We all know the old saying “Good things come in pairs”, and when you’re only examining one member of a dynamic duo, sometimes you can miss the key contribution of the trusty sidekick. Attesting to the above, a talented team from Stanford show off a clever new technique to simultaneously image 5mC and 5hmC on a single molecule, with only trace (50 pg) amounts of DNA.
Here’s how the dual-labelling imaging technology works to pull 5hmC out of 5mC’s shadow:
- The DNA is end-labelled with biotin, which immobilizes it on a microscope slide.
- 5hmC with is labelled with a red (Cy5) fluorophore via β-glucosytransferase (βGT) and an azide-modified glucose.
- 5mC is labelled with a green (Cy3) fluorophore in a one-pot reaction involving βGT and TET1.
- The fluorescent DNA is then tethered to a special microscope slide and imaged with single-molecule total internal reflection fluorescence (TIRF) microscopy.
Demonstrating the imaging power of this dual-labelling imaging technology, the team examined both young and old mouse brain (cerebellum) genomic DNA to gain insight into the role of 5hmC in development. They found that unlike 5mC, 5hmC levels changed with age.
The team then put the picture together by using single-molecule fluorescence resonance energy transfer (smFRET) analysis to measure the proximity between 5mC and 5hmC. Interestingly, smFRET analysis of a number of tissues and mouse embryonic stem cells (mESCs) revealed a previously undetectable epigenetic state.
In this new epigenetic state, one strand contains 5hmC, and the other strand maintains 5mC, which produces opposing hemi-hydroxymethylated/hemi-methylated CpGs known as 5hmC/5mCpGs. To further investigate this regulatory feature, they tested Methyl-CpG Binding Domain Protein 2 (MBD2) on synthetic DNA using an electrophoretic mobility shift assay (EMSA). This led to the observation that 5hmC/5mCpGs reduce the binding affinity of MBD2 when compared to standard 5mC/5mCpGs. The team hypothesizes that 5hmC/5mCpGs prevent repression of gene expression and serve as a new regulatory feature in the genome that activates gene expression.
Get the full methylation picture for only 50 pg in PNAS, March 2016