Weaning Induces Stress-Dependent DNA Methylation and Transcriptional Changes in Piglet PBMCs

Abstract

Changes to the epigenome, including those to DNA methylation, have been proposed as mechanisms by which stress can induce long-term physiological changes in livestock species. Pig weaning is associated with dietary and social stress, both of which elicit an immune response and changes to the hypothalamic-pituitary-adrenal (HPA) axis. While differential methylation following stress has been assessed in model organisms, it remains poorly understood how the pig methylome is altered by stressors in production settings. We quantified changes in CpG methylation and transcript abundance in piglet peripheral blood mononuclear cells (PBMCs) following weaning and also assessed differential patterns in pigs exhibiting high and low stress response as measured by cortisol concentration and lesion scores. Blood was collected from nine gilt piglets 24 h before and after weaning, and whole-genome bisulfite sequencing (WGBS) and RNA-sequencing were performed on six and nine animals, respectively, at both time points. We identified 2,674 differentially methylated regions (DMRs) that were enriched within promoters of genes associated with lymphocyte stimulation and transcriptional regulation. Stress groups displayed unique differential methylation and expression patterns associated with activation and suppression of T cell immunity in low and high stress animals, respectively. Differential methylation was strongly associated with differential expression; specifically, upregulated genes were enriched among hypomethylated genes. We observed post-weaning hypermethylation of the glucocorticoid receptor (NR3C1) promoter and a significant decrease in NR3C1 expression (n = 9, p = 6.1 × 10^-3). Our results indicate that weaning-associated stress elicits genome-wide methylation changes associated with differential gene expression, reduced T cell activation, and an altered HPA axis response.

Keywords: DNA methylation; NR3C1; epigenetics; gene expression; pig; weaning.

FIGURE 1

Differential methylation in post- versus pre-weaning PBMCs. (A) Volcano plot of methylation difference against –log10 (q-value). Red dots indicate significant differentially methylated regions (DMRs). (B) Enrichment of hypomethylated and hypermethylated DMRs for All, HS, and LS pigs in gene features. Horizontal line indicates expected relative proportion of DMRs in feature if no enrichment (i.e., 1). HS, high stress; LS, low stress.

FIGURE 2

Glucocorticoid receptor (NR3C1) gene methylation and expression in response to weaning. (A) Two CpG sites in the NR3C1 promoter (797 and 328 bp downstream of TSS) are significantly hypermethylated post-weaning (n = 6 animals/stage). (B) NR3C1 transcript abundance is significantly reduced post-weaning, as measured by RNA-sequencing and RT-qPCR (n = 9 animals/stage). *= < 0.05, **= < 0.01.