Effects of Sirt1 manipulation on gene expression in human Caco-2 cell line
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Simon Cockell
PROVIDER: E-GEOD-30486 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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).
2014-03-31 | E-GEOD-53569 | biostudies-arrayexpress
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.
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Project description:To compare gene expression in human intestinal cell line after Sirt1 manipulation by overexpression or targetted siRNA knockdown
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Project description:Mammalian SIRT1 is a central regulator of metabolism and aging. This project is to analyze global phosphorylation levels of mammalian SIRT1 in proliferating and senescence states using human lung fibroblast IMR90, in order to explore the post-translational regulation of SIRT1 protein upon cellular senescence and its potential roles in the regulatory mechanisms of SIRT1 homeostasis.
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2012-05-08 | E-GEOD-34175 | biostudies-arrayexpress
Project description:Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in satellite cells To establish the role of the deacetylase SIRT1 in skeletal muscle we examined the genome wide distribution of H4K16ac in quiescent (FI) and proliferating (Cul) satellite cells isolated from WT mice (C57Bl/6 background) and SIRT1mKO (generated via breeding of Pax7cre/+ knock-in mice with mice containing the floxed exon 4 SIRT1 allele). We also analyzed the distribution of SIRT1 in quiescent and proliferating FACS isolated WT satellite cells (two replicates). We generated the mRNA profiles (at least two replicate for each experiment) of FACS isolated quiescent, proliferating and differentiating (1 day in differentiation medium) satellite cells of WT mice and SIRT1mKO. The selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program.
2015-01-30 | E-GEOD-64379 | biostudies-arrayexpress
Project description:In humans, there are eleven subtypes of linker histones that exhibit cell- and tissue-specific expression. Linker histone H1 proteins bind to both the core histones and linker DNA of chromatin fibers; and not only participate in control of gene activity but also serve to stabilize higher order chromatin structure. To determine the potential roles of linker histones in differentiation, we examined the global distribution of linker histone subtype H1.5 in human IMR90 fibroblasts and H1 embryonic stem cells (hESCs). Surprisingly, H1.5 binds to and represses a large fraction of gene family clusters in fully differentiated cell types representing all three embryonic germ layers. Little or no H1.5 enrichment at gene family clusters was detected in undifferentiated hESCs or partially differentiated somatic cells. We also found that SIRT1 histone deacetylase and H3K9me2, a repressive histone modification, are also enriched at gene family cluster in IMR90 cells, likely generating a stably repressive chromatin domain. To find out the mechanism of H1.5 targeting, H1.5 or SIRT1 was depleted in IMR90 cells by siRNA, and the binding patterns of SIRT1 and H1.5 were examined. In H1.5 knockdown cells, SIRT1 binding pattern was changed dramatically, and this changed pattern highly correlates to SIRT1 distribution in hESC. However, depletion of SIRT1 could not change the global binding pattern of H1.5. Depletion of H1.5 or SIRT1 leads to up-regulation of ~50% gene family clusters. However, the sets of gene family clusters that are affected by these two factors are different, suggesting H1.5 and SIRT1 may regulate gene transcription via different pathways. Two-color microarrays. Two replicates for each sample.
2012-09-18 | E-GEOD-26856 | biostudies-arrayexpress
Project description:Resveratrol (RSV) is a small compound with many reported beneficial effects, including increased expression of genes involved in inborn errors of metabolism. We wanted to investigate the effects of RSV on gene regulation on a global scale in order to detect new target genes for RSV mediated up-regulation with respect to disease treatment. Because fibroblasts are widely used diagnostically and reflect many metabolically important tissues well, we chose to investigate normal fibroblasts treated with RSV. We performed this with and without knock down of sirtuin 1 (SIRT1) since SIRT1 has been reported as a key mediator of RSV effects. We performed Next Generation Sequencing of RNA on 2 normal fibroblast cell line with and without the respective treatments.
2018-11-12 | E-MTAB-7360 | biostudies-arrayexpress
Project description:BACKGROUND: Several in vitro assays have been used to identify “cancer stem cells” (CSC), including expression of cell surface markers and Hoechst dye efflux properties. However, each of these methods has potential pitfalls that complicate interpretation of the results. Focusing on colon cancers (CC), the CD133 antigen has been proposed as a marker of colon CSC. However, conflicting results have been reported in the literature indicating the need of a systematic analysis of CSC within CC and a complete validation of markers for the isolation of these cells. AIMS: Aim of this study was to confirm that CD133 expression is a valid method for isolating CSC in CC and verify if other antigens can increase the specificity of this marker for isolating CSC in CC. METHODS: CD133+ and CD133- cells were isolated from different human CC lines (CaCo-2, HT29, LOVO, HCT-116) by FACS sorter and the tumor-initiating potential of CD133+ cells was assessed in vitro, by soft-agar colony formation assay, and in vivo, upon transplantation into nude mice. Furthermore, the gene expression profile of CD133+ versus CD133- CaCo-2 cells was compared by the means of microarray analysis. Then, in the effort to identify a common “tumor stem cell” signature for CC, the most relevant transcripts resulting from gene expression profiling on CD133+ cells was assessed by real-time PCR on SP-fraction isolated by FACS sorter from the same CC cell lines. Finally, we deplete CD133 expression in the CaCo-2 cell line by the means of siRNA and verified by Western Blot analysis whether there was a functional correlation between CD133 and the target genes. Moreover, CaCo-2 and HCT116 cells were exposed to sodium butyrate (NaBu) for 72h. Colon cells differentiation was assessed by Alkaline phosphatase activity and expression of CD133 and target genes was tested by western blot. RESULTS: We confirmed that only CD133+ cells have a tumor-initiating potential in vitro and in vivo. Furthermore, microarray analysis of CD133+ versus CD133- CaCo-2 cells revealed a significant overexpression of various transcripts involved in cell proliferation, invasion and stemness in CD133+ cell fraction. Comparison of the transcripts by real-time PCR revealed that the genes of Endothelin-1 (END-1) and NR4A2 are highly expressed in both CD133 + cells and in SP fractions. Finally, when we deplete CD133 expression in Caco-2cells by siRNA, we observed a significant attenuation of END-1 and NR4A2 expression, thus demonstrating that CD133 is involved in the transcriptional regulation of these genes. Interestingly, we also showed that the expression of all three genes was inversely correlated with cell differentiation status as demonstrated by the fact that their expression decreases in a time- and dose-dependent manner after differentiation induced by NaBu. CONCLUSION: Overall, this study confirms the role of CD133antigen as CSC marker and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression, hypothesizing that CD133 is involved in the transcriptional regulation of these gene. Microarray analysis was performed on CD133+ and CD133- sorted CACO-2 cells. For both fractions, cells were sorted three independent times. Sample preparation was performed according to Affymetrix recommendations. A total of 6 arrays were hybridized, including 3 CD133+ replicates and 3 CD133- replicates.
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