When navigating complex environments, it’s always handy to have a good map. Although the human epigenome project has been pretty good at mapping the complexities of 5-methyl cytosine (5mC), and more recently 5-hydroxymethylation (5hmC), other oxidative derivatives of 5hmC, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have remained relatively uncharted terrain.
Researchers from North Carolina and Active Motif have been successful in detecting 5fC and 5caC using pull down techniques, but their techniques cannot be employed efficiently to detect these modifications at a global scale. Bisulfite sequencing is the gold standard when it comes to detecting 5mC and de-methylated intermediates. However, due to the harsh chemical reactions used, DNA degradation occurs, such that bisulfite-based chemical conversion cannot distinguish between the various intermediate de-methylated oxidative derivatives of 5mC.
Now a team of chemical biologists from Chicago and Beijing led by Chuan He and Chengqi Yi have designed a bisulfite-free method (called fC-CET) to detect 5fC based on selective chemical labeling. The researchers screened for a chemical label that would uniquely label 5fC (but not other de-methylated intermediates) and which could be read out as a C to T conversion during PCR.
Using this bisulfite free, base resolution analysis, the researchers obtained genome wide maps of 5fC at single base resolution and found that:
- Chemical conversion of 5fC to C and subsequently T specifically detected 5fC and not other oxidized derivatives of 5mC.
- There was limited overlap between 5fC and 5hmC sites in mESCs from TDG null mice (TDG is the enzyme involved in base excision repair), further hinting that these modifications have different biological roles.
- 5fC marks distinct regulatory elements and represents a more active marker than 5hmC.
- fC-CET technology could be employed for precious clinical samples.
This Bisulfite FREEway combined with other methylome sequencing could have wider implications in understanding the epigenome.
For a detailed landscape of 5fC, check out Nature Methods, November 2015.