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

Abstract

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-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.

Figure 1

Treatment of HepG2 cells with 2.5 μM 5-aza-dC results in DNA demethylation. Demethylated DNA is cleaved by Hpa II digestion and cannot be amplified; therefore the amount of methylated DNA is reflected in the amount of PCR product. (A) Electropherogram of genomic DNA from untreated HepG2 cells or cells treated with 5-aza-dC; DNA was extracted and digested with Rsa I + Hpa II and subjected to PCR using primers that flank the endogenously methylated Hpa II site at bp -2916 upstream of the ADH1B gene. (B) Quantitation of the PCR products produced in (A) using 2 X PicoGreen^® and an ABI PRISM^® 7700 Sequence Detection System. The order of the quantitation is the same as the order of the PCR products seen in (A).

Figure 2

Hierarchical cluster analysis of genes that vary across the experiment. Genes that varied across the experiment (CV ≥ 0.6) were selected and clustered [75] based upon Euclidean distance. Colors represent differences from the median expression of the gene across different conditions: the top 5% are yellow, then in declining order from red to green (median), then progressively more blue (below median) to blue (bottom 5%).