Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation

Luke O Dannenberg,Howard J Edenberg

doi:10.1186/1471-2164-7-181

Luke O Dannenberg, Howard J Edenberg

Open Access

https://doi.org/10.1186/1471-2164-7-181

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Abstract

BackgroundDNA 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.Results5-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/EBPα, C/EBPβ, and C/EBPγ) 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.ConclusionEpigenetics 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.

Highlights

DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation
Many genes were affected by the treatment of 5-aza-dC and/or Trichostatin A (TSA) in HepG2 cells (Table 1)
It is demonstrated by hierarchical clustering (Figure 2) in which the cells treated with 5-aza-dC, either alone or with TSA, segregated from cells not treated with TSA. 5-aza-dC treatment re-expressed 211 probe sets (188 genes) that had been silent in HepG2 cells [see Additional file 2], and up regulated more genes than were down regulated

Summary

Introduction

DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. Treatment with 5aza-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. 5-aza-2-deoxycytidine (5-aza-dC) exerts its demethylating function by sequestering DNA methyltransferase 1 (DNMT1) to 5-aza-dC-substituted DNA via the irreversible binding of the cysteine in the catalytic domain of DNMT1 to the 6 position of the cytidine ring (4,5). This decreases the cellular concentration of DNMT1, which leads to genomic DNA demethylation in the course of successive cell divisions [6,7,8]. Trichostatin A (TSA) is a reversible inhibitor of HDAC in vitro and in vivo, active at nanomolar concentrations, that causes a marked increase in histone acetylation [24]

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