DNA methylation signatures in development and aging of the human prefrontal cortex

Abstract

The human prefrontal cortex (PFC), a mastermind of the brain, is one of the last brain regions to mature. To investigate the role of epigenetics in the development of PFC, we examined DNA methylation in ∼14,500 genes at ∼27,000 CpG loci focused on 5' promoter regions in 108 subjects range in age from fetal to elderly. DNA methylation in the PFC shows unique temporal patterns across life. The fastest changes occur during the prenatal period, slow down markedly after birth and continue to slow further with aging. At the genome level, the transition from fetal to postnatal life is typified by a reversal of direction, from demethylation prenatally to increased methylation postnatally. DNA methylation is strongly associated with genotypic variants and correlates with expression of a subset of genes, including genes involved in brain development and in de novo DNA methylation. Our results indicate that promoter DNA methylation in the human PFC is a highly dynamic process modified by genetic variance and regulating gene transcription. Additional discovery is made possible with a stand-alone application, BrainCloudMethyl.

Figure 1

Depiction of Global DNA Methylation Patterns in Human Subjects across the Lifespan (A) Histograms of subject ages in the brain collection. All fetal samples (left panel) are between 14 and 20 gestational weeks. The following color scale is used: red, male fetal samples (n = 14); orange, female fetal samples (n = 16); light blue, male children (n = 10); green, female children (n = 5); blue, males older than 10 years (n = 32); and purple, females older than 10 years (n = 31). The same color scale is used in (B and C). (B) Global DNA methylation pattern examined by multidimensional scaling for autosomal CpG sites. Each DNA methylation sample is represented as a single point colored by the age of the subject. There is a tight cluster of the fetal samples, a progression of methylation levels from the fetal period to childhood, and another cluster for postchildhood. (C) Global DNA methylation pattern of CpG sites on the X chromosome examined by multidimensional scaling. Each DNA methylation sample is represented as a single point colored by the age of the subject and sex. Samples were distinctly segregated by sex. A progression of methylation was also observed from the fetal period to postchildhood within each sex group.

Figure 2

Depiction of Rates of DNA Methylation in Three Life Stages (A) Rates of methylation change with age during three developmental life stages (the fetal period, childhood, and postchildhood/adult) at each CpG site for 27,578 CpG loci. A rate of change is plotted on the x axis. The −log₁₀ p value is on the y axis, such that increasing values indicate more significant changes in methylation with age within a given stage. Red dots represent significant age-related methylation changes at FDR < 0.05. The fastest rates of methylation changes occurred during the fetal period, involving predominantly decreases in methylation (negative values on the x axis). In contrast, during the childhood and adulthood the changes were much slower and involved mainly increased methylation with aging (positive values on the x axis). (B) Average rates of significant methylation changes with age within three developmental stages (red = the fetal period, green = childhood, blue = postchildhood). The absolute rate of the methylation change per year for the significant age-related CpG loci (FDR < 0.05) is on the y axis. The fastest changes in methylation occurred during the prenatal period, followed by childhood and postchildhood. The error bars represent standard deviations. (C) Diagram showing the numbers of the CpG sites with significant age-related changes during the life stages. Of 27,578 CpG sites, significant age-related changes in DNA methylation (FDR < 0.05) were observed at 865 sites during the fetal period, 5,506 sites during childhood, and 10,578 sites during postchildhood. One hundred and thirty-eight sites out of the 252 sites that overlapped between the fetal period and the childhood and 188 sites out of the 3,529 sites that overlapped between children and postchildhood showed the opposite patterns of methylation (an increase followed by a decrease or vice versa) between these life stages. The transition from the fetal life to childhood is frequently associated with a reversal of direction in DNA methylation profile.

Figure 3

Depiction of Individual Loci's DNA Methylation Signatures across the Human Lifespan (A) Depiction of individual loci's DNA methylation signatures across the human lifespan, illustrating three patterns of DNA methylation across the fetal, child and adult life periods. Methylation levels are on the y axis, with age on the x axis. The CpG site of CDH22 (cg04640913) showed significant age-related increases during the fetal period, childhood, and postchildhood (p = 2 × 10⁻⁶, p = 1 × 10⁻³, and p = 2.3 × 10⁻⁴, respectively). The CpG site of SCGB3A2 (cg19530885) showed significant age-related decreases during the fetal period, childhood, and postchildhood (p = 4.5 × 10⁻¹³, p = 9.5 × 10⁻³, and p = 2.4 × 10⁻⁴, respectively).The CpG site of NOS1 (cg21006686) showed a significant age-related decrease during the fetal period (p = 8.6 × 10⁻⁴) and increases during childhood and postchildhood (p = 2 × 10⁻⁸ and p = 1.1 × 10⁻², respectively). (B) Depiction of individual loci's DNA methylation signatures across the human lifespan, illustrating sex differences. Females are red and males are blue. The CpG sites of MAOA (cg19441691) and TLE1 (cg15915418) showed significant sex differences (p = 1.04 × 10⁻⁶² and 1.27 × 10⁻⁶⁵, respectively).

Figure 4

Depiction of Age-Related Changes in DNA Methylation in Three Life Stages as a Function of CpGI Context Black lines show density distributions of regression coefficients for age term for the CpG loci located in the CpGIs. Red lines show density distribution of regression coefficients for age term for the CpG loci located outside the CpGIs. More loci are demethylated than hypermethylated during the fetal period, whereas increased methylation with age dominates in postnatal life (childhood and postchildhood). The effect sizes are larger for loci outside the CpGIs than those located in the CpGIs.

Figure 5

Depiction of Hierarchical Clustering with the Weighted Correlation Network Analysis (A) Hierarchical clustering dendrogram obtained with the weighted correlation network analysis. The first color row underneath (labeled Group) shows the module assignment determined by the Dynamic Tree Cut. The second color row (labeled chrX) represents autosomal (gray) or X chromosomal (red) location of the loci in the module. The third row (labeled CpGI) shows whether the loci are located in the CpGIs (red) or outside the CpGIs (gray). (B) Heat map visualization of DNA methylation status of six modules. A hierarchical clustering was conducted across 108 subjects shown in rows (a vertical color bar on the left indicates sex: red = females, blue = males; a vertical color bar on the right of the picture indicates age groups: red = fetal, green = childhood, blue = postchildhood) and six modules in columns (CpG loci were clustered into comethylation modules: groups 0–5, with group 0 containing unclassified loci). Color key on top indicates DNA methylation values: lowest, pink highest, blue lowest.

Figure 6

Depiction of Correlations between DNA Methylation and Gene Expression (A) Histogram of correlation coefficients for Pearson's correlations between DNA methylation and gene expression. Green line shows correlations of expression with methylation at the loci located in CpGIs, red line, with loci outside CpGIs. (B) An example of opposite patterns of DNA methylation (left) and expression (right) changes with age (x axis) for NNAT (neuronatin).

Figure 7

Depiction of Associations between the Genotype and DNA Methylation (A) Depiction of the mQTL for the CpG site cg01889448 and rs1063355 in HLA-DQB1 (p = 10⁻²⁹⁾. Three genotypes of rs1063355 are 1/1 in red (n = 21), 1/2 in green (n = 57), and 2/2 in blue (n = 30). The lines in the bar graph indicate the median, and the bars (whiskers) represent the minimum and maximum of the data after the removal of the outliers. (B) The relationship between the strength of association and the distance of the SNP from the CpG site. Only significant cis-SNPs associations are plotted (FDR < 0.05). The distance of SNPs from the CpG sites in base pairs is on the x axis and the −log₁₀ p values of SNP association with DNA methylation. Plotting p values against the distance of SNPs from each CpG site showed that the highest associations were with the SNPs located close to CpG site.