Unknown,Transcriptomics,Genomics,Proteomics

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Comprehensive DNA methylation and gene expression profiling in differentiating human adipocytes

ABSTRACT: Genome-wide DNA methylation profiling was performed of human mesenchymal stem cells (hMSCs) differentiating into adipocytes (day 10). The Illumina Infinium 450k Human DNA methylation Beadchip v1.2 was used to measure DNA methylation of hMSC before differentiation (day 0) and of adipocytes at day 10 of differentiation. Our aims were to 1) measure genome-wide DNA methylation changes during adipocyte differentiation in primary hMSCs and 2) investigate the relationship between DNA methylation and gene expression regulation in a panel of 84 adipocyte-related genes. Bisulphite converted DNA from four independent experiments was hybridised to the Illumina Infinium 450k Human Methylation Beadchip. The first comparison was made between undifferentiated hMSCs and differentiated adipocytes at day 10. As a positive control, differentiating cells were continuously exposed to 5-Aza-2â²-deoxycytidine (5-aza-dC). The second comparison was made between differentiated adipocytes at day 10 and 5-aza-dC exposed adipocytes at day 10.

ORGANISM(S): Homo sapiens

SUBMITTER: Myrthe van den Dungen

PROVIDER: E-GEOD-74609 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Expression profiling the response to inhibition of DNA methylation and histone deacetylation. Comparison of expression in HepG2 cells treated with 5-aza-dC, Trichostatin A, both, or none (control) to change methylation and acetylation status. Background:DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. We hypothesize that many genes in the human hepatoma cell line HepG2 are regulated by DNA methylation and histone deacetylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) to inhibit DNA methylation with and/or Trichostatin A (TSA) to inhibit histone deacetylation should allow us to identify genes that are regulated epigenetically in hepatoma cells. Results:5-aza-dC had a much larger effect on gene expression in HepG2 cells than did TSA, as measured using Affymetrix® HG-U133A Plus 2.0 microarrays. The expression of 1504 probe sets was affected by 5-aza-dC (at p < 0.01), 535 probe sets by TSA, and 1929 probe sets by the combination of 5-aza-dC and TSA. 5-aza-dC treatment turned on the expression of 211 probe sets that were not detectably expressed in its absence. Expression of imprinted genes regulated by DNA methylation, such as H19 and NNAT, was turned on or greatly increased in response to 5-aza-dC. Genes involved in liver processes such as xenobiotic metabolism (CYP3A4, CYP3A5, and CYP3A7) and steroid biosynthesis (CYP17A1 and CYP19A1), and CCAAT element-binding proteins (CEBPA, CEBPB, and CEBPG) were affected by 5-aza-dC or the combination. Many of the genes that fall within these groups are also expressed in the developing fetal liver. Quantitative real-time RT-PCR assays confirmed selected gene expression changes seen in microarray analyses. Conclusions:Epigenetics play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis in HepG2 cells. Many genes whose expression is normally silenced in these hepatoma cells were re-expressed by 5-aza-dC treatment. Many genes that are expressed in the fetal liver are up-regulated by demethylation, indicating that DNA methylation is a major factor in restricting the expression of fetal genes during liver development. Keywords: comparison of treatments

Project description:Expression profiling the response to inhibition of DNA methylation and histone deacetylation. Comparison of expression in HepG2 cells treated with 5-aza-dC, Trichostatin A, both, or none (control) to change methylation and acetylation status. Backgroubd: DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. We hypothesize that many genes in the human hepatoma cell line HepG2 are regulated by DNA methylation and histone deacetylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) to inhibit DNA methylation with and/or Trichostatin A (TSA) to inhibit histone deacetylation should allow us to identify genes that are regulated epigenetically in hepatoma cells. RESULTS: 5-aza-dC had a much larger effect on gene expression in HepG2 cells than did TSA, as measured using Affymetrix HG-U133 Plus 2.0 microarrays. The expression of 1504 probe sets was affected by 5-aza-dC (at p < 0.01), 535 probe sets by TSA, and 1929 probe sets by the combination of 5-aza-dC and TSA. 5-aza-dC treatment turned on the expression of 211 probe sets that were not detectably expressed in its absence. Expression of imprinted genes regulated by DNA methylation, such as H19 and NNAT, was turned on or greatly increased in response to 5-aza-dC. Genes involved in liver processes such as xenobiotic metabolism (CYP3A4, CYP3A5, and CYP3A7) and steroid biosynthesis (CYP17A1 and CYP19A1), and genes encoding CCAAT element-binding proteins (C/EBPalpha, C/EBPbeta, and C/EBPgamma) were affected by 5-aza-dC or the combination. Many of the genes that fall within these groups are also expressed in the developing fetal liver and adult liver. Quantitative real-time RT-PCR assays confirmed selected gene expression changes seen in microarray analyses. CONCLUSION: Epigenetics play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis in HepG2 cells. Many genes whose expression is normally silenced in these hepatoma cells were re-expressed by 5-aza-dC treatment. DNA methylation may be a factor in restricting the expression of fetal genes during liver development and in shutting down expression in hepatoma cells

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Comprehensive DNA methylation and gene expression profiling in differentiating human adipocytes

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