Project description:To compare gene expression in human intestinal cell line after Sirt1 manipulation by overexpression or targetted siRNA knockdown Triplicate biological replicates for Caco-2 control and Sirt1 overexpression (6 samples), duplicate biological replicates for Caco-2 control siRNA and two non-overlapping targetted Sirt1 siRNAs (6 samples)
Project description: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).
Project description: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 faction 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. MBD-Sequencing to ascertain effects of SIRT1 over & under expression on methylation, in presence and absence of TNF-alpha. One sample per condition.
Project description: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 faction 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.
Project description: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.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression. Two-condition experiment, Normoxic MSCs vs. Hypoxic MSCs.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.