Everybody loves a twofer, and now for the amount of time spent reading a single Nature paper ( or at least this EpiGenie summary), you can learn about two new methods for 5hmC analysis. A clever team of researchers has developed two independent approaches for the genome-wide mapping of 5hmC, neither of which relies on 5hmC antibodies:
- GLIB (glucosylation, periodate oxidation, biotinylation) precipitation uses T4 phage -glucosyltransferase to add a glucose molecule to each 5hmC. Then, researchers oxidize the glucose moiety with sodium periodate and add two biotin groups with an aldehyde-reactive probe. The biotin groups allow them to pull down 5hmC-containing DNA with streptavidin and then sequence it. This approach is similar to another recent method that also uses a sugar molecule to hit 5hmC’s sweet spot.
- CMS (cytosine 5-methylenesulfonate) precipitation uses sodium bisulfite to convert 5hmC into CMS. Then, researchers pull down 5hmC-containing DNA with anti-CMS antibodies. In dot blots, these antibodies were more sensitive and less dependent on 5hmC density than anti-5hmC antibodies.
Both techniques isolated 5hmC-containing DNA more efficiently than anti-hmC antiserum, and GLIB was able to precipitate more than 90% of 201-bp DNA fragments containing a single 5hmC.
When the researchers applied their method to genomic DNA from mouse embryonic stem cells, high-throughput sequencing revealed that 5hmC enrichment within exons and near transcriptional start sites. In particular, 5hmC was enriched at promoters bearing dual histone H3K27me3 and H3K4me3 modifications—a signature of “poised” chromatin states found in developmentally regulated genes.
With these promising methods added to the ever-expanding 5hmC analysis toolkit, we’re sure that the function of the 6th base won’t remain a mystery for long.
Check out this great deal for yourself at Nature, May 8, 2011.