It’s hard to tell some things apart—for instance, with her new haircut, Miley Cyrus could almost be mistaken for Pink. Well, almost. Anyhow, if you’re trying to tell 5-methylcytosines (5-mCs) from 5-hydroxymethylcytosines (5-hmCs) from each other, you’re in luck because there’s a new method for that—one that’s inexpensive and selective.
Sure, some methods for doing this on a genome-wide basis already exist, but they’re low-resolution, biased, or require boat-loads of pricey sequencing. That’s why a team from China decided to develop their own method.
Called “hydroxymethylation and methylation sensitive tag sequencing” (HMST-Seq), the new approach is cost-effective and high-res. Here are the basics:
- HpaII only cuts at unmodified Cs. MspI cuts at 5-mCs and 5-hmCs.
- β-glucosyltransferase can add a glucose to 5-hmC, producing “5-ghmC,” which MspI can’t cut.
- Analyzing three genomic libraries consisting of a regular MspI digestion, an MspI digestion of β-glucosyltransferase-treated DNA, and a regular digestion with HpaII can then tell you where the various cytosines are hiding out.
Differential 5hmC Action in Bivalent Domains
They then used this method on human embryonic stem cells and their differentiated embryoid bodies. They found lots of 5-hmC but low levels of 5-mC at transcription start sites and distal regulatory elements. Interestingly, the team saw that differential hydroxymethylation happens mostly at bivalent, primed genes with both activating and repressing histone modifications during differentiation.
“Our results support that hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decisions on choosing active or inactive states of these genes upon cellular differentiation,” they say.
Read all the details at Epigenetics, April 2013.