But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.
The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “It’s a pretty good method but the DNA gets pretty badly battered. The polymer can’t be copied by conventional polymerase easily and the strands are of a different sequence. Furthermore, bisulphite conversion does not distinguish 5mC and 5hmC,” which may hamper the analysis.
Several years ago, he explained, “We started looking at enzymes that can detect 5-methylcytosine. Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, and this unique microbial enzyme can decode the hydryoxmethylome of the mammalian genome. You easily can find out where the hydroxymethyl regions are.”
AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and cleaves at narrow range of distances away from the recognized modified cytosine. By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.
Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.
In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.
As a result of their studies, they propose that factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition including include chromatin compaction, nucleosome positioning, or TF binding. The most striking example, they said, is the regularly oscillating 5hmC profile around the CTCF-binding sites, suggesting 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment. They further proposed that some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.
“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.
And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.