Genome-wide Epigenetic Regulation by Early-Life Trauma

Figure 1. Chromosomal location and expression. A, Chromosomal distribution of differentially methylated probes in abused suicide completers (SAs). The first row represents the chromosomal location of probes with increased methylation (hypermethylated) in the SAs and the second row indicates the chromosomal location of probes with decreased methylation (hypomethylated) in the SAs. B, Inverse correlation between whole-genome expression and promoter methylation differences between SAs and controls. The mean promoter methylation differences (Meth) and gene expression (Expr) differences show that differential methylation is inversely correlated with differential gene expression across the genome (Pearson r = −0.19, P ≤ 4.0E⁻⁶). Data were obtained by summarizing promoter methylation and gene expression differences across regions of 1 megabase throughout the whole genome.

Figure 2. Pathway analysis and fluorescence-assisted cell-sorting summary. A, Functional annotation chart of the neuronal plasticity cluster in the abused suicide completer group determined from the differentially methylated gene sets after false discovery rate correction. Shown are the genes and associated annotations for functional annotation cluster 4 related to neuronal plasticity. Green represents corresponding gene-term associations previously reported, and black represents corresponding gene-term associations not yet reported. B, Total nuclei stained with NeuN generated a bimodal fluorescence intensity distribution. The intensity of fluorescence in the nonneuronal fraction (left peak) is less than in the neuronal fraction (right peak). C, Density plot representing the neuronal and nonneuronal cell fractions as a function of the intensity of fluorescence (FITC-A). SSC-A indicates side scatter (internal granulosity).

Figure 3. In vitro analysis of ALS2 promoter sequence methylation. A, ALS2 promoter sequence, showing the location of the CpG dinucleotides. The full-length (FL) (1050 basepairs [bp], black triangle, solid underline) and the truncated (Trunc) (543 bp, white triangle, broken underline) constructs are shown, along with the specific CpG dinucleotide that was hypermethylated as shown by EpiTYPER (circled, CpG4). The solid-lined box represents an upstream stimulating factor 1 putative binding site with the blue area indicating area investigated with EpiTYPER. Capitalized letters represent the beginning of exon 1. B, Mean (SE) levels of luciferase expression in Be(2)c cells for the FL and Trunc constructs. Results are expressed as mean luciferase expression normalized by renilla expression. Experiments were run with 6 replicates. C, Mean (SE) levels of luciferase expression in Be(2)c cells for the FL ALS2 promoter that was either unmethylated (FL No CH₃) or artificially methylated at CpG4 (FL CH₃). Results are expressed as mean luciferase expression normalized by renilla expression. Experiments were run with 6 replicates. D, Mean (SE) levels of ALS2 isoform 1 expression in the hippocampus. Results are expressed as mean ALS2 expression normalized with the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) expression. Abused suicide completer (SA) (n = 22), nonabused suicide completer (SNA) (n = 19), and control subjects (n = 12). E, Mean (SE) levels of ALS2 isoform 2 expression in the hippocampus. Results are expressed as mean ALS2 expression normalized with GAPDH expression. SA (n = 22), SNA (n = 17), and controls (n = 13). * P < .05. † P < .0001.

Figure 4. Methylation levels in ALS2 promoters in abused suicide completer (SA), nonabused suicide completer (SNA), and control groups. A, Total percentage of methylation in all CpGs for ALS2 in the neuronal cell fraction. B, Total percentage of methylation in all CpGs in ALS2 promoter in the nonneuronal cell fraction. C, Individual CpG methylation levels in the promoter of ALS2 in the neuronal cell fraction. Abused suicide completer (n = 24), SNA (n = 19), and controls (n = 16). For ALS2, methylation values for CpGs 7 and 8, as well as 16 and 17, are pooled. Values are given as the mean (SE) percentage of methylation.* P < .05. † P < .10.