Epigenetics and The Brain — 5-hydroxy-methocytosines


Detecting 5-hmCs

5-hydroxymethylcytosines (5-hmC) went undiscovered because they showed up like other 5-methoxycytosines through bisulfide sequencing. The neuroscientists behind a new study in Nature Neuroscience profiles 5-hmC across development using T4 bacteriophage B-glucosyltransferase to transfer an engineered glucose-azide moiety onto the the hydroxyl of 5-hmC. This moiety was then detected and used to map 5-hmCs across the genome of mouse cerebellar and hippocampal cells.

5-hmCs and the brain

5-hmCs are found overall in the brain at approximately 10x the levels found in stem cells, though it is variable between cell type and varies by age. Interestingly, the 5-hmc seems to be depleted from the X-chromosome of both males and females. Also, 5-hmC was found at lower levels in immature neurons of young and old mice, suggesting it plays a role in neuronal development.

5-hmCs and MECP2

This new study also shows that the bases differ in function from 5-methylcytosines (5-mCs) in that they are not bound by methyl CpG binding protein 2 (MECP2). Since MECP2 is mutated in the Rett syndrome, a severe neurodevelopmental disorder that results in seizures, usually complete verbal impairment, and mental retardation, this indicates that the accumulation of 5-hmC could have significant effects on brain function. Conversely, the authors wanted to see what affect MECP2 has on 5-hmC levels.  They found that MECP2 KOs showed a ~20% increase in 5-hmC and that MECP2-overexpressing animals showed a ~25% decrease in 5-hmC. They hypothesized that MECP2 binding to 5-mC may block the bases from being oxidized by TETs. They supported this hypothesis by adding the TET1 catalytic domain to 5-hmC DNA in vitro with or without the MECP2-methyl binding domain, and showed with a 1:1 ratio of Mecp2-MDB to 5-mC they could block 90% of the hydroxylation.

Future Directions

It is still unknown if proteins that specifically bind these epigenetic marks exist. Recently, polyclonal 5-hydroxymethylcytidine antibodies have been created , that can be used with DIPseq (DNA immunoperciptiation sequencing). I do not know if these enzymes can work with ChIP to isolate fragments enriched in 5-hmCs. I’m interested in the possibility of purifying proteins from ChIP and performing Mass-Spec on them to identify novel DNA-binding proteins. However, I’m not sure how hard it is to analyze mass-spec after the crosslinking and unlinking process.

I’d also be interested in generating TET-overexpressing mice to see if they could speed the process of 5-hydroxy-methylation in aging mice and see if there are behavioral or cellular phenotypes.

Finally it might be of interest to some scientists interested in studying 5-hmC modifications that there are restriction enzymes such as such as PVurts1I that cleave 5-hmC but not 5-mC DNA or unmethylated DNA, but I also have no personal experience with this enzyme.


If you’re interested in the epigenetic effects on neurons and the brain, check out: We know a lot about drugs – new data shows long-term epigenetic changes

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