Abstract
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.
Highlights
sirtuin 1 (SIRT1) is likely to play a role in the extension in healthspan induced by dietary restriction
We showed recently that manipulating the expression of the histone deacetylase SIRT1 in human cells affected promoter DNA methylation of a small panel of genes that we tested, selected on the basis that they have been reported to show an age-related change in DNA methylation and to be expressed differentially in response to dietary restriction (DR), an intervention shown robustly in multiple species to increase lifespan and/or healthspan [16]
For Caco-2 cells, DNA was enriched for the methylated fraction by methylated DNA immunoprecipitation (MeDIP) using an antibody recognising 5-methylcytidine (5mC), and efficacy was confirmed by measuring enrichment by qPCR of a lambda phage DNA added as a spike to all samples in both a demethylated and in vitromethylated form and of two loci known to be hypermethylated (H19 and L1.2) relative to a hypomethylated locus (UBE2B) [27]
Summary
SIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. 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 susceptible to age-related changes in DNA methylation. A notable feature of the age-related drift in DNA methylation is that hypermethylation clusters at the gene targets of polycomb group proteins (PCGTs), as observed in human whole blood from postmenopausal women [7], mouse intestine [4] and mouse haematopoietic stem cells [3]. PCGTs tend to be hypermethylated in cancer [13,14,15]
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