Differentially methylated plasticity genes in the amygdala of young primates are linked to anxious temperament, an at risk phenotype for anxiety and depressive disorders

Abstract

Children with an anxious temperament (AT) are at a substantially increased risk to develop anxiety and depression. The young rhesus monkey is ideal for studying the origin of human AT because it shares with humans the genetic, neural, and phenotypic underpinnings of complex social and emotional functioning. Heritability, functional imaging, and gene expression studies of AT in young monkeys revealed that the central nucleus of the amygdala (Ce) is a key environmentally sensitive substrate of this at risk phenotype. Because epigenetic marks (e.g., DNA methylation) can be modulated by environmental stimuli, these data led us to hypothesize a role for DNA methylation in the development of AT. To test this hypothesis, we used reduced representation bisulfite sequencing to examine the cross-sectional genome-wide methylation levels in the Ce of 23 age-matched monkeys (1.3 ± 0.2 years) phenotyped for AT. Because AT reflects a continuous trait-like variable, we used an analytical approach that is consistent with this biology to identify genes in the Ce with methylation patterns that predict AT. Expression data from the Ce of these same monkeys were then used to find differentially methylated candidates linked to altered gene regulation. Two genes particularly relevant to the AT phenotype were BCL11A and JAG1. These transcripts have well-defined roles in neurodevelopmental processes, including neurite arborization and the regulation of neurogenesis. Together, these findings represent a critical step toward understanding the effects of early environment on the neuromolecular mechanisms that underlie the risk to develop anxiety and depressive disorders.

Keywords: BCL11A; Ce; DNA methylation; JAG1; anxious temperament.

Figure 1.

Methylation differs across the regions of the genome (a–c) and CpG islands (d–f). Standard regions of the genome (a) were used for analyses of density and methylation. Specifically, methylation data distributions were classified depending on how they fell in regard to: the TSS, regions defined as 200 bp upstream of the TSS (TSS200), 1500 bp upstream of the TSS (TSS1500), the 5′ and 3′UTRs, the 10 kb upstream of the gene body (Up10K), and 10 kb downstream of the gene body (Down10K). Distributions of methylation data were binned into these groups based on both density of reads (b) and mean percentage DNA methylation (c), revealing genome region-specific methylation patterns. A similar strategy was used to assess patterns of methylation across CpG islands, which were defined as at least 200 bp stretch of DNA with a C+G content of >50% and an observed CpG/expected CpG in excess of 0.6. CpGs were grouped (d) as those that fell into: CpG island north and south shores (0–2 kb from the island edges), CpG island north and south shelves (2–4 kb from the island edges), and the “open sea” (>4 kb from any island edge). Distributions of methylation data were binned into these groups based on both density of reads (e) and mean percentage DNA methylation (f), revealing CpG-island specific methylation patterns.

Figure 2.

The distribution of AT-associated methylation levels across standard genomic structures. a, The percentage of all CpGs (blue), AT-methylated loci (red), and AT-demethylated loci (purple) for different methylation levels (x-axis) revealed a tendency for highly methylated sites to be more AT-related. Notably, more CpGs were negatively correlated with AT than positively correlated with AT (pie chart inset). b, Manhattan plot of AT-associated loci in the monkey Ce reveals AT-related loci to be distributed across the genome. Positively correlated loci are displayed with a positive log10 (p value), and negatively correlated loci are displayed with a negative log10 (p value). Significant loci are displayed as either AT-methylated (red) or AT-demethylated (purple; FDR < 0.05), whereas loci that are not significant alternate between black and gray to indicate each chromosome. p values shown are all aLIS p values. c, d, The percentage of all CpGs (blue) and AT-related within different genomic regions (c), and CpG islands (d) revealed a tendency for an enrichment in some genomic structures. *Permutation p value <0.05.

Figure 3.

The correlations between AT, DNA methylation, and gene expression for BCL11A (a–d) and JAG1 (e–h). For each of these genes, we identified methylation sites where the methylation levels were correlated with individual differences in AT (a, e; p values shown are aLIS p values) and gene expression (b, f). Moreover, expression levels of BCL11A and JAG1 were also correlated with individual differences in AT (c and g, respectively). Importantly, a cluster of CpGs demonstrated correlated methylation differences to AT (d, h; red represents positive; black represents negative) that reached statistical significance (FDR <0.05, dashed line; and FDR <0.01, solid line).