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Abstract
The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). 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). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. 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.
Highlights
Methylation on the 5-carbon of cytosine nucleotides in genomic DNA of eukaryotes is the most extensively studied epigenetic modification
Over 70,000 research papers, methods chapters, and review articles have been dedicated to the study of DNA methylation (5mC). 5mC provides diverse functionality in the regulation of gene expression, genome stability, chromatin compaction, and developmental timing [1]
Faithfully copied during DNA replication by the maintenance methylation machinery, DNA methyltransferase 1 (DNMT1) and Ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) [2–5]. 5mC patterning is conserved across most somatic tissues, with the most dynamics occurring at enhancers and other distal regulatory regions of the genome that influence gene expression [6, 7]
Summary
Methylation on the 5-carbon of cytosine nucleotides in genomic DNA of eukaryotes is the most extensively studied epigenetic modification. While recent evidence suggests that the oxidized forms of 5mC can act in a regulatory manner through the recruitment of reader proteins [16, 17], perhaps the most well-studied roles for the active DNA demethylation pathway are in the early stages of mammalian development [18] Following fertilization, both the paternal and maternal genomes undergo massive changes in DNA methylation patterning that occurs through both active and passive DNA demethylation, respectively [19–23]. As hypermethylation of tumor suppressor genes is a hallmark of cancer, significant effort has been devoted to developing therapies that induce DNA demethylation of these genes in order to restore their expression and function in cancer cells [27] Both passive and active DNA demethylation mechanisms are being targeted for combination cancer therapies with DNMT inhibitors like 5-aza-20-deoxycytidine (DAC) and with L-ascorbic acid (Vitamin C, VitC), a co-factor for TET dioxygenase activity [31–33]. We detail assays that can be used to determine relative global change in 5mC and 5hmC across gDNA samples, which we use to check samples prior to EPIC array
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