Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons - PubMed (original) (raw)
Comparative Study
Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons
Kazuya Iwamoto et al. Genome Res. 2011 May.
Abstract
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. We performed comprehensive DNA methylation analysis in neuronal and non-neuronal nuclei obtained from the human prefrontal cortex. Neuronal nuclei manifest qualitatively and quantitatively distinctive DNA methylation patterns, including relative global hypomethylation, differential enrichment of transcription-factor binding sites, and higher methylation of genes expressed in astrocytes. 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.
Figures
Figure 1.
Separation of neuronal and non-neuronal nuclei by FACS. (A) Typical example of the nuclei sorting based on Alexa Fluoro 488-conjugated anti-NeuN antibody. (B) Microscopic examination of isolated neuronal (NeuN+) and non-neuronal (NeuN−) nuclei. Note that the image did not reflect the yield because of the different levels of dilution.
Figure 2.
Global DNA methylation assay by LUMA. (Top) HpaII/MspI ratio of the standard samples. (Bottom) HpaII/MspI ratio of the bulk and sorted brain samples. Theoretically, when all CCGG sites are not methylated, the ratio of HpaII/MspI is close to 1, whereas the ratio is expected to be close to 0 when all sites are methylated. To test the accuracy of the methods, we used samples containing various amounts of methylated and unmethylated genomic DNA as standard samples. Values are mean ± SD.
Figure 3.
Differential epigenetic signatures between neuronal and non-neuronal nuclei and evolutionary conservation of methylation status. The assay allowed quantification of DNA methylation levels at 1505 individual CpG sites chosen from 807 genes (Bibikova et al. 2006). (A) Scatter-plot of average methylation levels of the 1505 CpG sites. (B) Statistical comparison of correlation. Values are mean ± SD. (C) Hierarchical clustering based on the 833 CpG sites that showed an identical sequence with regard to the probe region between human and chimpanzee.
Figure 4.
Promoter tiling array analysis. (A) Example of results of the methylation status of the imprinted gene, MEG3, and (B) commonly (DRD5, dopamine receptor 5) or differentially (NRGN, neurogranin) methylated genes. Exon structure of the MEG3 is illustrated in a collapsed manner. Individual MRs detected by the MAT analysis are shown in pink (neuronal) or light blue (non-neuronal) bars. Common MRs are shown in dark green. Supplemental Figure 5 shows the methylation status of other imprinted genes. (C) The results of bisulfite sequencing of DRD5 and NRGN. Our bisulfite sequencing analysis excluded the possibility that tiling array signals are derived from DRD5 pseudogenes. In addition, their methylation levels were separately analyzed (data not shown).
Figure 5.
Ontology and expression analysis of the genes associated with DNA methylation. (A) Venn diagrams of the RefSeq genes associated with MRs. Supplemental Tables 7–9 list the genes. (B) PANTHER ontology analysis of the genes associated with neuronal and non-neuronal MRs. The ontology terms are arbitrarily grouped based on their functions. (ns) Not significant; (BP) biologic process; (MF) molecular function.
Figure 6.
Module membership analysis of the genes associated with MRs. (A) Enrichment analysis for modules of coexpressed genes. Each color indicates one of 19 coexpression modules that was previously identified in the human cortical transcriptome (Oldham et al. 2008). Each module was examined at various levels of module membership significance, with the _P_-values on the _x_-axis corresponding to the maximum significance thresholds for the Pearson correlation between the expression level of a gene and a module's eigengene (i.e., the first principal component obtained by singular value decomposition). The significance of enrichment was calculated for each level of module membership using the Fisher exact test (_y_-axis). The red line indicates significance at P < 0.05, whereas the red dotted line indicates significant enrichment after applying a Bonferroni correction for multiple comparisons. (B) Average correlation analysis. Average correlation between each module eigengene (Oldham et al. 2008) and the expression levels of genes associated with neuronal MR (NeuN+), non-neuronal MR (NeuN−), and all brain-expressed genes (all probes). Dagger indicates significant difference (P < 0.05, one-way ANOVA, followed by the Dunnet test using all brain-expressed genes [Iwamoto et al. 2005a] as reference). (NA) Functional characteristics were not assigned to that module.
Figure 7.
Variations in neuronal and non-neuronal methylation. (A) Average correlation of pairwise comparisons of signal intensities of all probes on the tiling array. Green bars indicate the average correlation coefficient between NeuN+ and NeuN− samples from the same subject (n = three pairs). Blue and red bars indicate the average correlation coefficient between NeuN− samples (n = six combinations) and between NeuN+ samples (n = six combinations), respectively. The dagger indicates P < 0.05 by the Student's _t_-test. Values are mean ± SD. (B) Interindividual variations of the location of neuronal and non-neuronal MRs. Number of MRs detected in various conditions are shown. Single occurrence: MRs found in only one subject. Multiple occurrences: MRs shared by two or all subjects. Nonconserved: MRs in one subject or shared by two subjects. Conserved: MRs shared by all subjects. _P_-values are determined by the Fisher exact test. Note that the number of shared MRs in three subjects differed from that represented in the text because of the exclusion of common MRs from every individual MR for this analysis (Supplemental Fig. 3).
Figure 8.
Confirmation of higher variations in neurons compared with non-neurons. (A) Histogram of correlation determined in independent samples (n = 24 male control subjects). Pairwise correlation coefficients were plotted. Red, blue, and green bars indicate comparison between NeuN+ samples, between NeuN− samples, and between NeuN+ and NeuN− from the same subject, respectively. (B) Interindividual variations of the location of neuronal and non-neuronal MRs. Single occurrence: methylated probes found in one subject. Multiple occurrences: methylated probes shared by more than two subjects. Nonconserved: methylated probes in one subject or shared by up to 23 subjects. Conserved: MRs methylated probes shared by all subjects. _P_-values are determined by the Fisher exact test.
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