Chronic social stress induces DNA methylation changes at an evolutionary conserved intergenic region in chromosome X

Abstract

Chronic stress resulting from prolonged exposure to negative life events increases the risk of mood and anxiety disorders. Although chronic stress can change gene expression relevant for behavior, molecular regulators of this change have not been fully determined. One process that could play a role is DNA methylation, an epigenetic process whereby a methyl group is added onto nucleotides, predominantly cytosine in the CpG context, and which can be induced by chronic stress. It is unknown to what extent chronic social defeat, a model of human social stress, influences DNA methylation patterns across the genome. Our study addressed this question by using a targeted-capture approach called Methyl-Seq to investigate DNA methylation patterns of the dentate gyrus at putative regulatory regions across the mouse genome from mice exposed to 14 days of social defeat. Findings were replicated in independent cohorts by bisulfite-pyrosequencing. Two differentially methylated regions (DMRs) were identified. One DMR was located at intron 9 of Drosha, and it showed reduced methylation in stressed mice. This observation replicated in one of two independent cohorts. A second DMR was identified at an intergenic region of chromosome X, and methylation in this region was increased in stressed mice. This methylation difference replicated in two independent cohorts and in Major Depressive Disorder (MDD) postmortem brains. These results highlight a region not previously known to be differentially methylated by chronic social defeat stress and which may be involved in MDD.

Keywords: DNA methylation; Major depressive disorder; Methyl-Seq; postmortem brain; social defeat stress.

Figure 1.

Behavioral phenotypes, organ weight and corticosterone level of mice following 14-day social defeat stress (N = 16) compared to control mice (n = 16). (a–b) Open field test. (c–e) Elevated plus maze. (f) Sucrose preference test. (g–i) Organ weights. (a) Time spent immobile. (b) Time spent in inner zone. (c) Time spent in closed arm. (d) Time spent in center. (e) Time spent in open arm. (f) Consumption of sucrose solution. (g) Adrenal gland hypertrophy. (h) Splenic hypertrophy. (i) Thymic atrophy. (j) Basal corticosterone level. N.S.: not significant; *: P < 0.05; **: P < 0.0001. Figure shows mean ± S.E.M.

Figure 2.

Bisulfite-pyrosequencing of DMCs in the intergenic region on Chromosome X and intron 9 Drosha. Bisulfite-pyrosequencing of intergenic region on chromosome X in (a) cohort 2 (n = 7 stressed and 12 control mice) and (b) cohort 3 (n = 15 stressed and 15 control mice). Bisulfite-pyrosequencing in intron 9 of Drosha for (c) cohort 2 and (d) cohort 3. Figures shown are mean ± S.E.M. Data was analyzed using one-way ANOVA and corrected for multiple testing using Holm-Šídák test. ***: P < 0.001; *: P < 0.05; N.S.: not significant.

Figure 3.

Methylation differences of DMRs at intergenic region of chromosome X and intron 9 of Drosha. Methylation of CpGs detected by bisulfite-pyrosequencing were averaged across the region and compared between stressed and control mice. DMR at the intergenic region of chromosome X in replication (a) cohort 2 and (b) cohort 3. DMR at intron 9 of Drosha in replication (c) cohort 2 and (d) cohort 3. Figures shown are mean ± S.E.M of the DMR for each group. #: P = 0.052; *: P < 0.05; **: P < 0.01; N.S; not significant. P values were calculated using student’s t-test.

Figure 4.

Comparison of DNA methylation between MDD and controls at the evolutionary conserved intergenic region at chromosome X and intron 9 of DROSHA in Broadman Area 46. (a–c) Intergenic region of chromosome X. (d–f) Intron 9 of DROSHA. Linear mixed modeling was used to compare DNA methylation at the DMRs between (a, d) all MDD cases vs. controls, (b, e) MDD suicide cases vs. controls and (c, f) MDD non-suicide cases vs. controls. Figures shown are mean ± S.E.M of the DMR for each group. P values were calculated using likelihood ratio test as described in material and methods controlling for age, race, sex, postmortem interval, and pH. **: P < 0.01 [χ² (1) = ~7.3]; *: P < 0.05 [χ² (1) = ~4]; N.S.: not significant.