Expanded methyl-sensitive cut counting reveals hypomethylation as an epigenetic state that highlights functional sequences of the genome

Abstract

Methyl-sensitive cut counting (MSCC) with the HpaII methylation-sensitive restriction enzyme is a cost-effective method to pinpoint unmethylated CpGs at single base-pair resolution. However, it has the drawback of addressing only CpGs in the context of the CCGG site, leaving out the remainder of the possible 16 XCGX tetranucleotides in which CpGs are found. We expanded MSCC to include three additional enzymes to address a total of 5 of the 16 XCGX combinations. This allowed us to survey methylation at about one-third of all a mammalian genome's CpGs. Applied to mouse liver DNA, we correctly confirmed data reported with other methods showing hypomethylation to be concentrated at promoters and in CpG islands (CGIs), with gene bodies and intergenic regions being mostly methylated. Grouping unmethylated CpGs, characterized by high MSCC scores (7% false discovery rate), we found a large number of unmethylated regions not qualifying as CGIs located in intergenic and intronic regions, which are highly enriched in functional DNA sequences (open regulatory annotation database) as well as in noncoding yet highly conserved mammalian sequences thought to be important but with as yet unknown function. About 50% of MSCC-defined unmethylated regions do not overlap algorithm-defined CGIs and offer a novel search space in which new functionalities of DNA may be found in health and disease.

DNA methylation-based biomarkers in serum of patients with breast cancer

“…In order to enrich the CpG-containing regions, the digested fragments are subjected to size selection, followed by sequencing on the next-generation sequencing platform [61]. Methyl-sensitive cut counting (MSCC) with the HpaII methylation-sensitive restriction enzyme is a costeffective method to pinpoint unmethylated CpGs at single-basepair resolution [62].…”

Folate and DNA Methylation: A Review of Molecular Mechanisms and the Evidence for Folate's Role

“…Currently, new DNA sequencing technologies are beginning to provide novel insight into genome-wide patterns of DNA methylation (10)(11)(12)(13). Although high-resolution truly genome-wide studies have been limited to a very small sample size, a recent study described the DNA methylation level at 1505 individual sites (loci) in 808 genes in 1628 human genomic DNA samples (14).…”

A Minimal Set of Tissue-Specific Hypomethylated CpGs Constitute Epigenetic Signatures of Developmental Programming

“…Most of the research done to address this problem has focused on the function of promoters and their CpG islands (CGIs), shaping the perception that the CGIs are the hotspots for epigenetic regulation of tissue specific transcriptional activity [9] [10]–[14]. However, technological advances in methylome analysis have increasingly shown T issue S pecific D ifferentially M ethylated R egions (TS-DMR) as hypo-methylated loci existing outside promoters and CGIs, [15]–[18]. We recently showed that most of the hypo-methylated loci existing in the liver of an adult mouse are located in introns or intergenic regions and do not meet commonly accepted definitions of CGIs [15].…”