Fig. 6: Experimental validation of HNF4A as an upstream regulator of PRC2⁺-CGI genes through activation of distal enhancers.

a–c Focusing on the 33 HNF4A-binding enhancers, showing a HNF4A ChIP-Seq peaks (left) and ATAC-Seq peaks in EAC normal and tumor tissues (right), b TCGA ESCC and EAC tumor tissues, and c EAC cell lines. The ATAC-Seq signals in a are normalized with CPM method and those called as peaks are marked with a triangle. Differential analyses using DiffBind were performed to compare the difference between normal and tumors for each region in a and those with fold-change > 1.5 and FDR < 0.1 are marked with asterisks. TCGA ATAC-Seq signals were normalized by the TCGA consortium and the difference in ATAC-Seq signals between EAC and ESCC samples was calculated. Those genes with significant increase in EAC tumors were marked with asterisks (two-sided t-test, p-value < 0.05; The exact p-values are shown in Supplementary Data 4). d In EAC cells (OE19) with HNF4A knockdown, volcano plots show expression changes of either PRC2⁺-CGI (left) or PRC2⁻-CGI genes (right) that are linked to HNF4A-binding enhancers. e Promoter H3K27me3 signals were measured by ChIP-qPCR in both scramble and siHNF4A OE19 cells. n = 2 biological biologically independent experiments. f A summary graph illustrating the cancer-specific deregulation of both PRC2⁺-CGI and PRC2⁻-CGI genes, including the underlying molecular mechanisms and biological implications.