Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

Abstract

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500–1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

DNA in nanochannels: theory and applications

“…The short recognition site of a methyltransferase has been used to generate dense labels, so-called amplitude modulation profiles, along the DNA backbone (Grunwald et al, 2015) and methyltransferasebased labeling was utilized to obtain high-resolution optical maps also by Jeffet et al (2016). By combining nick labeling protocols with methyltransferase-based labeling hybrid genetic/epigenetic maps, providing both genomic and methylation profiles simultaneously, can be obtained, as illustrated by Sharim et al (2019) and Gabrieli et al (2022).…”

“…The latter has recently been demonstrated (Torstensson et al ., 2021), as briefly discussed above. Alternatively, labeling to obtain genetic information can be combined with other types of labels, such as epigenetic markers (Sharim et al ., 2019; Margalit et al ., 2020; Gabrieli et al ., 2022) or labeling of DNA damage (Müller et al ., 2019), thereby adding an additional layer of information to the optical DNA map. The development of labels that can be used to obtain such additional information that is not yet accessible remains a challenge in ODM.…”

Simultaneous global labeling (SiGL) of 5-methylcytosine and 5-hydroxymethylcytosine by DNA alkylation with a synthetic cofactor and engineered methyltransferase

“…CpG methyltransferases are blocked when the cytosine is methylated, and thus will label only um-CpGs (Figure 2a). We and others have shown that a double mutant of the CpG-specific DNA MTase M.Sssl can label unmodified cytosines with azide groups via the AdoYnAzide cofactor 37,38 . However, M.Sssl is difficult to express at high concentrations and it tends to aggregate upon expression.…”

Optical Genome and Epigenome Mapping of Clear Cell Renal Cell Carcinoma

“…Using fluorescence microscopy and designated chemistries, OGM can provide multilayered information from individual DNA molecules. Fluorescent labeling of different genomic features with different colors allows studying multiple epigenetic marks on the single-molecule level, creating a hybrid genetic/epigenetic map for every DNA molecule (19, 23, 24, 26).…”