Fig. 6
Identification of tumor-specific hypoDMRs. A Heatmaps showing DNA methylation levels for each EAC hypoDMR. Each column denotes one sample and the row was ordered by the delta mean methylation between EAC and NGEJ (left). EAC ts-hypoDMRs were identified using a one-tailed t test between EAC tumor and NGEJ samples (right) with the FDR cutoff < 0.05. B Stacked bar plots showing fractions of ts-hypoDMRs that overlap with different genomic features. C, D Cistrome-GO enrichment analyses using either EAC (C) or ESCC (D) ts-hypoDMRs and the upregulated genes in each subtype compared with corresponding nonmalignant samples. Top 15 most significant pathways are shown. The transcriptomic data of esophageal cancer from the TCGA consortium and GSE149609. E Scatter plots showing transcription-factor-binding sites that were enriched in EAC ts-hypoDMRs compared with cts-hypoDMRs. The X axis represents the expression fold change between EAC and matched nonmalignant GEJ samples. The Y axis shows the delta enrichment score of transcription-factor-binding sites between EAC ts- and cts-hypoDMRs. Expression data were from the TCGA and motif enrichment analyses were performed by the ELMER method. F EAC ts-hypoDMRs contained significantly more HNF4A-recognition motifs compared with cts-hypoDMRs. G More HNF4A peaks overlapped with ts-hypoDMRs than cts-hypoDMRs. Peaks were called from HNF4A ChIP-seq in ESO26 (GSE132813) and OE19 cell lines (E-MTAB-6858). H HNF4A was predicted to co-occupy with the AP-1 family in ts-hypoDMRs, while with FOXA1/2 in cts-hypoDMRs. Sequence motif analysis was performed using ts- vs. cts-hypoDMRs containing HNF4A motifs. Significant transcription factors with FDR < 0.05 are shown. OR value over 1 represents higher enrichment in ts-hypoDMRs, while below 1 represents higher enrichment in cts-hypoDMRs. I qPCR experiments measuring HNF4A mRNA expression in the scramble shRNA vs. shHNF4A group in ESO26 and OE19 cell lines. J, K FOSL1 ChIP-qPCR assays were performed in ESO26 (J) and OE19 (K) cells, in either the scramble shRNA or shHNF4A group. IgG was used as a negative control antibody. The number of biological replicates is 3. p-values were determined by a two-sided t test. ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant; nd, not detectable