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

Authors: CORBETT, RYAN - Michigan State University; LUTTMANN, ANDREA - Michigan State University; HERRERA-URIBE, JUBER - Iowa State University; LIU, HAIBO - Iowa State University; RANEY, NANCY - Michigan State University; GRABOWSKI, JENNA - Michigan State University; Loving, Crystal; TUGGLE, CHRISTOPHER - Iowa State University; ERNST, CATHERINE - Michigan State University

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2022
Publication Date: 8/11/2022
Citation: Corbett, R.J., Luttmann, A.M., Herrera-Uribe, J., Liu, H., Raney, N.E., Grabowski, J.M., Loving, C.L., Tuggle, C.K., Ernst, C.W. 2022. Assessment of DNA methylation in porcine immune cells reveals novel regulatory elements associated with cell-specific gene expression and immune capacity traits. Biomed Central (BMC) Genomics. 23(1). Article 575. https://doi.org/10.1186/s12864-022-08773-5.
DOI: https://doi.org/10.1186/s12864-022-08773-5

Interpretive Summary: Pigs are an important protein source supporting global food security. A major goal of biological research is using genetic information, or genotype, to predict the complex physical composition of an individual or individual cells, or phenotype. Genome sequencing and cataloging genes expressed in specific cell types is important in understanding genotype to phenotype, but it does not take into consideration the mechanisms that regulate gene expression. Deeper functional annotation of animal genomes is critical to further improvement of selection for desirable traits. One form of regulation that controls gene expression is methylation of DNA (or genome), which alters the ability of proteins to access the DNA and initiate gene expression. Cells in the blood are expected to represent the physiological state of an animal, and potentially predict outcomes when animals face stress or disease. Thus, gene expression in nine separate blood immune cell populations was paired with DNA methylation analysis to understand factors controlling immune cell gene expression. Gene expression in specific cell types was inversely correlated with methylation rates at expected DNA locations, indicating methylation limits accessibility to DNA for gene expression in immune cells, similar to other mammals. Overall, the results improve the functional annotation of the pig genome through identification of genes whose expression is likely controlled by DNA methylation, as opposed to other regulatory mechanisms.

Technical Abstract: 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 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 various epigenetic mechanisms that regulate mammalian gene expression, DNA methylation is the most ubiquitous epigenetic 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. 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 processed to remove red blood cells and isolate neutrophils and peripheral blood mononuclear cells (PBMCs). PBMCs were subjected to magnetic- and fluorescence-activated cell sorting to isolate eight cell types (in addition to neutrophils) for methylome analysis: 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 genes with enriched expression in 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.