SIRT1 affects DNA methylation of polycomb group protein target genes, a hotspot of the epigenetic shift observed in ageing.

BACKGROUND: SIRT1 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). RESULTS: The 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. CONCLUSIONS: SIRT1 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.

Effects of Sirt1 on DNA methylation and expression of genes affected by dietary restriction.

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 Sirt1 has effects on DNA methylation across the genome and affects, in particular, the expression of genes that respond to dietary restriction. Sirt1-mediated effects on DNA methylation and, consequently, gene expression may thus be one of the mechanisms underlying the response to dietary restriction.