Epigenetics and Fear Development

Epigenetics, the study of heritable DNA modifications and their impact on the regulation of gene expression, has been a field of scientific inquiry for nearly 80 years. In that time, however, the most commonly studied DNA modification has been DNA methylation, with other modifications known but infrequently studied. In a recent study by Timothy Bredy’s group from The University of Queensland, the researchers investigated the role of epigenetic modifications in the brain and their role in the regulation of fear.

Specifically, the researchers investigated N6-methyl-2’-deoxyadenosine (m6dA), or the methylation of adenosine at the nitrogen-6 position of the base. Previously, this modification had only been observed in prokaryotes and single-celled eukaryotes. Only recently was it shown to be present in multicellular eukaryotes, and it has been observed across the entire lifespan from the embryonic stage to adult animals. In this study, the researchers studied the expression pattern of m6dA in response to fear. 

First, the researchers isolated neurons from mouse embryos using the Arc Polyclonal Antibody and NeuN antibodies from Bioss, and used an enzyme that specifically cuts at methylated adenosines to determine whether the m6dA modification was present in these cells. Indeed, m6dA was observed in embryonic mouse neurons. In vitro depolarization of these neurons led to an increase of m6dA, indicating that it accumulates in response to activation.

The researchers next wanted to study a specific population of neurons present in a region of the brain known as the infralimbic prefrontal cortex that are responsible for the formation of an extinction response. Extinction response describes the phenomena of a gradual decrease in response to a stimulus that is repeated over time. In this study, the stimulus was a trigger of fear. In neurons in the infralimbic prefrontal cortex, isolated using the Bioss anti-Arc antibody, m6dA was increased at over 300,000 sites unique to this stimulus that were not observed in control mice or in repeat elements of the genome. Classification of these genes revealed that most are associated with the synapse/cell junction, dendrites, and postsynaptic membrane. In individual neurons, an accumulation of m6dA was correlated with an increase in expression of these genes.

N6amt1 is a methyltransferase responsible for the N6-methyl-2’-deoxyadenosine modification in prokaryotes. In this paper, the researchers showed that its expression increases in response to fear, and is the primary protein responsible for the m6dA modification in the neurons of the infralimbic prefrontal cortex. 72% of the genes that have the m6dA modification in these cells also have an accumulation of N6amt1.

Another gene frequently associated with the extinction response is Bdnf. The researchers next sought to determine whether m6dA is involved in Bdnf activity during this process, and found that the m6dA modification does accumulate at a particular site in the promoter region of the Bdnf gene. This modification led to the subsequent recruitment of transcriptional machinery to this region of the genome. Other genes also previously identified to be a part of the extinction response were transcriptionally activated following m6dA modification.

To show the functional control of m6dA in maintaining the extinction response to fear, the researchers finally used a system in which N6amt1 was knocked out in the infralimbic prefrontal cortex using shRNA. When N6amt1 was knocked out during the initial fear conditioning, these mice had an impaired extinction response. This was concurrent with an absence of transcriptional machinery binding to Bdnf and other related loci. This shows the importance of the m6dA modification, which is regulated by N6amt1, in the formation of the extinction response.

REFERENCE:

Li X. et al., "The DNA modification N6-methyl-2’-deoxyadenosine (m6dA) drives activity-induced gene expression and is required for fear extinction." Nature Neuroscience volume 22, pages534–544 (2019) https://www.nature.com/articles/s41593-019-0339-x