Assessment of DNA methylation in porcine immune cells reveals novel regulatory elements associated with cell-specific gene expression and immune capacity traits.

BACKGROUND: Genetics studies in the porcine immune system have enhanced selection practices for disease resistance phenotypes and increased the efficacy of porcine models in biomedical research; however limited functional annotation of the porcine immunome has hindered progress on both fronts. Among epigenetic mechanisms that regulate gene expression, DNA methylation is the most ubiquitous modification made to the DNA molecule and influences transcription factor binding as well as gene and phenotype expression. Human and mouse DNA methylation studies have improved mapping of regulatory elements in these species, but comparable studies in the pig have been limited in scope. RESULTS: We performed whole-genome bisulfite sequencing to assess DNA methylation patterns in nine pig immune cell populations: CD21+ and CD21- B cells, four T cell fractions (CD4+, CD8+, CD8+CD4+, and SWC6γδ+), natural killer and myeloid cells, and neutrophils. We identified 54,391 cell differentially methylated regions (cDMRs), and clustering by cDMR methylation rate grouped samples by cell lineage. 32,737 cDMRs were classified as cell lowly methylated regions (cLMRs) in at least one cell type, and cLMRs were broadly enriched in genes and regions of intermediate CpG density. We observed strong correlations between differential methylation and expression across immune cell populations, with cell-specific low methylation disproportionately impacting genes exhibiting enriched gene expression in the same cell type. Motif analysis of cLMRs revealed cell type-specific enrichment of transcription factor binding motifs, indicating that cell-specific methylation patterns may influence accessibility by trans-acting factors. Lastly, cDMRs were enriched for immune capacity GWAS SNPs, and many such overlaps occurred within genes known to influence immune cell development and function (CD8B, NDRG1). CONCLUSION: Our DNA methylation data improve functional annotation of the porcine genome through characterization of epigenomic regulatory patterns that contribute to immune cell identity and function, and increase the potential for identifying mechanistic links between genotype and phenotype.

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10-11, and the PI among siblings (PIsib) was 1.5 x 10-5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.

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

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.