Assessment of DNA methylation in porcine immune cells

The porcine immune system possesses a vast repertoire of broad-mammalian and species-enriched cell types that are critical in combatting infection. Genetics studies have enhanced pig selection practices for disease resistance phenotypes as well as increased the efficacy of the porcine model in biomedical research; however limited functional annotation of the porcine immunome has hindered progress on both fronts. Among various epigenetic mechanisms that regulate mammalian gene expression, DNA methylation is the most ubiquitous modification made to the DNA molecule and has been shown to influence 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. We performed whole-genome bisulfite sequencing in nine pig immune cell populations to assess cell-specific DNA methylation patterns and their associations with: 1) cell-enriched functions and gene expression, 2) transcription factor binding motifs, and 3) GWAS SNPs for immune capacity and disease traits. Whole blood was collected from two crossbred barrows and subjected to density gradient centrifugation to remove red blood cells and separate out neutrophils, and peripheral blook mononuclear cells underwent magnetic- and fluorescence-activated cell sorting into myeloid cells, natural killer (NK) cells, two B cell fractions (CD21+ and CD21-) and four T cell fractions (CD4+, CD8+, CD4+CD8+, and SWC6?d+). 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 (methylation<75% and z-score<-1), and cLMRs were broadly enriched in genic regions as well as regions of intermediate CpG density. Immune cDMRs exhibited methylation rates that were significantly correlated with local transcript abundance across cell types, with the majority of these correlations being negative. Furthermore, cell lowly methylated genes were overrepresented among expression-enriched genes for the same cell type, suggesting that low methylation is strongly associated with cell-specific gene activation. Motif analysis of cLMR sequences revealed cell type-specific enrichment of transcription factor binding motifs among B, T, myeloid, and NK cells, indicating that cell-specific methylation patterns may influence accessibility by trans-acting factors. Lastly, immune cell DMRs were specifically enriched for immune capacity GWAS SNPs; many such overlaps occurred within genes known to influence immune cell development and function and have previously been associated with immune phenotypes, including CD8B and NDRG1. Overall, 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.