Epigenetic modifications are chemical processes that affect gene activity without altering the DNA sequence. In mammalian genomes, two major epigenetic modifications called 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) play important roles in embryonic development and diseases such as cancer, but little is known about the interplay between these modifications due to a lack of suitable techniques for simultaneously revealing the locations of nearby 5mC and 5hmC sites.
Scientists developed an ultrasensitive single-molecule imaging technique to detect and quantify 5mC and 5hmC in the same DNA molecule. Using trace amounts of synthetic DNA and genomic DNA from different mouse tissues, the authors labeled 5mC and 5hmC with red and green fluorescent dyes, respectively, and imaged the molecules using a fluorescence microscope.
Next, the authors used a biophysical technique called single-molecule fluorescence resonance energy transfer to measure the distance between the labeled 5mC and 5hmC molecules. The findings revealed high levels of adjacent and opposing 5mC and 5hmC molecules at CpG sites—DNA regions consisting of a cytosine nucleotide followed by a guanine nucleotide.
According to the authors, these widespread methylated and hydroxymethylated CpG sites represent an important epigenetic state in the mammalian genome and could play previously unappreciated roles in gene regulation.