5-hydroxymethylcytosine’s (5-hmC) discovery last year in mammalian DNA (Kriaucionis & Heintz, Science, 2009. and Tahiliani et al., Science, 2009), left the epigenetics community more shaken than a 007 martini. Although its broader role (if any) in epigenetics remains a mystery today, early evidence suggests a few putative mechanisms that could have big implications:
- Conversion of methylcytosine (5-mC) to 5-hmC could displace methyl binding proteins (MBPs). MeCP2, for instance, will not bind to 5-hmC.
- 5-hmC may induce demethylation by interfering with the methylation maintainence of DNMT1 during cell division.
- 5-hmC may have its own specific binding proteins that alter chromatin structure or DNA methylation patterns.
A year later, there remain far more questions than answers and as more researchers set out to tackle some of these questions, they’re finding that studying 5-hmC might not be so straight forward. Two separate groups from Harvard Medical School and Western General Hospital in Edinburgh, Scotland have recently uncovered that some of the favorite methods the epigenetics community has been using to study 5-hmC’s popular relative, 5 methylcytosine, might not be able to differentiate between the two marks.
5-hmC and 5-mC are Doppelgangers
Like twins’ parents, only a few discerning enzymes can tell these two methylation marks apart which is complicating analysis. According to both papers, 5-hmC and 5-mC are virtually indistinguishable by DNAm weapons of choice (e.g. bisulfite-based apps.). Here is some of what they found:
- Enzymatic digestion approaches can’t tell 5-hmC from 5-mC.
- Bisulfite treatment of 5-hmC turns it into the intermediate, cytosine 5-methylenesulfonate (CMS), which resists deamination, protecting it from conversion to uracil, and making it appear like 5-mC in downtream analysis.
- This CMS branch also stalls Taq Polymerase during PCR somehow, especially when two or more 5-hmC marks are close together. This could prevent you from detecting some methylated loci where 5-hmC is also present.
Hydroxymethylcytosine and You
Why should you care about 5-hmC anyways? After all, it’s only been tied to a couple of cell types so far. Fair question. There are a few ways to look at it.
Glass half empty: The DNA methylation data that you worked so hard to generate might not be as accurate as you think, especially if you were studying ES cells or Purkinje cells where there’s a good chance that at least some of the methyl marks you found are actually hydroxymethyl.
Glass half full: 5-hmC might just be the reason your methylation data didn’t quite add up during your last set of experiments. Maybe it’s your next big opportunity knocking!
Either way, we’re betting that 5-hmC will be too tempting to ignore for most folks in the field which brings us to how these two marks are analyzed today.
Approaches for 5-hmC Analysis
Although 5-hmC might be able to pull a fast one on a few methods, there are a few reagents out there that appear to be specific for one mark or the other.
5-mC Specific Reagents: MeCP2 and 5meC antibody appear to be specific for 5-mC and can be accessed easily.
5-hmC Specific Reagents: Here there are few options. You can wait for reagents to hit the market (we hear there is a 5-hmC antibody from Diagenode coming out soon). Or you can score major points in the community by developing a new reagent that enables single nucleotide 5-hmC profiling. Any takers? We hope so.
The bottom line is that right now no one really knows how 5-hmC works, or how important it is. But, there’s only one way to find out. We’ll be following closely. Check out all of these pubs for the most recent analysis of 5-hmC at BioTechniques, April 2010 and PLoS ONE, January 2010.