Improved methodology in studying epigenetic DNA methylation

This 2015 New York human study was of:

“The two major populations of human prefrontal cortex neurons..the excitatory glutamatergic projection neurons and the inhibitory GABAergic interneurons which constitute about 80% and 20% of all cortical neurons, respectively.

Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG).

A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons.

Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation.”

The researchers performed numerous cross checks to test the results of their methodologies. This was necessary because, for example, studies such as A human study of changes in gene expression point out that current technologies such as the 450K array:

“Queries only 1.6% of all CpGs in the genome and the CpG selection is biased towards CpG islands.”

From the Discussion section:

“The higher abundance of hmCpG sites in GLU versus GABA neurons appears indicative of a difference in transcriptional potential between the neuronal subtypes. The increased hydroxymethylation could enable certain genes (e.g. activity-dependent genes) to be more readily induced in GLU versus GABA neurons.

These findings emphasize the importance of even subtle differences in the promoter CpG methylation for neuron subtype-specific gene expression. They also suggest that differences in CpG methylation within gene bodies and distal regulatory elements are not always directly reflected in differences in gene expression between neuronal subtypes.

The functional relevance of the association between gene expression and distal non-CpG methylation remains to be characterized.

Our data suggest that, compared to GABA interneurons, GLU projection neurons are characterized by more permissive chromatin state that is less constrained by repressive DNA methylation marks and is instead controlled by more dynamic means of transcription inhibition, such as non-coding RNAs and/or histone modifications.”

This study was similar to A problematic study of DNA methylation in frontal cortex development and schizophrenia in examining:

“If common risk variants determined by the recent genome wide associated studies (GWAS) for several neuropsychiatric diseases including schizophrenia (SCZ), autism spectrum disorder (ASD), major depressive disorder (MDD), and Alzheimer’s disease (AD) significantly overlap.

These findings strongly suggest an association between the epigenetic specification of both GABA and GLU neurons and SCZ. Risk variants associates with ASD, MD, or AD were not enriched.

An alternative explanation of our negative results could be the involvement of different developmental stages and/or brain regions in different diseases.”

The current study performed more detailed analyses, but on fewer subjects. The emphasis was on demonstrating an improved methodology.

Both studies’ findings regarding disease were of effects, not causes. That both study designs were limited to the postmortem prefrontal cortex reminded me of the old joke about looking for lost keys under the street light because the light was better there. At least the current study acknowledged the existence of other areas to search.

http://nar.oxfordjournals.org/content/early/2015/11/25/nar.gkv1304.full “Substantial DNA methylation differences between two major neuronal subtypes in human brain”

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