Epigenetic repression via DNA methylation and trimethylation of H3K27 alters gene expression in esophageal adenocarcinoma [ChIP-Seq]. Homo sapiens

Epigenetic modifications in the form of altered DNA or histone methylation can influence gene expression patterns critical for neoplastic initiation and progression. The fact that abnormal gene expression can be driven by epigenetic alterations led us to examine the correlation between DNA methylation, trimethylation of histone 3 lysine 9 (H3K9me3) and lysine 27 (H3K27me3), and gene expression in esophageal adenocarcinoma (EAC). Using genome-wide approaches (chromatin immunoprecipitation/sequencing (ChIP-Seq) and methylation arrays), we identified gene targets that were enriched with the chromatin-repressive marks H3K9me3, H3K27me3, and/or DNA hypermethylation across patients with EAC. Using RNA-Seq, we found genes involved in cellular morphology and movement, epithelial cell differentiation, epithelial junction signaling, as well as genes involved in epithelial-mesenchymal transition (EMT) were down-regulated in patients with poorly differentiated EAC. Additionally, comparative analyses of ChIP-Seq, DNA methylation, and RNA-Seq data allowed us to identify a group of genes downregulated in EAC is associated with aberrant H3K27me3 enrichment or a combination of H3K27me3 and DNA hypermethylation in the poorly differentiated EAC cases. Furthermore, by comparison to TCGA data sets, H3K27me3 enrichment is associated with aberrant DNA methylation across numerous EAC cases, suggesting that dysregulation of H3K27me3 and DNA methylation is crucial in the pathogenesis of EAC. Overall design: Esophageal adenocarcinoma specimens from two patients (both poorly differentiated) and normal human esophageal tissues were crosslinked in 1.1% formaldehyde before chromatin shearing using the Diagenode Bioruptor. The resulting sheared 200-500 bp chromatin fragments were incubated with H3K9me3 (Millipore #07-473) or H3K27me3 (Millipore #07-449) antibody-conjugated Protein A Dynabeads (Life Technology #10002D) overnight. For normalization, an aliquot of sheared chromatin fragments were incubated with antibody against unmodified H3 antibody-conjugated Protein A Dynabeads. Subsequently, enriched chromatin fragments were eluted, de-crosslinked, and purified for library preparation (Illumina Library Kit, Cat#15026486) for the Illumina HiSeq2000 sequencer. DNA obtained from each ChIP pull-down was sequenced to high depth of 150 million tags with 50 basespair-end sequencing.