Genome-wide epigenetic regulation by early-life trauma

Abstract

Context: Our genome adapts to environmental influences, in part through epigenetic mechanisms, including DNA methylation. Variations in the quality of the early environment are associated with alterations in DNA methylation in rodents, and recent data suggest similar processes in humans in response to early-life adversity.

Objective: To determine genome-wide DNA methylation alterations induced by early-life trauma.

Design: Genome-wide study of promoter methylation in individuals with severe abuse during childhood. PATIENTS, SETTING, AND MAIN OUTCOME MEASURES: Promoter DNA methylation levels were profiled using methylated DNA immunoprecipitation followed by microarray hybridization in hippocampal tissue from 41 French-Canadian men (25 with a history of severe childhood abuse and 16 control subjects). Methylation profiles were compared with corresponding genome-wide gene expression profiles obtained by messenger RNA microarrays. Methylation differences between groups were validated on neuronal and nonneuronal DNA fractions isolated by fluorescence-assisted cell sorting. Functional consequences of site-specific promoter methylation were assessed by luciferase assays.

Results: We identified 362 differentially methylated promoters in individuals with a history of abuse compared with controls. Among these promoters, 248 showed hypermethylation and 114 demonstrated hypomethylation. Validation and site-specific quantification of DNA methylation in the 5 most hypermethylated gene promoters indicated that methylation differences occurred mainly in the neuronal cellular fraction. Genes involved in cellular/neuronal plasticity were among the most significantly differentially methylated, and, among these, Alsin (ALS2) was the most significant finding. Methylated ALS2 constructs mimicking the methylation state in samples from abused suicide completers showed decreased promoter transcriptional activity associated with decreased hippocampal expression of ALS2 variants.

Conclusion: Childhood adversity is associated with epigenetic alterations in the promoters of several genes in hippocampal neurons.

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.