5hmC was originally discovered way back in the 50’s in bacteriophages and was first observed in mammals in the 70’s, but research into this was set back when these studies failed to replicate. And going back to the future, in 2009 two independent research groups, publishing on the same pages in science, found that this “unknown molecule” was abundant in brains and present in embryonic stem cells.

When it comes to location and function it really seems that 5hmC is a mark of the brain and development, and while originally disregarded in the groovy 70’s as an oxidative stress by-product due to some “questionable” experimentation it was quickly shown to be a true functional mark related to chromatin regulation and function.

Researchers found that 5hmC wasn’t only present in conditions of oxidative stress but was rather part of active DNA demethylation that uses oxidation in its enzymatic reactions and is DNA replication independent. Since the historic 6th base was brought into the world of epigenetics a lot has been done to characterize 5hmC and determine its function, including uncovering a role in cancer.

Meet the TET Family

The TET enzyme family actively causes DNA demethylation by oxidizing 5mC and creating 5hmC, along with intermediates 5fC and 5caC, which may represent epigenetic marks of their own with a role for priming the epigenome.

TET has been caught demethylating DNA via x-ray crystallography and the enzymes split their DNA demethylation labour across the protein family, with differing dynamics, just like the DNMTs. But the craziness doesn’t end there, since just like methylation isn’t loyal to only RNA, it seems that demethylation follows along too, as the TETs also like to play the field and catalyze reactions on Thymine.

This fundamental process creates genome-wide 5hmC profiles that are essential to a cell’s function as a differentiated individual and resets genomic imprinting across generations. Now researchers show that by examining the 5hmC profiles of distinct cancer mutants, you can find some intimate molecular associations.

Assaying 5hmC

Bisulfite conversion has always been a solid choice for assaying DNA methylation, but 5hmC creates some interesting challenges for assaying DNA hydroxymethylation. The reason behind this is that conventional bisulphite sequencing doesn’t discriminate between 5mC and 5hmC, so its read out is actually a mix of the two signals.

Two techniques have risen to the challenge to overcome this by using an additional reaction to read the true levels of one mark and infer the other, by comparing it to the total signal:

Oxidative Bisulfite Sequencing (OxBS-seq) uses an additional oxidation to discriminate between the two marks. TET-assisted Bisulfite Sequencing (TAB-seq) takes advantage of the TET enzymes for discrimination.

It doesn’t just have to be sequencing, as the 450K array has been hacked to utilize OxBS as well. These technologies have allowed for characterization of genomic 5hmC profiles and their key players via some clever experimental designs.

TET’s Protein Posse

The epigenome never likes to act alone, and TET is no exception, with researchers uncovering a number of binding partners. One active group has shown that mutations causing epigenomic dysregulation, like those in IDH1/IDH2, can induce cancer on their own. In their previous study the team found that mutations in IDH1 and IDH2 impair TET2’s ability to convert 5mc to 5hmC.

In their most recent report, by assaying 5hmC and 5mC in the bone marrow of acute myeloid leukemia (AML) subtypes they found that:

In a number of mutants there is a similar pattern of altered genome-wide and locus-specific 5hmC, with these mutations being related to the hydroxymethylation pathway.

WT1 mutations negatively correlate with TET2/IDH1/IDH2 mutations in AML, leading the group to hypothesize that WT1 impacts TET2 function.

AML patients with a WT1 mutation have reduced levels of 5hmC, mirroring what happens in AML patients with mutations in TET2/IDH1/IDH2.

Differential 5hmC is more reflective of altered gene expression than differential 5mC in the mutant cells.

Taking it in vitro, they found that: Changing WT1 expression levels changes global 5hmC levels. WT1 physically interacts with TET2 and TET3. WT1 loss of function mutants show a similar differentiation phenotype to TET2 mutants.



Author Altuna Akalin concludes that “WT1-mutant AMLs have similar hydroxymethylation alterations to IDH1/2 and TET2 mutants which are shown to be mutated not only in AMLs but multiple other cancers such as gliomas and melanomas. In addition to that, [this] paper shows that WT1 interacts with TET2. All these indicate WT1 mutation as a possible new addition to class of mutations causing epigenetic dysregulation in AML and possibly other cancers.”

Learn more about TET’s interactions in Cell Reports, January 2015