One of the great things about science is that whenever a new problem presents itself, there are always some members of the community who set out to find innovative solutions. In this case, it’s been a bear to detect and quantify 5-hydroxymethylcytosine (5-hmC) but some cagey researchers at the Ludwig Maximilians University (LMU) in Munich, Germany, have established a simple and accurate method to quantify global levels of genomic 5-hmC.
5-methylcytosine (5-mC) is considered the “fifth base” and a predominant epigenetic mark in eukaryotic genomes. Recent work from the labs of Anjana Rao and Yi Zhang showed that 5-mC can be further modified to 5-hmC by a family of TET hydroxylases. Cumulative evidence supports a role for 5-hmC as an intermediate of DNA demethylation, earning it the nickname “the sixth base”, but at present its functional relevance in mammalian genomes is not well understood mainly due to the lack of methods that discriminate it from the more abundant 5-mC.
The new assay by the LMU researchers is based on an enzyme from bacteriophage T4 that specifically transfers glucose to the hydroxymethyl group of 5-hmC (all cytosines of this phage are modified to glucosylated 5-hmC). By transferring tritiated glucose, genomic 5-hmC can be specifically and quantitatively labeled. “Our preliminary tests hold promise for substantial improvement on the sensitivity of the assay. We envisage that chemical derivatization of the glucose with small reactive groups may allow indirect labeling of 5-hmC with popular haptens such as biotin. This would allow not only non-radioactive versions of our glucosylation assay, but also very selective enrichment of 5-hmC containing DNA by standard hapten affinity purification” said Fabio Spada, co-leader of the work with Heinrich Leonhardt.
Using their method the LMU team determined 5-hmC levels in various adult mouse tissues and differentiating embryonic stem cells (ESCs). They showed that, after initial reduction, 5-hmC levels pick up again during differentiation of ESCs as embryoid bodies (EBs). At the same time they observed a switch in the relative transcript levels for Tet1 and Tet2/3, the enzymes that catalyze the 5-mC to 5-hmC conversion. While Tet1 transcripts are preponderant in undifferentiated ESCs, Tet2 and especially Tet3 mRNA dominates in late EBs, a situation similar to that found in most adult tissues. Thus, the switch in tet gene expression seems to be an early event during specification of somatic cell lineages.
Learn more about the the LMU assay and how to use it at Nucleic Acids Research, August 2010.
Thanks to Fabio Spada for contributing this Method. Next round’s on us Fabio!