Fetal glucocorticoid receptor (Nr3c1) deficiency alters the landscape of DNA methylation of murine placenta in a sex-dependent manner and is associated to anxiety-like behavior in adulthood

Abstract

Prenatal stress defines long-term phenotypes through epigenetic programming of the offspring. These effects are potentially mediated by glucocorticoid release and by sex. We hypothesized that the glucocorticoid receptor (Gr, Nr3c1) fashions the DNA methylation profile of offspring. Consistent with this hypothesis, fetal Nr3c1 heterozygosity leads to altered DNA methylation landscape in fetal placenta in a sex-specific manner. There was a significant overlap of differentially methylated genes in fetal placenta and adult frontal cortex in Nr3c1 heterozygotes. Phenotypically, Nr3c1 heterozygotes show significantly more anxiety-like behavior than wildtype. DNA methylation status of fetal placental tissue is significantly correlated with anxiety-like behavior of the same animals in adulthood. Thus, placental DNA methylation might predict behavioral phenotypes in adulthood. Our data supports the hypothesis that Nr3c1 influences DNA methylation at birth and that DNA methylation in placenta correlates with adult frontal cortex DNA methylation and anxiety-like phenotypes.

Fig. 1. Anxiety-like behavior measured by the dark–light-box.

a Increased latency to enter the light department in Gr (Nr3c1)^+/− animals (p = 0.011, F_3,44 = 7.047). b Increased latency to explore the end of the compartment in Gr (Nr3c1)^+/− animals (p = 0.008, F_3,44 = 7.798). c Significant decrease in the number of exits in Gr (Nr3c1)^+/− animals (p = 0.013, F_3,44 = 6.646). d Decreased duration of time spent in the light area in Gr (Nr3c1)^+/− animals (p = 0.001, F_3,44 = 14.059). Means ± SEM

Fig. 2. Sex-dependent effects of Nr3c1 deficiency.

a Number of differentially methylated CpG sites for the comparisons of female vs male Nr3c1^+/+, female Nr3c1^+/+ vs female Nr3c1^+/−, male Nr3c1^+/+ vs Nr3c1^+/−, and female vs male Nr3c1^+/− as depicted in the x-axis. Numbers directly above the graph bars represent absolute numbers of differentially methylated CpG sites. b Global methylation of CpG, CHH, and CHG sites in wildtype and Nr3c1 knockouts in both sexes. Numbers depicted directly on the graphs represent the percentage of CpG sites (see also y-axis to the left). The percentage of CHH and CHG sites are illustrated on the right y-axis. c Effects of Nr3c1 loss on hyper- and hypomethylation in males and females. d Genome-wide methylation tracks for CpG sites using Integrative Genomics Viewer (Broad Institute). Comparison is shown for female vs male wildtype, female wild type vs female Nr3c1 heterozygous, male wild type vs male Nr3c1 heterozygous, and male vs female Nr3c1 heterozygous animals. e DNA methylation landscape of Nr3c1 deficiency in male and female placental DNA. Heatmap (row distance metric: Pearson correlation, average linkage) depicting the clustering of 6017 CpGs that were differentially methylated (q < 0.2) between placentae of Gr^+/+ and Gr^+/− fetuses of both sexes. Rows correspond to CpGs and columns to animals’ genotype and sex. Here, group number 1–3 indicates three pooled samples of Gr^+/+ males, number 4–6 stands for three pooled samples of Gr^+/− males, 19–21 for three pooled samples of Gr^+/+ females, and 22–24 for three pooled samples of Gr^+/− females. Red indicates higher methylation in a row and blue indicates lower methylation. See also Table S4-S9

Fig. 3. Ingenuity pathway analysis.

Selected canonical pathways for the comparison of methylation changes in males and females after Nr3c1 loss. See also Table S11 and S12

Fig. 4. Pearson correlation between methylation ratio and anxiety as measured by the time spent in the light department of the dark–light-box.

a–e Depicted are line charts for the genes Slc1a7, Tspo, Itgb7, Tiam2, and 9530052E02Rik of male animals. fCorrelation between time spent in the light and Trpm1 methylation in female animals. All values are FDR-corrected p-values

Fig. 5. Ingenuity pathway analysis of genes that are differentially methylated in frontal cortex from Nr3c1 antisense animals and placentae from Nr3c1 heterozygotes.

Selected canonical pathways that are differentially methylated in NR3C1 ^{+ /−} mice in fetal placental and adult Nr3c1 antisense mice frontal cortex. See also Table S13-S15