An investigation of roles for SIRT1 and dietary polyphenols in modulating the ageing process through DNA methylation Laura Jane Ions Thesis submitted for the degree of Doctor of Philosophy Institute for Cell and Molecular Biosciences, Newcastle University, UK September 2011 Declaration I certify that this thesis is my own work, except where stated, and has not been previously submitted for a degree or any other qualification at this or any other university. Laura J Ions September 2011 Thank you to Professor Dianne Ford and Dr Luisa Wakeling whose support, guidance and enthusiasm have been invaluable over the last four years. Abstract Dietary restriction (DR) can increase lifespan across evolutionarily distinct species, from yeast to rodents. The NAD+-dependent (class III) histone deacetylase SIRT1 in mammals, and its ortholog in other species, may play a major role in this response, but may affect ‘healthspan’ (number of years of good health), rather than lifespan per se. Ageing is accompanied by changes in genome methylation, which may be causal in the ageing process. Since histones are one of the many substrates that are deacetylated by SIRT1, we hypothesised that epigenetic effects of SIRT1 activity – and specifically effects on DNA methylation, which is associated closely with histone acetylation – mediate some of the beneficial effects of DR that contribute to increased healthspan. We also propose that dietary polyphenols may act at the cellular level in a similar way. To test this hypothesis, we first investigated effects of altering SIRT1 expression, by overexpression of a transgene or siRNA-mediated knockdown, on global DNA methylation (methylation of the LINE-1 element) in the human intestinal cell line, Caco-2. We also measured effects on global DNA methylation of dietary isoflavones and resveratrol, under control conditions as well as conditions of SIRT1 overexpression. Measured effects on global DNA methylation revealed possible complex interactions between SIRT1 and these dietary polyphenols but were dependent on the assay used to measure methylation at the LINE-1 element (COBRA or pyrosequencing), so were not considered to be robust observations. We investigated factors that may affect SIRT1 expression – specifically we examined SIRT1 promoter activity in response to polyphenols, effects of promoter methylation and effects of age. Treatment of Caco-2 cells with dietary polyphenols had no effect on the SIRT1 promoter in a promoter-reporter construct. In contrast, methylation of the SIRT1 promoter reduced reporter gene expression in this model. Age did not appear to change the levels of SIRT1 protein expressed in mouse intestinal tissue when comparing young and older mice. An in silico analysis was carried out to investigate if overlaps between groups of genes compiled from published and publically-available data found to i) associate with SIRT1, ii) show altered expression in response to DR, and iii) show altered methylation with ageing were greater than expected by chance, which would support the hypothesis, and provided targets to investigate possible site-specific effects of SIRT1 on DNA methylation. Ten genes were found to fit into the ‘three way’ overlap, which was statistically greater than expected by chance. Pyrosequencing assays could be optimised for only 8 of these 10 genes, so we focused further investigations on this sub-set. Significant effects of SIRT1 overexpression and/or knockdown on methylation were observed on at least one CpG site in the I promoters of six of these genes (CDC7, EIF5, IRX3, KLF3, PTPRG, TBX3) and expression at the mRNA level of all of the eight genes (also PCYT1A and SLC39A4) was affected significantly. To gain a more comprehensive view of the extent to which DNA methylation at specific loci may be affected by SIRT1 expression levels microarray-based analysis of the methylation pattern across the genome in Caco-2 cells was carried out under conditions where SIRT1 was overexpressed or where expression was reduced by siRNA. In parallel, we measured the response to expressing SIRT1 at different levels at the level of the transcriptome. Overlaps that were statistically greater than expected by chance between these data and the lists of genes compiled from published and publically-available data used for the in silico analysis were found to exist between the complied list of genes reported to respond to dietary restriction and the set of genes we found to show altered expression in response to changing the level of SIRT1 expression and the set of genes we found to be differentially-methylated in response to reducing the level of SIRT1 expression. This observation is in broad support of our overarching hypothesis. The findings of this study indicate that effects of SIRT1 on methylation of specific genes may correspond with altered expression under conditions of dietary restriction. The data reveal a large number of gene targets for which causal links between these modifications roles in modulating the ageing process could be investigated. II Abbreviations A Adenine A260 Absorbance reading at 260nm Acetyl-CoA Acetyl-coenzyme A AceCS2 Acetyl-coenzyme A Synthetase 2 ADP Adenosine diphosphate AROS Active regulator of SIRT1 ASP Adenosine 5' phosphosulfate ATCC American Tissue Culture Collection ATP Adenosine triphosphate Bax Bcl2-associated bp Base pair BSA Bovine serum albumin C Carbon C Cytosine Caco-2 Colonic adenocarcinoma cAMP Cyclic adenosine monophosphate CCD Charged coupled device CDC7 Cell division cycle 7 CDK Cyclin dependent kinase cDNA copy DNA C/EBPα CCAAT/enhancer binding protein alpha ChREBP Carbohydrate response element binding protein COBRA Combined bisulfite restriction analysis CPRG Chlorophenol red-β-D-galactopyranoside CPS-1 Carbamoyl phosphate synthetase 1 CR Calorie restriction CREB cAMP response element binding protein CTα CTP:phosphocholine cytidylyltransferase alpha CtBP C-terminal binding protein CTP cytidine 5’-triphosphate D Aspartic acid DBC-1 Deleted in breast cancer 1 DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DNMT1 DNA methyltransferase 1 III dNTP Deoxynucleotide triphosphate DR Dietary restriction E Glutamic acid EDTA Ethylenediaminetetraacetic acid EIF5 Eukaryotic translation initiation factor 5 ERC Extrachromosomal circles FOXO Forkhead box type O G Guanine GAP GTPase accelerating protein GDH Glutamate dehydrogenase GDI GDP dissociation inhibitor GDP Guanosine diphosphate GSTP-1 Glutathione S-transferase 1 GTP Guanosine triphosphate H Histone HAT Histone acetyltransferase HIC-1 Hypermethylated in cancer 1 HDAC Histone deacetyltransferase HeLa Henrietta Lacks IF Intermittent fasting IGF-1 Insulin-like growth factor 1 IgG Immunoglobulin G IIS Insulin/IGF-1 signalling INHAT Inhibitor of histone acetyltransferases InR Insulin receptor IRX3 Iroquois related homeobox 3 JNK c-Jun N-terminal kinase K Lysine kb Kilo bases KLF3 Krüppel-like factor 3 LB Luria Bertoni LDLR Low density lipoprotein receptor LINE-1 Long interspersed nucleotide element 1 LUMA Lumometric methylation assay MCK Muscle creatine kinase MCM2-7 Minichromosome maintenance 2-7 MeCP2 Methyl CpG binding protein 2 IV MeDIP Methylated DNA immunoprecipitation MGMT O6-methylguanine DNA methyltransferase miR Micro RNAs MMLV RT Moloney murine leukaemia virus reverse transcriptase mRNA Messenger ribonucleic acid MYH10 Myosin, heavy chain 10 NaDC3 Na+-dependent dicarboxylate cotransporter NAD(H) Nicotinamide adenine dinucleotide (hydrogen) NM II Non muscle myosin II NMHC II NM II heavy chain isoform NMNAT-1 Nicotinamide mononucleotide adenylyltransferase 1 p Phosphate p Probability PBS Phosphate buffered saline PCR Polymerase chain reaction PCYT1A Phosphate cytidylyltransferase 1 choline alpha isoform PGC-1α PPAR-gamma coactivator 1 alpha PI3K Phosphoinositide 3 kinase PIC Pre-initiation complex PPAR γ Peroxisome proliferator-activated receptor gamma PPi Pyrophosphate PTPRG Protein tyrosine phosphate receptor type G PVDF Polyvinylidene difluride RARβ Retinoic acid receptor beta r.f. Representation factor RISC RNA-induced silencing complex RNA Ribonucleic acid RNAi Ribonucleic acid interference ROS Reactive oxygen species RPTC Renal proximal tubular cells RT-PCR Reverse transcriptase-polymerase chain reaction RT-qPCR Reverse transcriptase-quantitative polymerase chain reaction S Serine SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM Standard error of the mean SIR Silent information regulator siRNA Short interfering ribonucleic acid V SLC30A5 Solute carrier 30, member 5 SLC39A4 Solute carrier 39, member 4 SMRT Silencing mediator of retinoid and thyroid hormone receptor STAC Sirtuin activating compound T Thymine TBX3 T-box3 transcription factor TDR Transcriptional repressor domain TEMED N,N,N’,N’-Tetramethylethylenediamine tk Thymidine kinase TOR Target of rapamycin TSA Trichostatin A UCP2 Uncoupling protein 2 UV Ultra violet WAT White adipose tissue VI Contents Abstract .................................................................................................................................................... I Abbreviations ........................................................................................................................................ III Contents................................................................................................................................................ VII List of Figures ........................................................................................................................................