Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: Beneficial effects of SIRT1 on healthspan are likely to be pleiotropic and may include effects on DNA methylation. We demonstrated recently that manipulating SIRT1 in human cells affected DNA methylation of a panel of test genes, and that genes with expression modified by dietary restriction corresponded with genes that underwent changes in DNA methylation during ageing. Here we tested the hypothesis that genes particularly susceptible to SIRT1-induced effects on DNA methylation across the genome map to genes for which DNA methylation changes during ageing. We increased or reduced SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA, respectively. Effects on DNA methylation were measured by enriching for the methylated fraction then either sequencing (HuVEC) or hybridising to a human promoter microarray (Caco-2). Effects using these two different cell lines and techniques for analysis were remarkably consistent. Genes with a DNA methylation status affected by SIRT1 manipulation were enriched for those that undergo age-dependent changes in DNA methylation, thus supporting our hypothesis. Polycomb group protein target genes (PCGTs), which are suppressed by epigenetic mechanisms in stem cells and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation, were over-represented within the set of genes showing altered DNA methylation in response to SIRT1 manipulation in both cell lines. We thus propose that effects of SIRT1 to extend healthspan include influences on the DNA methylation status of genes affected during ageing, in particular PCGTs. Analysis of methylation profiles of Caco-2 cells meeting the following conditions: SIRT1 overexpressed by transfection with pCMV6-ENTRY-SIRT1 (2 replicates), corresponding vector control (2 replicates), each of the siRNAs that target SIRT1 (1 & 2 replicates, respectively) and control siRNA (2 replicates).

ORGANISM(S): Homo sapiens

SUBMITTER: Simon Cockell

PROVIDER: E-GEOD-53569 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:N-nitroso compounds (NOC) may be implicated in human colon carcinogenesis, but the toxicological mechanisms involved have not been elucidated. Since it was previously demonstrated that nitrosamines and nitrosamides, representing two classes of NOC, induce distinct gene expression effects in colon cells that are particularly related to oxidative stress, we hypothesized that different radical mechanisms are involved. Using ESR spectroscopy, we investigated radical generating properties of genotoxic NOC concentrations in human colon adenocarcinoma cells (Caco-2). Cells were exposed to nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosurea) or nitrosamines (N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine). Nitrosamines caused formation of reactive oxygen species (ROS) and carbon centered radicals which was further stimulated in presence of Caco-2 cells. N-methyl-N-nitrosurea exposure resulted in a small ROS signal, and formation of nitrogen centered radicals (NCR), also stimulated by Caco-2 cells. N-methyl-N'-nitro-N-nitrosoguanidine did not cause radical formation at genotoxic concentrations, but at increased exposure levels, both ROS and NCR formation was observed. By associating gene expression patterns with ROS formation, several cellular processes responding to nitrosamine exposure were identified, including apoptosis, cell cycle blockage, DNA repair and oxidative stress. These findings suggest that following NOC exposure in Caco-2 cells, ROS formation plays an important role in deregulation of gene expression patterns which may be relevant for the process of chemical carcinogenesis in the human colon, in addition to the role of DNA alkylation. Keywords: Nitrosamines, nitrosamides, N-nitroso compounds, free radicals, toxicogenomics, colon carcinogenesis The study investigated differential gene expression in Caco-2 cell line mRNA following 1, 6 or 24 hours of exposure to six different N-nitroso compounds. Two biological replicates per sample compound. One compound per array, hybridized against vehicle control. Dye-swap between biological replicates.

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

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