22 March 2007. Silencing DNA by methylation ensures that patterns of gene activity are passed from one cell generation to the next during mitosis. But two recent papers support the idea that DNA methylation in terminally differentiated neurons is linked to synaptic plasticity and the formation of memory. Writing in the March 15 Neuron, Courtney Miller and David Sweatt of the University of Alabama, Birmingham, report that DNA methylase activity is boosted in animals when new memories are being formed and that this leads to silencing of genes that suppress memories. They also report that activation of the gene for reelin, a protein that helps remodel synaptic connections and which has been linked to the pathology of schizophrenia, is increased during memory formation by none other than demethylases—enzymes that remove methyl groups from DNA. The findings suggest that methylation and demethylation play a key role in how memories are formed and stored. That idea is supported by data from Erminio Costa and colleagues at the University of Illinois, Chicago, who found that demethylation of genes for reelin and another protein, the 67 kDa glutamic acid decarboxylase, can be induced in mice by administering small molecules that interfere with the packaging of DNA in the nucleus. That finding is reported in the March 11 PNAS online. These new studies may not only change how we think about memory formation, but they suggest that DNA methylation, once considered permanent, is dynamic in neurons and might be exploited for therapeutic benefit.
Methylation and Memory
The idea that covalent modification of the chromatin structure of DNA is involved in memories is not new. It has been established that acetylation of histone proteins, which form the chromatin scaffold upon which DNA is tightly wrapped, is linked to synaptic signal transduction. Sweatt and colleagues previously showed that activation of NMDA-type glutamate receptors leads to acetylation of histone H3 (Levenson et al., 2004), a modification that weakens the affinity of histones for nucleic acids and allows other proteins, such as those involved in gene activation, to access DNA. In fact, the histone acetyl transferase (HAT) activity of CREB binding protein, a key neuronal transcription factor, has been linked to that protein's effects on memory (see SRF related news story), while boosting histone acetylation by inhibiting histone deacetylases enhances long-term memory as well.
Because histone-linked gene silencing may help regulate memory, Miller and Sweatt wondered if methylation of DNA might have similar effects. Though DNA methylation has generally been viewed as an epigenetic mechanism that preserves patterns of gene activation through mitosis and development, methylase activity is high in the adult mammalian brain even though most of the cells there are non-dividing. And since DNA methylation can silence genes, in part by recruiting histone deacetylases, there is reason to believe that methylases may be involved in regulatory processes in neurons.
To specifically test the role of methylation in memory, Sweatt and colleagues inhibited DNA methyl transferases (DNMTs) in hippocampal slices, and found that this prevents induction of long-term potentiation, the activity-dependent strengthening of synapses that is crucial for learning and memory. They also found that these inhibitors reduced methylation of reelin DNA, an indication that methylation is reversible. These experiments were described last year (see Levenson et al., 2006). Now, Miller and Sweatt advance those observations by looking at methylation patterns in live mice during contextual fear conditioning, a paradigm where animals learn to associate a particular environment with an unpleasant stimulus, such as a mild shock.
The researchers report that levels of mRNA for methylases DNMT3A and DNMT3B, which are believed to be involved in de-novo methylation, are significantly increased in the hippocampus following contextual fear training. Furthermore, mice given DNMT inhibitors seemed to have trouble making memories, because when placed back into the fear context, they froze in place much less frequently than did control animals.
How might DNA methylation affect mouse memories? Any number of methylation-prone DNA regions could be involved, so to narrow things down Miller and Sweatt looked at methylation of genes known to play key roles in memory. First they looked at the memory suppressor protein phosphatase 1 (PP1) on the premise that silencing that gene might boost memory. Indeed, the researchers found that 1 hour after contextual fear training methylation of the PP1 promoter region was increased by over 100-fold and mRNA levels of PP1 in the CA1 region of the hippocampus were slightly, though significantly reduced. For this effect, the animals had to have both the new context and the mild foot shock; alone, neither had any effect on methylation, indicating that a true memory must be formed for PP1 methylation to take place. Interestingly, DNMT inhibitors dramatically increased the fraction of PP1 promoters that were not methylated, again suggesting that demethylation may be just as important for regulation as methylation.
To address the role of demethylation, Miller and Sweatt measured how reelin DNA is altered by the learning paradigm. They found that after 1 hour of contextual fear training, reelin promoter methylation was decreased and reelin mRNA levels increased almost twofold. DNMT inhibitors led to an even greater demethylation of reelin DNA. Though DNA methylation has generally been considered a permanent modification, these results suggest that in neurons, at least, the process may be more dynamic.
Role of Histones
In the second paper, Costa and colleagues describe a slightly different approach to studying reelin demethylation. They studied downregulation of reelin and the 67 kDa glutamic acid decarboxylase (GAD67) in mice treated chronically with the methyl donor methionine. The suppression of the two genes under these conditions is attributed to enhanced methylation, leading to the recruitment of histone deacetylases (HDACs), which in turn increase histone affinity for DNA and prevent it from binding proteins necessary to initiate transcription. First author Erbo Dong and colleagues wondered what might happen if they prevented that histone deacetylation.
After treating mice with methionine for a week, Dong and colleagues then gave the animals HDAC inhibitors and followed the pattern of DNA methylation. The researchers discovered that in the presence of these inhibitors, demethylation of the two genes was accelerated as judged by the reduction in number of promoters that immunoprecipitated with MeCP2, a protein that binds to methylated DNA. The researchers suggest that the rapid demethylation could be due to either inhibition of a methylase or stimulation of putative demethylase activity, but they favor the latter scenario because a DNMT inhibitor had no effect on the rate of demethylation.
All told, these findings point to a dynamic methylation/demethylation process that is linked to synaptic plasticity and memory formation. “An as yet unknown signaling pathway targets the nucleus and activates demethylases and DNMTs. This results in the demethylation of positive regulators of memory, such as reelin. HATs are then free to acetylate demethylated genes, releasing them from the transcriptional silencing induced by methylation. This leads to transcriptional activation of reelin and, likely, other memory-enhancing genes. Simultaneously, DNMTs target negative regulators of memory, such as PP1, for transcriptional silencing,” write Miller and Sweatt.
This new, though poorly understood regulatory mechanism may also yield new clues to various neurologic and psychiatric diseases such as fragile X mental retardation, Rett syndrome, and autism, which have been linked to DNA methylation. It may also lead to new avenues of exploration for schizophrenia, since there is some evidence for increased methylase activity, methylation of the reelin and GAD67 promoters, and loss of the two proteins in the prefrontal cortex in schizophrenia patients (see Guidotti et al., 2000; Grayson et al., 2006). A better understanding of methylation/demethylation dynamics could lead to novel therapeutics, as recently suggested by Costa and colleagues (see Kundakovic et al., 2007) and Jonathan Levenson at the University of Wisconsin (Levenson, 2007).—Tom Fagan.
Miller CA, Sweatt JD. Covalent modification of DNA regulates memory formation. Neuron. 2007, March 15;53:857-869. Abstract
Dong E, Guidotti A, Grayson DR, Costa E. Histone hyperacetylation induces demethylation of reelin and 67-kDa glutamic acid decarboxylase promoters. PNAS. 2007, March 13;104:4676-468. Abstract