Miller, C. A. and Sweatt, J. D. (2007). Covalent modification of DNA regulates memory formation. Neuron, 53:857–869. DOI 10.1016/j.neuron.2007.02.022.
The article by Miller and Sweatt (2007) examines the possible role of DNA methylation as an epigenetic mechanism in the regulation of memory in the adult central nervous system. The authors sought to know whether memory regulation in this case occurs through control of transcription in a gene-specific manner in the hippocampus. The specific methylation process under examination was the cytosine-5’ methylation which is a covalent modification aided by the enzyme DNA (cytosine-5’) methyltransferases (DNMTs). The study utilized adult male Sprague-Dawley rats as animal models and the rats were placed under contextual fear conditioning. Cannula implantation and administration of DNMT inhibitors were done on the animals while real time RT-PCR and DNA methylation assays were used to quantify RNA as a way of assessing gene transcription. Several statistical tests were used including one-sample t test, one-way ANOVA and Tukey-Kramer test as a post-hoc test with all tests being performed at a significance level of .05.
The study revealed that for memory to be built, the activity of enzyme DNA methyltransferase is necessary. This conclusion was arrived by infusing some animals with 5-AZA, a DNMT inhibitor while the comparison group animals were vehicle-treated whereby DNMT-inhibited animals had less freezing compared to the vehicle-treated animals. It was evident that memory consolidation is blocked by DNMT inhibition in a plastic manner as evidenced by ability of formation of fear in the test animals later in the experimentation. From this study, Miller and Sweatt (2007) were able to identify that contextual fear conditioning leads to an increase in DNA methylation of a memory suppressor gene and this process is enhanced as training progresses. It was observed that silencing of a memory suppressor gene was prevented by DNMT inhibition since increase in gene methylation that would lead to fear conditionings are inhibited.
Another finding from this study was that the process of a memory promoting gene transcription is controlled by rapid demethylation. This was determined using reelin gene since it enhances LTP induction. Further, the study revealed that continued demethylation of reelin is brought about by DNMT inhibition in fear conditioning situation. The c-fos as well as DNMT1 confirmed gene-specificity of DNA methylation. The c-fos was identified as a good example of demonstrating interactions that occur in the hippocampus during DNA methylation thus adding to reelin. Finally, Miller and Sweatt (2007) confirmed that the changes in the process of DNA methylation occurring in the hippocampus are usually highly dynamic. It was confirmed that the adult CNS experiences changes in DNA methylation but the changes are not permanent and instead they can be both reversible as well as dynamic. This was confirmed by looking at the levels of methylation of reelin and PP1 24 hours post-training followed with euthanizing of the animals after 24 hours where the levels of both genes went back to baseline.
Koshibu, K., Graff, J., Beullens, M., Heitz, F. D. and Berchtold, D. et al. (2009). Protein Phosphatase 1 regulates the histone code for long-term memory. The Journal of Neuroscience, 29(41):13079 –13089.
Graff et al (2009) sought to know the role of PP1 in chromatin remodeling as well as its possible contribution to epigenetic regulation of memory and learning. This investigation was to be accomplished by coming up with transgenic mice which would facilitate selective inhibition of PP1 in the nucleus of forebrain neurons. The authors of this study worked on the hypothesis that histone posttranslational modification (PTMs) in neurons is altered by PP1 inhibition in nucleus of forebrain thus impacting on gene transcription thus altering long-term memory.
The authors prepared transgenic mice (NIPPI*-EGFP double transgenic mice). Control mice did not have the fragment contained in the transgenic mice. The researchers then conducted several procedures including: reverse-transcription polymerase chain reaction (RT-PCR), immunohistochemistry, protein phosphate assay, coimmunoprecipitation, in vitro phospholylation assay, HDAC activity assay, western blotting, quantitative RT-PCR, preparing lentivirus and injecting the same in hippocampal slices, and chromatin immunoprecipitation. The behavior of the mice was assessed by testing for novel object recognition for five minutes in five sessions followed by memory testing where the mice was introduced to a novel object in addition to two already familiar objects. Discrimination ration was then calculated as well as molecular analysis of the killed mice. Several statistical analyses were conducted including analysis of variance (ANOVA) with appropriate post hoc tests such as Tukey’s test, and 2-tailed paired t-test. Statistical significance was determined at .05, .01 and .001 levels.
It was identified that NPP1* was dominantly expressed mainly in the cortex and hippocampal formation but it was marginally present in the amygdala. Non-forebrain structures lacked the nuclear inhibitor NPP1*. In addition, NPP1* was only present in nucleus of neurons thus it only inhibition of PP1 was limited to the nucleus. Graff et al (2009) identified that nuclear PP1 forms an important component of the brain’s epigenetic machinery since inhibition of PP1 interfered with association of PP1 and histone-modifying enzymes. Western blot analyses identified that multiple histone posttranslaton modifications are altered by nuclear PP1 inhibition. The authors of this study further noted (by aid of ChIP assays) that inhibiting nuclear PP1 leads to altered gene expression (transcription to be specific) and this leads to altered memory formation since the genes responsible for memory formation are affected. In addition, the study revealed that all long-term memories depending on hippocampal structures as well as cortical structures are enhanced by nuclear PP1 inhibition. Lastly, Graff et al (2009) revealed that long-term memory induces specific histone PTMs including H4K5 acetylation and H3K36 trimethylation, all of which enhance CREB expression. Overall, the study shows that PP1 that is pooled in the nucleus of neurons in adults is crucial in epigenetic regulation of memory formation.
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