Fig. 1
HCC methylome analysis identifies a recurrent hypomethylated enhancer targeting a liver-enriched transcriptional factor C/EBPβ. a A circos plot showing genomic regions that are significantly hypomethylated (4528 regions, in red) or hypermethylated (2654 regions, in green) in the tumor group as compared to matched non-tumors based on MBDCap-seq data. b Enrichment of DMRs in annotated functional elements, defined as (Lmi/Lm)/(Li/L), where Lmi is the total length of DMRs overlapping with the annotated functional elements of type i and Lm is the total length of all DMRs. Li is the length of the annotated functional elements of type i, and L is the total length of all annotated functional elements combined. c Genome-wide methylation patterns of differentially methylated FANTOM5 enhancers in HCC tumor (orange) and normal liver (blue) tissues based on WGBS. d Enhancer methylation levels of HCC tumor and normal liver tissues. e Relative methylation levels of 894 DMEs between HCC tumor and normal liver tissues. f ChIP-seq tracks of H3K27ac, H3K4me1, H3K4me3, and H3K27me3 at the 1-kb C/EBPβ enhancer locus (chromosome 20: 48,900,221–48,901,229) in HCC tumor tissues. g–i Methylation levels of 13 CpG sites within the C/EBPβ enhancer in 48 pairs of HCC tumor and non-tumor tissues as determined by pyrosequencing. j qRT-PCR analysis of HCC tumor and non-tumor tissues (33 pairs). C/EBPβ mRNA levels were calculated by the 2−ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average value of the non-tumor group. k Correlation between C/EBPβ enhancer methylation and expression in 33 pairs of HCC tumor and non-tumor tissues. C/EBPβ mRNA levels are ΔCt values using 18s rRNA as internal control. l, m Kaplan–Meier survival analysis of 48 HCC patients according to their C/EBPβ hypomethylation statuses (relative methylation of tumor vs. non-tumor). Patients with strong hypomethylation (top 24) show poorer (l) overall and (m) disease-free survival rates than those with weak hypomethylation (bottom 24). Data are presented as mean ± SD. **P < 0.01; ****P < 0.0001 as calculated by Wilcoxon signed-rank test (d), paired two-tailed Student’s t-test (i, j), Pearson correlation test (k) and Kaplan–Meier survival analysis (l, m)
