Prenatal Glucocorticoid Exposure Results in Changes in Gene Transcription and DNA Methylation in the Female Juvenile Guinea Pig Hippocampus Across Three Generations

Abstract

Synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery, to mature the fetal lung and decrease neonatal morbidity. sGC also profoundly affect the fetal brain. The hippocampus expresses high levels of glucocorticoid (GR) and mineralocorticoid receptor (MR), and its development is affected by elevated fetal glucocorticoid levels. Antenatal sGC results in neuroendocrine and behavioral changes that persist in three generations of female guinea pig offspring of the paternal lineage. We hypothesized that antenatal sGC results in transgenerational changes in gene expression that correlate with changes in DNA methylation. We used RNASeq and capture probe bisulfite sequencing to investigate the transcriptomic and epigenomic effects of antenatal sGC exposure in the hippocampus of three generations of juvenile female offspring from the paternal lineage. Antenatal sGC exposure (F₀ pregnancy) resulted in generation-specific changes in hippocampal gene transcription and DNA methylation. Significant changes in individual CpG methylation occurred in RNApol II binding regions of small non-coding RNA (snRNA) genes, which implicates alternative splicing as a mechanism involved in transgenerational transmission of the effects of antenatal sGC. This study provides novel perspectives on the mechanisms involved in transgenerational transmission and highlights the importance of human studies to determine the longer-term effects of antenatal sGC on hippocampal-related function.

Figure 1

(A) Venn diagram illustrating the number of genes that are significantly differentially expressed in the HPC of young female F₁-F₃ offspring following prenatal synthetic glucocorticoid treatment of the F₀ pregnancy and the number of genes that overlap between generations (Veh F₁ (n = 6), F₂ (6), F₃ (6); sGC F₁ (5), F₂ (6), F₃ (7)). (B) Heatmap of the 18 genes that are differentially expressed in F₁ and F₃ female offspring. Values indicate the log-fold change in gene expression in sGC animals relative to control, color further indicates the direction of change (green: significantly down-regulated; red: significantly up-regulated; grey: not significant).

Figure 2

Heatmaps showing key hippocampal genes driving the gene set enrichment differences between sGC exposed offspring lineages and controls (Veh F₁ (n = 6), F₂ (6), F₃ (6); sGC F₁ (5), F₂ (6), F₃ (7)) in (A) F₁ juvenile females, the corticosterone response pathway was significantly down-regulated (NE > 1.6, p < 0.01, FDR < 0.05); (B) F₂, the vesicle docking pathway was significantly down-regulated (NES > 1.6, p < 0.01, FDR < 0.05); (C) F₃, the neurotransmitter receptor activity pathway was down-regulated (NES > 1.6, p < 0.01, FDR < 0.05). Green: decreased transcription; red: increased transcription.

Figure 3

Heatmaps showing significantly differentially methylated regions (DMRs) in the hippocampus between sGC exposed offspring lineages and controls (Veh F₁ (n = 6), F₂ (6), F₃ (7); sGC F₁ (6), F₂ (6), F₃ (5)) in (A) F₁ juvenile females, a total of 406 regions were differentially methylated; 184 demethylated, 222 hypermethylated (>10%, FDR < 0.05); (B) In F₂, 139 regions were differentially methylated; 74 demethylated, 65 hypermethylated (>10%, FDR < 0.05); (C) In F₃, 380 regions were differentially methylated; 170 demethylated, 210 hypermethylated (>10%, FDR < 0.05). Green decreased methylation, red increased methylation; (D) Venn diagram illustrating the number of regions related to genes that are significantly differentially methylated in the HPC from F₁-F₃ female PT (Paternal Transmission) and the number of regions that overlap between generations.

Figure 4

Visualization of significant differences in expression and DNA methylation, overlaid with genomic loci in (A) F₁ and (B) F₃. Top row represents significant changes in gene expression associated with prenatal sGC. Red indicates significantly increased expression (FDR < 0.05), green indicates significantly decreased expression (FDR < 0.05). Second row represents significant changes in methylation. Blue indicates significantly decreased methylation (>10%, FDR < 0.05), red indicates significantly increased methylation (>10%, FDR < 0.05). Enhancer row identifies which regions of the genome were captured as enhancers. Gene tracks are represented above the gene names in blue, with horizontal lines representing intron sequences and coding exons represented by blocks.

Figure 5

CpG distributions with respect to transcription start sites (TSS) in (A) F₁ distribution of all CpG sites captured with respect to distance to TSS. (B) Distribution of significantly differentially methylated CpGs in F₁ (p < 0.05, FDR < 0.05) with respect to distance to TSS. (C) F₂ distribution of all CpG sites captured with respect to distance to TSS. (D) Distribution of significantly differentially methylated CpGs in F₂ (p < 0.05, FDR < 0.05) with respect to distance to TSS. (E) F₃ distribution of all CpG sites captured with respect to distance to TSS. (F) Distribution of significantly differentially methylated CpGs in F₃ (p < 0.05, FDR < 0.05) with respect to distance to TSS. Red indicates increased methylation, while blue indicates decreased methylation. Veh F₁ (n = 6), F₂ (6), F₃ (7); sGC F₁ (6), F₂ (6), F₃ (5).