Patterns of gene expression and DNA methylation in human fetal and adult liver

Abstract

Background: DNA methylation is an important epigenetic control mechanism that has been shown to be associated with gene silencing through the course of development, maturation and aging. However, only limited data are available regarding the relationship between methylation and gene expression in human development.

Results: We analyzed the methylome and transcriptome of three human fetal liver samples (gestational age 20-22 weeks) and three adult human liver samples. Genes whose expression differed between fetal and adult numbered 7,673. Adult overexpression was associated with metabolic pathways and, in particular, cytochrome P450 enzymes while fetal overexpression reflected enrichment for DNA replication and repair. Analysis for DNA methylation using the Illumina Infinium 450 K HumanMethylation BeadChip showed that 42% of the quality filtered 426,154 methylation sites differed significantly between adult and fetal tissue (q ≤ 0.05). Differences were small; 69% of the significant sites differed in their mean methylation beta value by ≤0.2. There was a trend among all sites toward higher methylation in the adult samples with the most frequent difference in beta being 0.1. Characterization of the relationship between methylation and expression revealed a clear difference between fetus and adult. Methylation of genes overexpressed in fetal liver showed the same pattern as seen for genes that were similarly expressed in fetal and adult liver. In contrast, adult overexpressed genes showed fetal hypermethylation that differed from the similarly expressed genes. An examination of gene region-specific methylation showed that sites proximal to the transcription start site or within the first exon with a significant fetal-adult difference in beta (>0.2) showed an inverse relationship with gene expression.

Conclusions: Nearly half of the CpGs in human liver show a significant difference in methylation comparing fetal and adult samples. Sites proximal to the transcription start site or within the first exon that show a transition from hypermethylation in the fetus to hypomethylation or intermediate methylation in the adult are associated with inverse changes in gene expression. In contrast, increases in methylation going from fetal to adult are not associated with fetal-to-adult decreased expression. These findings indicate fundamentally different roles for and/or regulation of DNA methylation in human fetal and adult liver.

Fig. 1

Analysis of gene expression in fetal and adult liver. Microarray data were analyzed by GSEA (a) and IPA (b). Blue bars and red bars reflect higher expression in the fetal and adult samples, respectively. Results are stratified based on level of significance. GSEA data are shown as the FDR q-value. IPA results show the unadjusted p-value. The threshold for significance of the IPA results was determined using fetal (blue arrow) and adult (red arrow) control data sets, as described in Methods

Fig. 2

Distribution of methylation data. Methylation sites were stratified based on the average methylation level. a A histogram showing the number of CpGs at any given methylation level was generated using intervals of 0.05 for the range of 0 to 1. All methylation sites were included in the determination of the mean of triplicate samples for each site. Fetal results are shown in blue and adult in red. b The difference in methylation between fetal and adult data sets, calculated as the average delta beta for each site, is shown as a histogram. For this analysis, sites were included only if there was a q-significant difference between fetal and adult

Fig. 3

Distribution of DNA methylation based on gene region. Triplicate fetal (blue) and adult (red) samples were analyzed using all CpGs that were found to be methylated. The solid lines represent sites within 1,500 base pairs of the transcription start site or within the first exon, and the dashed lines those sites that are within the gene body, 5’UTR or 3’UTR

Fig. 4

The relationship between DNA methylation and gene expression. Results are shown as the average methylation for each gene (mean beta value for the sites on that gene) versus the mean fetal:adult expression ratio for each gene. Data for the three fetal samples (blue) and the three adult samples (red) were filtered using various parameters to generate the input for the analysis. a All genes for which methylation was significantly different for fetal versus adult (q < 0.05). b Genes for which the fetal:adult difference in methylation (dβ) was at least 0.2. c Significant methylation; methylation restricted to TSS/Ex1 CpGs. d Significant methylation; methylation restricted to TSS/Ex1 CpGs for which dβ was at least 0.2

Fig. 5

Methylation of genes overexpressed in fetal liver. Overexpression was defined as a q-significant 5-fold difference in mean expression level between fetal and adult. a Fetal (blue) and adult (red) TSS/Ex1 methylation (FDR q < 0.05 with dβ >0.2) results are shown juxtaposed against one another. Also shown are methylation results for fetal (b) and adult (c) liver relative to data generated using randomly selected gene sets for which fetal and adult liver expression was similar (fetal:adult ratio of −1.1 to +1.1; gray bars)

Figure 6

Methylation of genes overexpressed in adult liver. Overexpression was defined as a q-significant 5-fold difference in mean expression level between fetal and adult. a Fetal (blue) and adult (red) TSS/Ex1 methylation (FDR q < 0.05 with dβ >0.2) results shown juxtaposed against one another. Also shown are methylation results for fetal (b) and adult (c) liver relative to data generated using randomly selected gene sets for which fetal and adult liver expression was similar (fetal:adult ratio of −1.1 to +1.1; gray bars)

Fig. 7

Distribution of gene region-specific methylation for genes overexpressed in fetal or adult liver. For all graphs, the density of fetal methylation is shown in blue and adult in red. Solid lines represent the density plots for genes that are 5-fold overexpressed in fetal liver (left) or adult liver (right). Results for genes that were equally expressed in fetal and adult liver (fetal:adult ratio of −1.1 to +1.1) are shown for comparison purposes as dotted lines. Methylation data were filtered based on a significant (q < 0.05) difference of at least 0.2. The graphs are grouped in rows based on gene regions: CpGs within 200 base pairs of the transcription start site (TSS200), within 1,500 base pairs of the transcription start site (TSS1500), or within the 5’UTR, 1st exon, gene body, or 3’UTR