Abstract Background Enhancers are distal cis -regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity – however, most predicted enhancer regions remain to be functionally tested. Results Analyzing 128 epigenomic histone modification profiles of primary GC samples, normal gastric tissues, and GC cell lines, we report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are tumor-associated in vivo (&gt;50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans -acting TFs involved in GC expression. Specifically, we identified cancer-relevant genes (e.g. ING1 , ARL4C ) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans -acting factor associated with GC enhancer heterogeneity. Conclusions Our study indicates that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, and provides insights into the relative contribution of genetic ( cis ) and regulatory ( trans ) mechanisms to GC enhancer functional heterogeneity.

Integrative Epigenomic and High-Throughput Functional Enhancer Profiling Reveals Determinants of Enhancer Heterogeneity in Gastric Cancer