DNA Hydroxymethylation and Neuron Development
It has been well known for many years that DNA methylation plays many roles in gene regulation and can direct different cell pathways such as apoptosis, differentiation, cancer, and aging. Although DNA methylation can be extremely stable – able to persist through multiple generations – it can also be quite dynamic and able to change in response to a cell’s internal and external environment. More recently, an additional DNA modification, hydroxymethylation, has received much attention. Methylcytosine (5mC) can be oxidized to generate hydroxymethylcytosine (5hmC), which some researchers think is an intermediate in the DNA de-methylation pathway. Unlike 5mC, which is found in approximately equal levels throughout all tissues in the body, 5hmC is most abundantly expressed in the brain and CNS compared to other tissues in the body. This has led many to speculate that 5hmC is more than just a passive mark, and may have its own unique biological functions.
To investigate 5hmC’s role in the brain, Hahn et al. looked at changes in global levels of this modification during neurogenesis to see how these changes tracked with 5mC abundance and histone modifications. Immunostaining and LC/MS quantification showed an increase in 5hmC levels during neuronal differentiation. If 5hmC was only a transient intermediate in the de-methylation process, then a corresponding decrease in 5mC levels would be expected to follow an increase in 5hmC. However, the group found that despite the rise in hydroxymethylation, there was no change in global methylation levels between differentiated and undifferentiated cells.
To determine where in the genome 5hmC was accumulating, the authors performed a hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by a tiling array assay. They found that promoter regions and gene bodies were enriched for 5hmC during differentiation. Genes containing increased levels of 5hmC included those important for neuronal differentiation, migration, and axon guidance. These genes also showed increased transcription when profiled for their expression, suggesting that they may be activated by the presence of 5hmC. Targeted bisulfite sequencing of a selected few of these regions showed that 10 of 11 did not have the subsequent demethylation that might be expected! The authors believe that this is evidence that 5hmC is not only an intermediate state, but is a much more stable epigenetic mark than previously believed.
Next, the authors investigated changes in levels of histone modifications, including the repressive H3K27me3 mark, within the intragenic regions in which they found substantial 5hmC changes. They found that accumulation of 5hmC was associated with a loss of the H3K27me3 modification, and conversely, that a gain in H3K27me3 levels was associated with a loss of 5hmC. They suggest that this 5hmC-histone modification relationship is a shared signature of the many genes which become activated during neuronal differentiation.
The role of 5hmC in biology and whether this epigenetic mark has unique and specific functions in the genome remain unresolved. The authors show that 5hmC levels increase with neuronal differentiation without resulting in a decrease of 5mC. Additionally, 5hmC appears to be more stable than previously thought. The authors argue that these results provide evidence that 5hmC may have important and undiscovered roles in gene regulation – perhaps being able to better antagonize H3K27me3 accumulation, which has been shown to inhibit gene transcription, than its 5mC counterpart.
Hahn MA, Qiu R, Wu X, Li AX, Zhang H, Wang J, Jui J, Jin SG, Jiang Y, Pfeifer GP, & Lu Q (2013). Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis. Cell reports, 3 (2), 291-300 PMID: 23403289