While the cellular and molecular mechanisms of learning and memory have long been a central focus of neuroscience, it is only in recent years that attention has turned to the epigenetic mechanisms behind the dynamic changes in gene transcription responsible for memory formation and maintenance. Epigenetic gene regulation often involves the physical marking (chemical modification) of DNA or associated proteins to cause or allow long-lasting changes in gene activity. Epigenetic mechanisms such as DNA methylation and histone modifications (methylation, acetylation, and deacetylation) have been shown to play an important role in learning and memory.
DNA methylation DNA methylation involves the addition of a methyl group to a 5' cytosine residue. This usually occurs at cytosines that form part of a cytosine-guanine dinucleotide (CpG sites). Methylation can lead to activation or repression of gene transcription and is mediated through the activity of DNA methyltransferases (DNMTs). DNMT3A and DNMT3B regulate de novo methylation of CpG sites, while DNMT1 maintains established methylation patterns. S-adenosyl methionine acts as the methyl donor.
One current hypothesis for how DNA methylation contributes to the storage of memories is that dynamic DNA methylation changes occur temporally to activate transcription of genes that encode for proteins whose role is to stabilize memory. Another hypothesis is that changes in DNA methylation that occur even early in life can persist through adulthood, affecting how genes are able to be activated in response to different environmental cues.
The first demonstration about the role of epigenetics in learning in memory was the landmark work of Szyf and Meaney (PMID 15220929) where they showed that licking and grooming by mother rats (maternal care) prevented methylation of the glucocorticoid receptor gene. When these pups become adults, they respond better to stressors than rats who, as pups, were not licked and groomed by their mothers and instead had a buildup of methylation in the glucocorticoid receptor gene.
DNMTs and memory Miller and Sweatt demonstrated that rats trained in a contextual fear conditioning paradigm had elevated levels of mRNA for DNMT3a and DNMT3b in the hippocampus. Fear conditioning is an associative memory task where a context, like a room, is paired with an aversive stimulus, like a foot shock; animals who have learned the association show higher levels of freezing behavior when exposed to the context even in the absence of the aversive stimulation. However, when rats were treated with the DNMT inhibitors zebularine or 5-aza-2′-deoxycytidine immediately after fear-conditioning, they demonstrated reduced learning (freezing behavior). When treated rats were re-trained 24 hours later, they performed as well as non-treated rats. Furthermore, it was shown that when these DNMT inhibitors were given 6 hours after training, and the rats were tested 24 hours later, the rats displayed normal fear memory, indicating that DNMTs are involved specifically in memory consolidation. These findings reveal the importance of dynamic changes in methylation status in memory formation.
Feng et al. created double conditional knock out (DKO) mice for the genes DNMT3a and DNMT1. These mice were shown to have significantly weakened long-term potentiation (LTP) and much more easily stimulated long-term depression (LTD) in the hippocampus. When tested in the Morris water navigation task, which is used to study hippocampus-dependent spatial memory, the DNMT3a/DNMT1 DKO mice took longer to find the platform than control mice. Single knock-out mice (SKO) for either DNMT3a or DNMT1 performed normally. DKO mice were also unable to consolidate memory after fear-conditioning. Since SKO mice did not exhibit the same learning and memory defects as the DKO mice, it was concluded that DNMT3a and DNMT1 play redundant roles in regulating learning and memory.
When DNMTs are inhibited in the prefrontal cortex, recall of existing memories is impaired, but not the formation of new ones. This indicates that DNA methylation may be circuit-specific when it comes to regulating the formation and maintenance of memories.
DNA methylation targets The memory suppressor gene, protein phosphatase 1 (PP1), was shown to have increased CpG island methylation after contextual-fear conditioning. This corresponded to decreased levels of PP1 mRNA in the hippocampus of the trained rats. When DNMTs were inhibited, increased methylation at the PP1 gene was no longer observed. These data suggest that during memory consolidation in associative learning tasks, CpG methylation is used to inhibit the expression of PP1, a gene that negatively inhibits memory formation.
Demethylation and memory While DNA methylation is necessary to inhibit genes involved in memory suppression, DNA demethylation is important in activating genes whose expression is positively correlated with memory formation. Sweatt and Miller also showed that the gene reelin, which is involved in long term potentiation induction, had a reduced methylation profile and increased reelin mRNA in fear-conditioned versus control rats. Brain-derived neurotrophic factor (BDNF), another important gene in neural plasticity, has also been shown to have reduced methylation and increased transcription in animals that have undergone learning. While these studies have been linked to the hippocampus, recent evidence has also shown increased demethylation of reelin and BDNF in the medial prefrontal cortex (mPFC), an area involved in cognition and emotion.
The mechanism behind this experience-dependent demethylation response was previously not fully understood, with some evidence showing that DNMTs may be involved in demethylation. It was also suggested that members of the DNA damage repair GADD45 family may contribute to this demethylation process. However, more recently, the pathways illustrated in the Figure below, titled "Demethylation of 5-Methylcytosine (5mC) in neuron DNA," especially the TET dependent pathway, have been confirmed as pathways of DNA demethylation. A role for GADD45 has also recently been indicated, since GADD45 physically interacts with thymine-DNA glycosylase (TDG) and GADD45 may promote the activity of TDG in its role(s) during conversion of 5mC to cytosine.
Methyl-binding domain proteins (MBDs) Mice that have genetic disruptions for CpG binding protein 2 (MeCP2) have been shown to have significant problems in hippocampus#Role in memory-dependent memory and have impaired hippocampal LTP.
Methylation and learning and memory disorders Changes in expression of genes associated with post-traumatic stress disorder (PTSD), which is characterized by an impaired extinction of traumatic memory, may be mediated by DNA methylation. In people with schizophrenia, it has been shown that reelin is down-regulated through increased DNA methylation at promoter regions in GABAergic interneurons. DNMT1 has also been shown to be upregulated in these cells.
Histone methylation Methylation of histones may either increase or decrease gene transcription depending on which histone is modified, the amino acid that is modified, and the number of methyl groups added. In the case of lysine methylation, three types of modifications exist: monomethylated, dimethylated, or trimethylated lysines. The di- or trimethylation of histone H3 at lysine 9 (H3K9) has been associated with transcriptionally silent regions, while the di- or trimethylation of histone H3 at lysine 4 (H3K4) is associated with transcriptionally active genes.
Histone 3 lysine 4 trimethylation and memory formation The hippocampus is an important brain region in memory formation. H3K4 trimethylation is associated with active transcription. In contextual fear conditioning experiments in rats, it was found that levels of H3K4 trimethylation increases in the hippocampus after fear conditioning. In these experiments by Gupta et al., a connection was made between changes in histone methylation and active gene expression during the consolidation of associative memories. In this same study, it was also found that these histone methylations were reversible, as the levels of trimethylation of H3K4 returned to basal levels after a period of 24 hours. This indicated that active demethylation was occurring following memory consolidation. To further explore the role of methyltransferases in long-term memory formation, this study applied the same fear conditioning tests on rats deficient in Mll, a H3K4-specific methyltransferase. The rats with a heterozygous mutant Mll+/− gene showed a significant reduction in their ability to form long-term memories compared to normal rats with an intact Mll gene. Therefore, H3K4 methyltransferases, such as Mll, must have an essential role in long-term memory formation in the hippocampus.
The change in methylation state of histones at the location of specific gene promoters, as opposed to just genome-wide, is also involved in memory formation. Zif268 and BDNF genes are critical for memory consolidation. H3K4 trimethylation increases around both of the Zif268 and BDNF promoters following contextual fear conditioning, when these genes are transcriptionally active. This demonstrates that at the time of memory consolidation, the transcription of memory formation genes such as Zif268 and bdnf is regulated by histone methylation.
Histone 3 lysine 9 dimethylation and memory formation Histone H3 lysine 9 dimethylation is associated with transcriptional silencing. The G9a/G9a-like protein (GLP) complex is a methyltransferase specific for producing this modification. One study examined the role of G9a/GLP-mediated transcriptional silencing in the hippocampus and entorhinal cortex (EC) during memory consolidation. It was found that the inhibition of G9a/GLP in the EC, but not in the hippocampus, results in the enhancement of long-term memory formation. In addition, G9a/GLP inhibition in the entorhinal cortex altered histone H3 lysine 9 dimethylation in the Cornu Ammonis area 1 of the hippocampus, suggesting the importance of this complex in mediating connectivity between these two brain regions. Therefore, the G9a/GLP complex plays an important role in histone methylation and long-term memory formation in the hippocampus and the EC.
Histone methylation and other epigenetic modifications Histone methylation marks are also correlated with other epigenetic modifications, such as histone deacetylation and DNA methylation, in the context of learning and memory. Reduced histone deacetylation is correlated with an increase in H3K9 dimethylation, a modification associated with transcriptional silencing. Therefore, histone deacetylase inhibitors may be applied to increase histone acetylation and suppress H3K9 dimethylation, thereby increasing gene transcription. In the case of DNA methylation, it was found that increases in H3K4 trimethylation correlate with altered DNA methylation of CpG sites at the promoter of Zif268, a gene involved in memory formation, after fear conditioning. Gupta et al. showed that DNA methylation at the Zif268 promoter increased after fear conditioning, correlating with an increase in Zif268 gene expression. This finding was surprising, since it was previously thought that DNA methylation resulted in transcriptional silencing.
Histone acetylation Acetylation involves the replacement of a hydrogen with an acetyl group. In a biological context, acetylation is most often associated with the modification of proteins, specifically histones. The acetylation reaction is most often catalyzed by enzymes that contain histone acetyltransferase (HAT) activity.
Histone acetyltransferases (HATs)