Life was simpler back when there were only four bases. Nowadays researchers studying DNA methylation have to keep track of four methylCytosine derivatives alone, and the research is just starting to heat up.
The last couple of years the TET family of proteins has proven effective at catalyzing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), apparently as part of a demethylation pathway. Things got even more interesting earlier this year when 5-formylcytosine(5fC) and 5-carboxylcytosine (5caC) was found to be a part of that same pathway. Now, some recent work out Yi Zhang’s lab at the University of North Carolina suggests that 5fC and 5caC appear to be more than just intermediates.
The most recent data produced by Zhang’s lab, pioneers in the 5hmC and 5fC/5caC space, surfaced when the team set out to take a closer look how the 7th & 8th bases work.
Pulling Down 5fC & 5caC
The restriction enzyme & thin-layer chromatography techniques that helped the Carolina crew first identify 5fC and 5caC weren’t so useful for functional analysis, so they teamed up with Active Motif, an early player in the 5hmC antibody game, and developed polyclonal antibodies that pull down 5fc and 5caC.
“The 5-formylcytosine and 5-carboxylcytosine antibodies were generated by our R&D group, and the UNC researchers assisted with the validation and specificity testing. We’re excited to add these new antibodies to our existing portfolio of antibodies specific for DNA modifications, including 5-methylcytosine and 5-hydroxymethylcytosine,” says Active Motif’s Jim Bone.
The new abs were put to work in immunostaining experiments to examine 5fC and 5caC in mouse zygotic development. Here’s what they found:
- Much like 5hmC, 5fC and 5caC appear in the paternal pronucleus as 5mC disappears.
- 5fC and 5caC are not quickly degraded, but undergo “replication-dependent dilution” and appear stable during preimplantation development.
Taken together with previous data, the authors now believe that this demethylation mechanism works something like this: 5mC is converted by Tet proteins to 5hmC, then 5fC and finally 5caC which is then followed by replication-dependent dilution that effectively removes paternal DNA methylation prior to zygotic development. The scientists also suspect that since 5fC and 5caC are relatively stable, it’s possible that they have some functional role in development and germ cell programming.
See what else is new with these DNA methylation mods in Cell Research, November 2011.