An investigation of roles for SIRT1 and dietary polyphenols in modulating the ageing process through DNA methylation

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 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.