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

Abstract

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.

Integrative profiling of gene expression and chromatin accessibility elucidates specific transcriptional networks in porcine neutrophils

“…Lowly methylated regions (LMRs) were highly enriched among neutrophil SEGs ( p = 2.4 × 10 -3 ). In total, 119 neutrophil SEGs showed LMRs in comparison with other porcine immune cell methylomes reported previously ( Supplementary File S6 ) ( Corbett et al, 2022 ). LMRs across SEGs gene features showed that promoters and transcription termination sires (TTS) have higher enrichment with the expression of SEGs ( Figure 6B ).…”

“…We used published DNA methylation data generated from samples collected from the same pigs in parallel ( Corbett et al, 2022 ) to determine whether neutrophil DNA methylation was related to gene expression and accessible chromatin regions. Also, we tested the significant enrichment between differentially methylated regions (DMR) (calculated across the same immune cell populations used in this study) and SEGs and HEGs.…”

“…We compared neutrophil gene expression and chromatin accessibility data with previously published DNA methylation data from the same cell populations ( Corbett et al, 2022 ). Average DNA methylation profiles for neutrophil-enriched genes and neutrophil ATAC peaks were generated using deepTools ComputeMatrix command ( Ramírez et al, 2016 ) with default parameters and including 2-kb regions flanking these genes and peaks.…”

Pleiotropic architectures of porcine immune and growth trait pairs revealed by a self‐product‐based transcriptome method

“…Three‐color flow cytometry analyses were performed to distinguish CD4+ and CD8+ T cell subpopulations at the Medical Laboratory Animal Center (Hubei Medical College, China). Briefly, peripheral blood mononuclear cells were labeled with biotin‐labeled anti‐porcine CD3 ε , and the CD3 ε ‐positive fraction was further separated into CD4+ T cell subsets (CD4+; CD3 ε + CD8 α − CD4+) and CD8+ T cell subsets (CD8+; CD3 ε + CD8 α + CD4−) (Corbett et al, 2022). The monoclonal antibodies CD3‐SprD (PE‐CY5), anti‐piglet CD8‐PE and anti‐piglet CD4‐FITC were purchased from Southern Biotech.…”

Pig fetal skeletal muscle development is associated with genome-wide DNA hypomethylation and corresponding alterations in transcript and microRNA expression

“…DNA was isolated using PureLink Genomic DNA kit (Invitrogen), and quality and quantity were assessed on a Qubit fluorometer (Invitrogen). WGBS was performed at the Michigan State University (MSU) Research Technology Support Facility (RTSF) Genomics Core using methods previously reported by our group (Corbett et al 2022). Briefly, DNA samples were spiked with unmethylated lambda phage DNA (5 ng lambda DNA/1 μg sample DNA) to assess bisulfite conversion rates.…”