Epigenome information in mammalian brain cells reflects their developmental history, neuronal activity, and environmental exposures.Studying the epigenetic modifications present in neuronal cells is critical to a more complete understanding of the role of the genome in brain functions.
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Non-neuronal nuclei showed indistinguishable DNA methylation patterns from bulk cortex and higher methylation of synaptic transmission-related genes compared with neuronal nuclei.
We also found higher variation in DNA methylation in neuronal nuclei, suggesting that neuronal cells have more potential ability to change their epigenetic status in response to developmental and environmental conditions compared with non-neuronal cells in the central nervous system. In addition, epigenetic modifications are affected by environmental conditions, including chemical, nutritional, and social factors (Meaney and Szyf 2005; Maze et al. Unraveling the epigenetic status of neuronal cells would, therefore, be critical to understand the molecular basis of brain functions.
In mammals, DNA methylation primarily occurs at the fifth position of the cytosine residue in Cp G dinucleotides. Mutations in lead to Rett syndrome (Chahrour and Zoghbi 2007), and those in the imprinted genes are also implicated in some mental disorders (Wilkinson et al. Accumulating evidence suggests that DNA methylation also has an important role in the function of adult post-mitotic neurons and behavior. Blocking DNA methyltransferase activity results in the loss of long-term potentiation (Levenson et al. At the behavioral level, fear conditioning induces changes in DNA methylation (Miller and Sweatt 2007), and increased nurturing behavior of rat mothers alters DNA methylation in the promoter of in the hippocampus of offspring (Weaver et al. Taken together, the epigenome information in mammalian brain cells reflects their developmental history and activity (Borrelli et al. However, despite its importance, epigenetic profiling in the brain, especially in the human brain, has been sparsely explored.
Aberration of DNA methylation is associated with various diseases, as clearly seen in tumorigenesis (Bird 2002; Feinberg 2007). DNA methylation status varies globally or locally across different brain regions (Ladd-Acosta et al. 2006) and a decrease in the frequency of miniature excitatory synaptic current (Nelson et al. In addition, membrane depolarization can induce changes in DNA methylation in the promoter region of the (Chen et al. Many challenges remain, including the heterogeneity of cell types, such as neurons and glia.
DNA methylation also plays an important role in the development and cellular function within the brain. Here we performed comprehensive DNA methylation analysis in neuronal and non-neuronal nuclei obtained from the human prefrontal cortex by using a cell sorter-based separation method.
For example, changes in DNA methylation are associated with neuronal and glial differentiation from neural stem cells (Takizawa et al. The neural stem cells of mice lacking have reduced neuronal differentiation (Zhao et al. Aberrations of DNA methylation are also involved in human neurologic and psychiatric diseases (Iwamoto et al. Using the extracted DNA from separated fractions, we quantified both global and site-specific DNA methylation and examined genome-wide promoter methylation status.
We separated fresh-frozen prefrontal cortex cells into neuronal and non-neuronal nuclei by fluorescence-activated cell sorting (FACS) based on Neu N, which is a well-known nuclear antigen specific for neurons in mammals (Mullen et al. We considered Neu N-positive (Neu N+) and -negative (Neu N−) fractions as neuronal and non-neuronal nuclei, respectively (Fig. We typically obtained We conducted several lines of methylation experiments using genomic DNA extracted from various sources (Supplemental Table 1).
Because of the limitation of the specimen and the aim of each assay, not all assays were performed for all material (Supplemental Table 2). We found significantly lower DNA methylation levels in Neu N+ samples compared with bulk cortical samples () Hpa II/Msp I ratio of the bulk and sorted brain samples.
We measured global DNA methylation levels using the luminometric methylation assay (LUMA) method (Fig. Theoretically, when all CCGG sites are not methylated, the ratio of Hpa II/Msp I is close to 1, whereas the ratio is expected to be close to 0 when all sites are methylated.