5-hydroxymethylcytosine (5-hmC) has been hiding out in the genomic shadows too long, but now researchers armed with a new technique, can track 5-hmC not just to the specific tissue it’s holed up in, but also the developmental stage as well. Or as Martha and the Vandellas put it, there’s “nowhere to run to, baby, nowhere to hide”.
The team from New England Biolabs and UCLA kicked off their project by developing a method to detect and measure 5-hmC. “Our goal was to build a robust yet simple technique for 5-hydroxymethylcytosine identification and quantification that could be used successfully in virtually any molecular biology lab” said author and NEB R&D Scientist Sriharsa Pradhan. What they built was an enzymatic approach that can interrogate any CCGG loci within a genome.
5-hmC Restriction Digestion and Detection
Basically the protocol begins by labeling 5-hydroxymethylcytosine with glucose via enzymatic glucosylation reaction, which then sets up a scenario where glucosylated 5-hmC will give differential digestion patterns when treated with restriction enzymes (MspI and HpaII) than either cytosine (C) or 5-methylcytosine (5-mC, the precursor of 5-hmC). The results can then be easily analyzed by a method like qPCR. “One of the big benefits of this approach is that it enables you to measure both 5-hmC and 5-mC modifications in the same location, at the same time”, added Pradhan. Putting the new glucosylation methodology to work, the NEB crew found that:
- 5-hmC is pretty common in human and murine brain and heart genomic DNA.
- NIH3T3 and HeLa cell lines both showed little to no 5-hmC at the examined loci.
- 5-hmC levels in mouse embryonic stem cell DNA first increased then slowly tapered off during differentiation to embryoid bodies, while 5-mC gradually rose over time.
- Some VANGL1 and EGFR gene body methylation is indeed hydroxymethylation.
“The technique we’ve developed really enabled us to take the next step in 5-hmC detection. We were able to determine the percentages of unmodified cytosine, 5- mC and 5-hmC at CCGG sites by coupling it to quantitative PCR, plus demonstrate dynamic, gene and tissue-specific 5-hmC distribution, and examine 5-hmC’s role in gene body methylation as well” explained Pradhan.
The authors conclude that their efforts have only scratched the surface, and that more studies needed to pin down the exact function of 5-hmC within various tissues and different genomic regions.
If you’d like to join the search for 5-hmC, you can find the techniques used in this paper packaged in the EpiMarkTM 5-hmC and 5-mC Analysis Kit from New England Biolabs.
Find out more about where 5-hmC hangs out at Journal of Biological Chemistry, May 2011.