DETECTION OF DNA MODIFICATIONS IN THE BRAIN
Since 1925, 5mC was found as a minor base in various genomes (Johnson & Coghill, 1925). However, it was not identified in the brain until 1974, whenVanyushin, Nemirovsky, Klimenko,Vasiliev, & Belozersky (1972), using a thin-layer chromatography method, quantified methylated DNA in neocortex, hippocampus, and cerebellum from adult rats. Using a light conditioned reflex model, they determined a global increase in 5mC in hippocampus and neocortex; thus, this study provided the first evidence that DNA modification is involved in learning and memory processes. Surprisingly, the role of DNA methylation in modulating behavior was not investigated again for almost three decades.
CANDIDATE GENE APPROACH
Weaver et al. (2004) implicated DNA methylation was involved in modulation of adult stress responses by early life experiences. In rats, several behavioral characteristics of good mothering, including arched-back nursing (ABN) and licking and grooming (LG) of the offspring, are displayed with varying frequency by different dams. Offspring of attentive mothers exhibit higher expression of the glucocorticoid receptor (GR) in the hippocampus as adults, and they are better able to cope with stress. In contrast, low levels of maternal attention as measured by ABN and LG behavior are associated with reduced expression of the GR in hippocampus, and reduced ability to cope with stress, in adulthood. By using bisulfite sequencing, Weaver and colleagues mapped the methylation level of individual CpG site within the exon 17 promoter of the GR gene. When they compared results from the two cohorts of offspring, they found that rats with less attentive mothers exhibited higher level of methylation at 15 different CpG sites within the exon 17 promoter, resulting in reduced expression of the GR gene and abnormal behavior. This has become the first definitive evidence that site-specific CpG methylation could arise in response to experience or the environment and could modulate behavior. The same approach was later used to define a cortical DNA methylation pattern at specific genomic loci contributes to maintenance of remote memory in adult rats (Miller et al., 2010; Miller & Sweatt, 2007).
Because of massive reductions in the cost of genome-wide sequencing approaches, after 2010, few studies have moved away from the candidate gene approach in search of higher output, less biased methods to identify differentially methylated genes.