IDDF2019-ABS-0303 Methylation profiles of phantom 5 enhancer and open chromatin in colorectal cancers

Background Colorectal cancer (CRC) contributes around 1.36 million of the total cases worldwide and it has become evident over the past two decades that epigenetic alterations also play key roles in CRC pathogenesis. Majority of the research epigenetic alterations has examined the promoter regions, while other loci such including enhancers and open chromatins are not yet well described. Hence, this study aims to specifically profile the methylome of enhancers and open chromatins in CRC Methods Genomic DNA and total RNA were extracted from cancer-adjacent normal colonic tissues and subjected to bisulfite conversion and cDNA synthesis, respectively. DNA methylation analysis was performed using Human Infinium Epic Beadchip Array which includes >23,000 enhancers and >461,000 open chromatins. Microarray data were analyzed using Genome Studio V1.8 and Bioconductor-ChAMP V2.8.1. The differentially methylated regions were validated via bisulfite conversion, cloning, and sequencing of individual clones. In order to correlate the effect of DNA methylation at the specific loci, the gene expression of the differentially methylated loci was analysed using quantitative real-time PCR Results We identified 342 significant differently methylated enhancers and 2187 significant differentially methylated open chromatin. There were 192 hypermethylated and 150 hypomethylated enhancers compared to 1110 hypermethylated and 1076 hypomethylated open chromatins. Pathway enrichment analysis reveals that the majority of differentially methylated genes in the enhancer region are involved in the cancer and focal adhesion pathway. In addition, the pathways in cancer and PI3K-Akt signaling are significantly enriched in the differentially methylated open chromatin loci. Significant differentially methylated enhancer and open chromatin loci, OPLAH cg26256223 (hypermethylated open chromatin) and LYN cg08621168 (hypomethylated enhancer) were selected for further validation. qPCR analysis further confirmed the decrease of OPLAH gene expression level and vice versa for the LYN gene Conclusions This is the first insight on the enhancers and open chromatins methylation profile in Malaysian CRC patients. The new knowledge from this study can be utilized to further increase our understanding of CRC methylomics, particularly on the enhancers and open chromatins. The functional roles of OPLAH cg26256223 and LYN cg08621168 warrant future investigations