Early life adversity alters normal sex-dependent developmental dynamics of DNA methylation

Abstract

Studies in rodents, nonhuman primates, and humans suggest that epigenetic processes mediate between early life experiences and adult phenotype. However, the normal evolution of epigenetic programs during child development, the effect of sex, and the impact of early life adversity on these trajectories are not well understood. This study mapped the genome-wide DNA methylation changes in CD3+ T lymphocytes from rhesus monkeys from postnatal day 14 through 2 years of age in both males and females and determined the impact of maternal deprivation on the DNA methylation profile. We show here that DNA methylation profiles evolve from birth to adolescence and are sex dependent. DNA methylation changes accompany imposed weaning, attenuating the difference between males and females. Maternal separation at birth alters the normal evolution of DNA methylation profiles and targets genes that are also affected by a later stage maternal separation, that is, weaning. Our results suggest that early life events dynamically interfere with the normal developmental evolution of the DNA methylation profile and that these changes are highly effected by sex.

Figure 1.

(Color online) Developmental changes of DNA methylation levels in CD3+ T cells of male and female mother-reared monkeys over the first 2 years of life. DNA methylation differences across gene promoters were calculated by averaging the methylation differences of significantly affected probes (q < 0.2) of each gene promoter region. (a) Clustering (Jaccard distance, average linkage) of genes whose promoters were differentially methylated in CD3+ T cells of mother-reared, control (MR) male (M) and female (F) monkeys at later stages of development, that is, before their imposed weaning (BW) at 6–7 months of age, after their weaning (AW) at 9–10 months of age, or after 2 years (2y) at 26–30 months of age as compared with their first weeks of life (D14: Day 14–30 samples). (b) Number of genes whose promoters were hypermethylated (indicated in red online only) or hypomethylated (indicated in blue online only) in CD3+ T cells of MR monkeys (M, F, common) in later developmental stages (BW, AW, and 2y) as compared to their first weeks of life (D14). The shading indicates whether the state of methylation was altered at the specific time point compared to BW (new) or remained the same since BW. (c) Top six canonical pathways associated with stable differentially methylated genes, that is, whose promoter methylation levels changed between the first weeks of life (D14) and 6 months of age, before the weaning period, and remained different till their second year of life (2y) in both male and female monkeys’ CD3+ T cells.

Figure 2.

(Color online) DNA methylation differences between males and females in CD3+ T cells of mother-reared (MR) monkeys over the first 2 years of life. MR, Control monkeys, D14: Days 14–30 samples; BW, before weaning (6–7 months); AW, after weaning (9–10 months); 2y, after 2 years (26–30 months). The differences between males and females (M-F) are presented. (a) Clustering (row distance metric: Jaccard distance, average linkage) of genes whose promoters were differentially methylated (q < 0.2) between M and F MR monkeys’ CD3+ T cells over their first 2 years of life. The colors in the online version correspond to the levels of differences (log 2). DNA methylation differences of gene promoters were calculated by averaging the methylation differences of significantly affected probes (q < 0.2) of each gene promoter region. (b) Number of probes differentially methylated (q < 0.2) between M and F MR monkeys’ CD3+ T cells. The observed and expected (p < .05) distributions of probes between autosomal and sex chromosomes are indicated. (c) Number of genes whose promoters were hyper- and hypomethylated between M and F monkeys’ CD3+ T cells over their first 2 years of life. The percentages above the bars at each time point indicate the proportion of genes whose promoters were already differentially methylated during their first month of life. The shading indicates whether the state of methylation was altered at the time point (new) or remained the same since Days 14–30. (d) Pearson product-moment correlation coefficients between averaged promoter methylation differences (q < 0.2) detected between M and F monkeys’ CD3+ T cells during the sampled developmental stages (D14, BW, AW, and 2y). Significant coefficients (p < .05) are in bold. (e) Number of genes whose promoters were differentially methylated between M and F monkeys’ CD3+ T cells BW and that remained differentially methylated AW. The numbers next to the arrows indicate the numbers of genes whose promoters were differentially methylated (q < 0.2) BW but not AW in either M or F monkeys. Blue and red arrows correspond to decreases and increases in methylation AW compared to BW, respectively. (f) Clustering (row distance metric: Pearson correlation, average linkage) of genes whose promoters were differentially methylated (q < 0.2) between M and F monkeys’ CD3+ T cells BW but not AW. The colors correspond to the average methylation levels of the probes, for each promoter. (g) Top upstream regulators associated with genes differentially methylated between M and F monkeys BW but not AW (first row). Top canonical pathways associated with genes differentially methylated between M and F monkeys during their first 2 years of life in the same or opposite directions at 2 years old compared to the first month. The numbers at the right of the bars indicate the numbers of differentially methylated genes that are associated with the pathways or are known to be regulated by the upstream regulator.

Figure 3.

(Color online) DNA methylation differences between mother-reared (MR) and surrogate-peer reared (SPR) monkeys’ CD+ T cells over the first 2 years of life. (a) Clustering (row distance metric: Jaccard distance, average linkage) of genes whose promoters were differentially methylated between the SPR and MR monkeys’ CD3+ T cells. Female (F) and male (M) monkeys were analyzed separately at all sampled developmental stages (D14, first month; BW, before weaning; AW, after weaning; 2y, after 2 years). DNA methylation differences of gene promoters were calculated by averaging the methylation differences of significantly affected probes (q < 0.2) of each gene promoter region. (b) Number of genes whose promoters were hyper- and hypomethylated between SPR and MR monkeys’ CD3+ T cells over their first 2 years of life (M, F, and common). The percentages above the bars at each time point indicate the proportion of genes whose promoters were already differentially methylated during their first month of life. The shading indicates whether the state of methylation was altered at the time point (new) or remained the same since Day 14. (c) Number of genes whose promoters were differentially methylated between MR and SPR M monkeys BW and that remained differentially methylated AW. The numbers next to the arrows indicate the numbers of genes whose promoters were differentially methylated (q < 0.2) BW and AW in either M or F monkeys. Blue and red arrows in the online version correspond to decreases and increases in methylation AW compared to BW, respectively. (d) Clustering (row distance metric: Pearson correlation, average linkage) of genes whose promoters were differentially methylated (q < 0.2) between MR and SPR male monkeys’ CD3+ T cells BW but not AW. The colors online correspond to the average methylation levels of probes differentially methylated BW, for each promoter. (e) An example of canonical pathways associated with genes whose promoters were differentially methylated (q < 0.2) between MR and SPR M monkeys BW but not AW. The red and blue colors online indicate hyper- and hypomethylation in SPR monkeys compared to MR monkeys, respectively. (f) Overlaps between genes whose promoters were differentially methylated between SPR and MR monkeys during their first month of life (D14/SPR-MR) or between before and after the imposed weaning periods within MR monkeys (MR/AW-BW).

Figure 4.

(Color online) Validation of DNA methylation differences. (a,b) Relative DNA methylation enrichment (normalized m-C bound fraction) of the whole genome amplified pooled samples used for the methylated DNA immunoprecipitation (MeDIP) arrays by quantitative MeDIP analysis (mean+SEM), **p < .005, *p < .05, and #p < .1 at Student t test. (c,d) DNA methylation levels of mother-reared and surrogate-peer reared groups from the first and last sampled developmental stages (Day 14 and 2 years) measured by quantitative MeDIP (normalized m-C bound fraction) and by pyrosequencing (mean methylation per rearing group per CG-site with the SEM), **p < .005, *p < .05, and #p < .1 at Student t test. In the online version, blue indicates male groups, red indicates female groups, and shading indicates surrogate-peer reared groups.

Figure 4.

(Color online) Validation of DNA methylation differences. (a,b) Relative DNA methylation enrichment (normalized m-C bound fraction) of the whole genome amplified pooled samples used for the methylated DNA immunoprecipitation (MeDIP) arrays by quantitative MeDIP analysis (mean+SEM), **p < .005, *p < .05, and #p < .1 at Student t test. (c,d) DNA methylation levels of mother-reared and surrogate-peer reared groups from the first and last sampled developmental stages (Day 14 and 2 years) measured by quantitative MeDIP (normalized m-C bound fraction) and by pyrosequencing (mean methylation per rearing group per CG-site with the SEM), **p < .005, *p < .05, and #p < .1 at Student t test. In the online version, blue indicates male groups, red indicates female groups, and shading indicates surrogate-peer reared groups.