The roles of DNA(cytosine-5) methyltransferase1 in carcinogenesis related to cellular factors, virus and chemicals Vinay Badal (BSc, National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements I am most grateful to my supervisor, Associate Professor Benjamin F.L.Li, for the having faith in my ability to undertake graduate studies and his constant support, guidance and encouragement throughout it I would like to thank Associate Professor Uttam Surana and Associate Professor Thomas Leung, members of my supervisory committee for their comments, advices and discussions over the years I would like to extend my sincere thanks to: Prof Hans-Ulrich Bernard for his assistance in obtaining the clinical samples as well as his insights on HPV biology Dr Linda Chuang for patiently teaching me all these years most of the techniques I know as well as her help in reading and editing my thesis To Eileen and Wan Lin for all their help, reagents and putting up with my nonsense To Dr Oh Hue Kian, without whom the lab wouldn’t function, Claire our F&B manager, Zou Hao for her help with the adenovirus work and to all the past member of our lab for their assistance To Dr Roland Degenkolbe for sharing his ideas on HPV I would also like to thank my wife for her great love, support and understanding, making my years of studying fly by Finally I would like to dedicate my work to my parents without whom none of this would have been possible i Table of contents Acknowledgements i Table of contents ii Abbreviations viii Summary xii Chapter 1: Introduction 1.1 DNA Methylation 1.1.1 Role of DNA Methylation 1.1.2 Mammalian Methyltransferase 1.1.3 How does methylation suppress transcription? 1.1.4 Imprinting 10 11 1.2 DNA methylation and Cancer 1.2.1 Hypomethylation in cancer 11 1.2.2 Hypermethylation in cancer 13 1.2.3 Loss of imprinting 14 1.2.4 Role of DNMT1 in oncogenesis 14 1.2.5 DNMT1 depleting agents 15 1.3 Relationship of DNMT1 with replication 17 1.3.1 Expression of DNMT1 during S phase 17 1.3.2 DNMT1 binding to DNA 18 18 1.4 Role of PCNA in replication 1.4.1 Structure of PCNA 19 1.4.2 Interacting partners of PCNA 20 21 1.5 Role of Hsp70 in replication 1.5.1 Structure and sequence of Hsc70 23 1.5.2 Reaction cycle of Hsc70 24 1.6 Role of DNA methylation in Virus related carcinogenesis 28 1.6.1 EBV 28 1.6.2 HBV 29 ii 1.6.3 HIV 29 1.6.4 SV40 30 1.6.5 HPV 31 31 1.7 HPV 1.7.1 Classification 31 1.7.2 Cervical Cancer 32 1.7.3 Genome organization 32 1.7.4 Early proteins 33 1.7.5 Late proteins 38 1.7.6 Role of p97 promoter (early gene promoter) 39 1.7.6 Late gene promoter 41 1.8 Research Objective 43 Chapter 2: Materials and Methods 44 2.1 Cell lines 44 2.2 Antibodies 45 2.3 Bacterial strain and media 45 2.4 Drugs and Chemicals 45 2.5 Clinical specimens 45 2.6 Analysis of the DNA of cell lines 46 2.7 Oligonucleotides 47 2.8 Reverse transcription and PCR 49 2.9 PCR 50 2.9.1 HPV-16 and HPV-18 genomic walk through 50 2.9.2 LCR, G3 and G4 dissection 50 2.9.3 HPV-16 RT-PCR 50 2.9.4 Hsc70 fragments 51 2.10 Bisulfite sequencing 51 2.11 Cell lysis and Western Analysis 52 2.12 Flow Cytometry 52 2.13 Chromatin Immunoprecipitation Assay (ChIP) 53 iii 2.14 DNA manipulation 54 2.15 Expression of recombinant PCNA 54 2.16 PCNA purification - Gel filtration of 65% ammonium sulphate protein 55 precipitate 2.17 PCNA purification - FPLC chromatography on Mono Q exchanger 55 2.18 Purification of recombinant proteins 55 2.19 In-vitro binding experiments 56 2.20 Effect of ATP 57 2.21 Time dependent binding 57 2.22 Cell staining 57 2.22.1 Staining of Hsc70-PCNA 57 2.22.2 Staining of transfected DNMT1 and Hsc70 58 2.22.3 Staining of DNMT1 and YY1 58 2.22.4 Staining of DNMT1 and Hsc70 in 5AzadC treated MRC5SV40 59 2.23 ATPase assay 59 2.24 Immunoprecipitation experiments (IP) 59 2.24.1 PCNA IP 60 2.24.2 Hsc70 IP 60 2.25 Purification of Hsp40 60 2.26 p21Waf1 competition assay 61 Chapter 3: Results 62 3.1 Role of methylation in HPV 62 3.1.1 Copy number quantification of SiHa and CaSki cell lines 62 3.1.2 Methylation status of HPV16 and HPV18 genomes using McrBc cleavage 64 3.1.2a HPV-16 methylation status 65 3.1.2b HPV-18 methylation status 70 3.1.3 Study of promoter methylation status in HPV16 SiHa and CaSki cell lines 73 3.1.3a HpaII/MspI 73 3.1.3b McrBc 75 iv 3.1.3c Bi-sulphite analysis 78 3.1.4 Clinical analysis of HPV-16 infected clinical samples 84 3.1.4a Mapping of meCpG by McrBc digestion of the HPV-16 promoter 85 3.1.4b Mapping of meCpG by McrBc digestion of the HPV-16 genome 91 3.1.4c Mapping of meCpG of the HPV-16 promoter by Bi-sulphide modification 94 3.1.5 Discussion 97 3.2 Effect of 5AzadC on DNMT1 in HPV-16 cell lines 101 3.2.1 Effect of 5AzadC on CaSki cell line from ATCC 101 3.2.1a Protein expression levels and flow cytometric analysis 101 3.2.1b Transcriptional and genomic analysis 105 3.2.2 Recovery of CaSki ATCC cells treated with 5AzadC 108 3.2.2a Protein expression levels and flow cytometric analysis 108 3.2.2b Transcriptional and genomic analysis 110 3.2.3 Discussion 116 3.3 Effect of 5AzadC on CaSki variants 123 3.3.1 6h and 24h treatment of 5AzadC on CaSki cell lines 123 3.3.2 Genomic methylation analysis of the CaSki variants treated with 5AzadC 125 3.3.3 Effect of high dose of 5AzadC on CaSki Old cell line 127 3.3.3a Protein expression levels and flow cytometric analysis 127 3.3.3b Transcriptional and genomic analysis 130 3.3.4 Discussion 132 3.4 Effect of alkylating carcinogen MMS on DNMT1 in HPV-16 cell lines 134 3.4.1 MMS depletes DNMT1 in SiHa and CaSki old cells 134 3.4.1a CaSki old is more sensitive to MMS than SiHa 134 3.4.1b MMS leads to cell death in CaSki old but not SiHa cells 136 3.4.2 Loss of DNMT1 leads to extensive de-methylation of HPV-16 genome in CaSki old cells 137 3.4.2aMcrBc scanning of the genome 137 3.4.2b Bi-sulphite analysis of the CaSki genome after MMS treatment 139 3.4.3 De-methylation leads to up regulation of the late genes v 141 3.4.4 De-methylation leads to possible instability of the hPV-16 genome 142 3.4.5 Discussion 144 3.5 Effect of TSA on HPV-16 cell lines 148 3.5.1 TSA transiently down-regulates HPV-16 transcription in CaSki but not SiHa cells 149 3.5.2 Association of YY1, DNMT1 and HDAC1 with p97 increases with TSA 154 3.5.3 Discussion 159 3.6 An interacting partner of PCNA: Hsc70 164 3.6.1 Purification of recombinant PCNA: Gel filtration 164 3.6.2 Mono Q fractionation of recombinant PCNA 166 3.6.3 Co-localization of Hsc70 with PCNA at the replication foci 168 3.6.4 Immuno-precipitation of Hsc70 and PCNA 170 3.6.5 Discussion 171 3.7 Characterization of PCNA binding domain in Hsc70 172 3.7.1 PCR cloning and expression of recombinant Hsc70 truncated proteins 172 3.7.2 In vitro binding of PCNA to Hsc70 174 3.7.3 In vitro binding of Hsc70 to PCNA 175 3.7.4 Effect of ATP on the interaction between PCNA and Hsc70 175 3.7.5 Effect of PCNA on the ATPase activity of Hsc70 177 3.7.6 Discussion 179 3.8 Characterization of Hsc70 binding domain in DNMT1 182 3.8.1 DNMT1 binding domain in Hsc70 182 3.8.2 Co-localization of endogenous DNMT1 and Hsc70 183 3.8.3 Co-localization of transfected DNMT1 with Hsc70 184 3.8.4 Hsc70 binding domain in DNMT1 186 3.8.5 In-vitro binding of DNMT1b with Hsc70 189 3.8.6 Immuno staining of DNMT1b 191 3.8.7 Function of Hsc70 interaction with DNMT1 and PCNA 193 3.8.8 Discussion 195 vi Chapter 4: Conclusion 199 Chapter 5: References 203 Chapter 6: Appendix 226 List of publications 226 Patent filed 226 vii Abbreviations 5meC 5-methyl-2’-deoxycytidine 6meA 6-methyl-adenine 5AzadC 5-aza-2'-deoxycytidine, Decitabine 5AzaC 5-aza-2'-cytidine aa amino acids ADP adenosine diphosphate ACI, ACIII Albuquerque CIN I, CIN III samples AN Albuqurque normal samples AT Albuquerque tumour samples ATCC American Type Culture Collection ATP adenosine 5’-triphosphate BL Burkitt's lymphoma BN Brazilian normal samples bp base pair BRCA1 breast cancer susceptibility gene BrdU bromodeoxyuridine BSA bovine serum albumin BT Brazilian tumour samples CDP CCAAT-displacement protein ChIP Chromatin Immunoprecipitation Assay CIN cervical intraepithelial neoplasia CpA cytosine-adenine dinucleotides CpG cytosine-guanine dinucleotides CpT cytosine-thymine dinucleotides CML chronic myelogenous leukemia DAM DNA adenine methylase DME Dubelco’s Minimum Essential Medium DMS dimethylsulphate DnaJ bacterial homolog of heat shock protein 40 DnaK bacterial homolog of heat shock protein 70 viii DNMT DNA (cytosine-5) methyltransferase dDnmt2 Drosophila melanogaster DNA (cytosine-5) methyltransferase dNTPs deoxy-nucleotide triphosphates DTT dithiothreitol E2B E2 binding site EBV Epstein–Barr virus E.coli Escherichia coli ECL enhanced chemiluminescence EDTA ethylenediamine tetra-acetic acid EGF epidermal growth factor EGFR epidermal growth factor receptor FBS fetal bovine serum FITC fluorescein isothiocyanate GAPDH glyceraldehydes-3-phosphate dehydrogenase GAP glyceraldehydes-3-phosphate dehydrogenase GFP green fluorescent protein GSH reduced Glutathione GST Glutathione S-transferase HBV Hepatitis B virus HbsAg HBV surface antigen HCV Hepatitis C virus HDAC histone deacetylase HEPES N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid HIV Human immunodeficiency virus HMBP HIV-1 methylation binding protein HPV Human papilloma virus Hsc heat shock cognate gene Hsp heat shock protein HTLV human T cell leukemia virus ICF Immunodeficiency-Centromeric instability-Facial anomalies IgG Immunoglobin type G ix ... proteins E1 protein E1 protein is mainly involved in the replication of the virus by binding to the origin of replication (ori) in the 3’ end of the LCR (Ustav et al., 1991) It is shown to possess... dependent binding 57 2.22 Cell staining 57 2.22.1 Staining of Hsc70-PCNA 57 2.22.2 Staining of transfected DNMT1 and Hsc70 58 2.22.3 Staining of DNMT1 and YY1 58 2.22.4 Staining of DNMT1 and Hsc70 in. .. Cterminus substrate-binding domain The binding site of Hsc70 was localized to aa141-152, probably to the PXPXP sequence at the N-terminus of DNMT1 The presence of an additional 17aa inserted in the