5Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence Nucleic Acids Res 28: 415765 Zhu WG, Srinivasan K, Dai Z, Duan W, Druhan LJ, Ding H et al (2003) Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter Mol Cell Biol 23: 4056-65 Zhu YM, Huang Q, Lin J, Hu Y, Chen J, Lai MD (2007) Expression of human DNA methyltransferase in colorectal cancer tissues and their corresponding distant normal tissues Int J Colorectal Dis 22: 661-6 189 Chapter 7: APPENDIX List of Publications Tan H.H and Porter A.G (2009) DNA methyltransferase I is a mediator of doxorubicin-induced genotoxicity in human cancer cells Biochem Biophys Res Commun, 382(2): 462-467 Tan H.H and Porter A.G (2009) p21(WAF1) negatively regulates DNMT1 expression in mammalian cells Biochem Biophys Res Commun, 382(1): 171-176 Chuang L.S., Tan E.H., Oh H.K and Li B.F (2002) Selective depletion of human DNA-methyltransferase DNMT1 proteins by sulfonate-derived methylating agents Cancer Res, 62(6): 1592-1597 Badal V., Chuang L.S., Tan E.H., Badal S., Villa L.L., Wheeler C.M., Li B.F and Bernard H.U (2003) CpG methylation of human papillomavirus type 16 DNA in cervical cancer cell lines and in clinical specimens: genomic hypomethylation correlates with carcinogenic progression J Virol, 77: 6227-6234 190 ... over-expression of p21WAF1 inhibits DNMT1 73 3 .1. 1.7 TSA -mediated induction of p21WAF1 results in inhibition of 76 DNMT1 3 .1. 1.8 TSA -mediated induction of p21WAF1 is independent of p53 85 3 .1. 2 Transcriptional... between DNMT1 and p21WAF1 in the 63 cell cycle 3 .1. 1 .1 3 .1. 1.2 DNMT1 expression in the cell cycle 63 WAF1 in DNA 67 p21WAF1 68 Transient over-expression of DNMT1 does not inhibit 71 Inverse relationship... methylase 17 1. 3.3b DNMT1 interacts with Polycomb Group (PcG) proteins 17 1. 3.3c DNMT1 interacts with UHRF1 18 Transcriptional suppression 19 1. 3.4 iv 1. 4 DNMT1 in the Cell Cycle 1. 4 .1 21 1.4.2 E2F1/RB