Changes in DNA methylation across the life course may contribute to the ageing process. We hypothesised that some effects of dietary restriction to extend lifespan and/or mitigate against features of ageing result from changes in DNA methylation, so we determined if genes that respond to dietary restriction also show age-related changes in DNA methylation. In support of our hypothesis, the intersection of lists of genes compiled from published sources that (1) were differentially expressed in response to dietary restriction and (2) showed altered methylation with increased age was greater than expected. We also hypothesised that some effects of Sirt1, which may play a pivotal role in beneficial effects of dietary restriction, are mediated through DNA methylation. We thus measured effects of Sirt1 overexpression and knockdown in a human cell line on DNA methylation and expression of a panel of eight genes that respond to dietary restriction and show altered methylation with age. Six genes were affected at the level of DNA methylation, and for six expressions were affected. In further support of our hypothesis, we observed by DNA microarray analysis that genes showing differential expression in response to Sirt1 knockdown were over-represented in the complied list of genes that respond to dietary restriction. The findings reveal that

BackgroundSIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. Actions of SIRT1 are pleiotropic, and effects on healthspan may include effects on DNA methylation. Polycomb group protein target genes (PCGTs) are suppressed by epigenetic mechanisms in stem cells, partly through the actions of the polycomb repressive complexes (PRCs), and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation. We hypothesised that SIRT1 would affect DNA methylation particularly at PCGTs. To map the sites in the genome where SIRT1 affects DNA methylation, we altered SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA. DNA was enriched for the methylated fraction then sequenced (HuVEC) or hybridised to a human promoter microarray (Caco-2).ResultsThe profile of genes where SIRT1 manipulation affected DNA methylation was enriched for PCGTs in both cell lines, thus supporting our hypothesis. SIRT1 knockdown affected the mRNA for none of seven PRC components nor for DNMT1 or DNMT3b. We thus find no evidence that SIRT1 affects DNA methylation at PCGTs by affecting the expression of these gene transcripts. EZH2, a component of PRC2 that can affect DNA methylation through association with DNA methyltransferases (DNMTs), did not co-immunoprecipitate with SIRT1, and SIRT1 knockdown did not affect the expression of EZH2 protein. Thus, it is unlikely that the effects of SIRT1 on DNA methylation at PCGTs are mediated through direct intermolecular association with EZH2 or through effects in its expression.ConclusionsSIRT1 affects DNA methylation across the genome, but particularly at PCGTs. Although the mechanism through which SIRT1 has these effects is yet to be uncovered, this action is likely to contribute to extended healthspan, for example under conditions of dietary restriction.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-015-0036-0) contains supplementary material, which is available to authorized users.

Some effects of the polyphenol resveratrol mimic responses to dietary restriction. Dietary resveratrol may thus have the potential to prolong lifespan and reduce the risk of age related diseases, such as cardiovascular disease, diabetes and cancer. Actions of the histone deacetylase Sirt1 are one of several pathways believed to play a role in responses to dietary restriction. Controversy remains over whether or not resveratrol is an activator of SIRT1 and thus over if SIRT1 is the conduit for the beneficial actions of resveratrol.Previous work in human intestinal Caco-2 cells, based on DNA microarray analysis, indicated that resveratrol and SIRT1 affect distinct sets of genes, although genes affected by dietary restriction were significantly over-represented in both groups. The aim was to investigate if resveratrol and SIRT1 act independently to affect the expression of different genes with functions relevant to ageing.Clusterin and WWP1 were selected from the group of genes previously found to be affected by resveratrol and dietary restriction. Clusterin has been detected at high levels in Alzheimer's disease and modulates plaque solubility. WWP1 is a ubiquitin ligase that increased lifespan when overexpressed in C. elegans and when mutated led to abolition of the longevity response to dietary restriction. LAMP2, a key player in chaperone-mediated autophagy, which modulates cellular ageing, was selected from the group of genes that were affected by SIRT1 and dietary restriction. RT-qPCR was used to measure mRNA levels of all three genes and of SIRT1in human intestinal Caco-2 cells after treatment with resveratrol or reduction of SIRT1 expression using siRNA.Results are shown in Figure 1. SIRT1 knockdown increased Clusterin and WWP1 mRNA and decreased LAMP2 mRNA. Resveratrol decreased WWP1 mRNA and increased SIRT1 mRNA. Resveratrol also increased transcription from a SIRT1 promoter-reporter construct. Fig. 1. A. mRNA levels of WWP1, Clusterin (Clu) and LAMP2 relative to GAPDH in response to SIRT1 knockdown in Caco-2 cells B. mRNA levels of WWP1, Clusterin, LAMP2 and SIRT1 relative to GAPDH in response to treatment of Caco-2 cells over 24 h with 10 mM resveratrol. C. SIRT1 promoter-reporter assay to investigate the effect of resveratrol on the transcription of the SIRT1 gene. Data are mean, SEM (n = 3). *P < 0.05; ***P < 0.001 by Student's t-test.The observed response of the WWP1 gene to SIRT1 knockdown and to resveratrol is consistent with resveratrol acting via SIRT1. The observed effects of resveratrol on SIRT1 expression indicate that resveratrol can affect the expression of genes through increasing SIRT1 transcription. Thus effects of resveratrol on gene expression are not all independent of actions on SIRT1.