Distinguishing active versus passive DNA demethylation using Illumina MethylationEPIC BeadChip microarrays

The 5-carbon position on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). Genomic locations enriched for 5mC contribute to the regulation of chromatin structure and gene expression. While largely stable, massive waves of DNA demethylation are observed in key developmental windows in mammals. Additionally, DNA methylation is therapeutically targeted in cancer via DNA methyltransferase inhibition, in part to reverse abnormal silencing of tumor suppressor genes. DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These oxidized cytosine forms have reported function in gene regulation and also serve as intermediates in an active 5mC removal process that involves the base excision repair pathway. 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. Distinguishing active versus passive 5mC removal on a genome-wide scale remains a challenge. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis. Cells were treated with 1X PBS, 1 mM 5'aza-2'deoxycytidine (DAC), or 1 mM DAC plus 57 mM Vitamin C (VitC). DAC treated cells were pulsed with DAC for 24 hours and then with 1X PBS for the remaining 48 hours. VitC treated cells were treated every 24 hours. Cells were harvested at 72 hours post initial treatments. Extracted gDNA was converted with upstream modifications to bisulfite conversion to aid in distinguishing 5mC from 5hmC. Tet-assisted bisulfite (TAB) conversion was done to observe 5hmC, oxidative bisulfite (OxBS) conversion was done to observe 5mC, and bisulfite conversion (BS) was done to observe both 5mC and 5hmC. ZYMO indicates bisulfite conversion was done with the ZYMO EZ DNA methylation kit (Catalog #:D5001) and NUGEN indicates bisulfite conversion was done with NuGEN TrueMethyl oxBS Module, Tecan Genomics, Inc. (Catalog #: 0414-32).