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This means there is a precedent for removing DNA methylation in non-dividing cells. Perhaps there’s a similar mechanism in neurons. There’s still a lot of debate about how DNA methylation is actively removed, even in the well-established events in early development. There’s even less consensus about how it takes place in neurons. One of the reasons this has been so hard to investigate is that active DNA demethylation may involve a lot of different proteins, carrying out a number of steps one after another. This makes it very difficult to recreate the process in a lab, which is the gold standard for these kinds of investigations.

Silencing the silencer

As we’ve seen repeatedly, scientific research often throws up some very unexpected findings and so it happened here. While many people in epigenetics were looking for an enzyme that removed DNA methylation, one group discovered enzymes that added something extra to methylated DNA. This is shown in Figure 12.3. Very surprisingly, this has turned out to have many of the same consequences as demethylating the nucleic acid.

^{Figure 12.3 Conversion of 5-methylcytosine to 5-hydroxymethylcytosine. C: carbon; H: hydrogen; N: nitrogen; O: oxygen. For simplicity, some carbon atoms have not been explicitly shown, but are present where there is a junction of two lines.}

A small molecule called hydroxyl, consisting of one oxygen atom and one hydrogen atom, is added to the methyl group, to create 5-hydroxymethylcytosine. This reaction is carried out by enzymes called TET1, TET2 or TET3[223].

This is highly relevant to the question of DNA demethylation, because it’s the effects of DNA methylation that make this change important. Methylation of cytosine affects gene expression because methylated cytosine binds certain proteins, such as MeCP2. MeCP2 acts with other proteins to repress gene expression and to recruit other repressive modifications like histone deacetylation. When an enzyme such as TET1 adds the hydroxyl group to the methylcytosine to form the 5-hydroxymethylcytosine molecule, it changes the shape of the epigenetic modification. If a methylated cytosine is like a grape on a tennis ball, the 5-hydroxymethylcytosine is like a bean stuck to a grape stuck to a tennis ball. Because of this change in shape, the MeCP2 protein can’t bind to the modified DNA any more. The cell therefore ‘reads’ 5-hydroxymethylcytosine in the same way as it reads unmethylated DNA.

Many of the techniques used until very recently looked for the presence of DNA methylation. They often couldn’t distinguish between unmethylated DNA and 5-hydroxymethylated DNA. This means that many of the papers which refer to decreased DNA methylation may actually have been detecting increased 5-hydroxymethylation without knowing it. It’s currently unproven, but it may be that instead of actually demethylating DNA, as reported in some of the behavioural studies, neurons really convert 5-methylcytosine to 5-hydroxymethylcytosine. The techniques for studying 5-hydroxymethylcytosine are still under development but we do know that neurons contain higher levels of this chemical than any other cell type[224].

Remember, remember

Despite these controversies, research is continuing into the importance of epigenetic modifications in brain function. One area that is attracting a lot of attention is the field of memory. Memory is an incredibly complex phenomenon. Both the hippocampus and a region of the brain called the cortex are involved in memory, but in different ways. The hippocampus is mainly involved in consolidating memories, as our brains decide what we are going to remember. The hippocampus is fairly plastic in the way that it operates, and this seems to be associated with transient changes in DNA methylation, again through fairly uncharacterised mechanisms. The cortex is used for longer-term storage of memories. When memories are stored in the cortex, there are prolonged changes in DNA methylation.