Transcriptome And Genome-Wide DNA Methylome Analysis Of Porcine Intestinal Epithelial Cells Upon Deoxynivalenol Exposure

Deoxynivalenol(DON),which is also called vomitoxin,is a kind of fungal toxin that occurs predominantly in wheat,barley,oats,corn and other cereal crops.DON is absorbed from gastrointestinal tract through digestion of contaminated food and then reaches the blood compartment and whole body via blood circulation.DON can restrain protein synthesis and disturb immune and metabolic functions,resulting in toxic symptoms such as vomiting,anorexia,malnutrition,leukocytosis,and diarrhea.DON is chemically stable and resistant to high temperature and acidic conditions.DON contamination poses a risk to food safety and the health of humans and farm animals.High dose of DON can lead to the increase of morbidity and mortality,and low doses can damage the intestinal and immune system functions.Pig is highly sensitive to DON contamination,which can result in adverse reactions such as anorexia even in very low doses.The gene expression changes and underlying regulatory mechanisms in porcine intestinal cells upon DON exposure remain unclear.DNA methylation is a kind of epigenetic modification that play important roles in regulating gene expression.In addition,how DNA methylation regulate the expression of genes involved in DON-induced cytotoxic responses has not been reported.To better understand the molecular effects of DON exposure,we performed genome-wide comparisons of DNA methylation and gene expression from porcine intestinal epithelial cell IPEC-J2 upon DON exposure using reduced representation bisulfite sequencing and RNA-seq technologies.Integrative analyses were then performed to detect key genes involved in DON-induced cytotoxic effects.The main findings were as follows:(1)The cell viability was measured under different concentrations(0?300,500,1000,2000,2500,and 3000 ng/ml)of DON and cultured for 24 h,48 h,and 72 h.CCK-8 cell viability assay indicated that the cell viability tended to decrease over time with increasing DON dose.At 48 h of incubation and under the DON concentration of 1000 ng/ml,cell viability was sharply decreased.Cells treated in abovementioned condition were collected for transcriptome and genome-wide DNA methylation analysis.(2)Transcriptomic analyses were conducted for 4 DON-treated samples and four control samples.A total of 609.3 million raw reads were generated,and 597.2 million clean reads were kept after quality control,with an average of 74.6 million clean reads per sample.Alignment analysis showed that about 552 million reads were mapped to the pig genome,of which 528.6 million reads were uniquely mapped.Reads distribution across the genomic regions demonstrated that most of the reads(>86%)were mapped to exons.In total,3226 differentially expressed annotated genes(|log2 fold change|>1.5,corrected P<0.05)were identified,comprising 1004 upregulated and 2222 downregulated genes.Functional annotation indicated that genes significantly associated with pathways such as valine,leucine and isoleucine degradation,drug metabolism by cytochrome P450,glutathione metabolism were downregulated in DON-treated samples.The upregulated gene sets were significantly enriched in pathways including ribosome biogenesis in eukaryotes,mRNA surveillance pathway,RNA transport,and TNF signaling pathways.The DMR length was mainly distributed in the range from 50 to 200 bp.DMR distribution analysis demonstrated that the majority of DMRs were located at the CGI,CGI shore,exon,intron,and repeat regions.(3)Genome-wide DNA analyses were conducted for 4 DON-treated samples and four control samples.An average of 38.5 million clean reads per sample were yielded,and 67.4%on average could be mapped to the pig reference genome.The distribution of CpG cytosine methylation level in samples was shown to be the bimodal distribution.CpG methylation changes at distinct genomic contexts displayed that the promoter and 5'UTR regions exhibited lower methylation levels than other genomic contexts and the methylation level tended to sharply decrease toward to transcription start sites and increase toward to gene bodies.Differential methylation analysis identified a total of 3030 DMRs,of which 2093 DMRs showed differential higher methylation levels and 937 differential lower methylation levels in the DON-treated group.Integrative analysis revealed 29 DMRs at the promoter regions displayed inverse correlations with differential expression of the corresponding genes,which include ASAP3,ST3GAL5,and SLC4A11 that essential for cell growth and cell proliferation.Moreover,we identified a total of 258 differentially expressed genes were differentially methylated at their gene bodies.The methylation level of these genes were classified into three groups,low(0-0.4),medium(0.4-0.7)and high(0.7-1)groups,and the changes of these gene expression levels in the three groups displaying similar trend with the gene bodies methylation changes.This result supported the idea that DNA methylation at gene bodies is usually positively correlated with gene expression.Altered DNA methylation and expression of various genes suggested their roles and potential functional interactions upon DON exposure.Our data set offers a layer of epigenetic insights for comprehensively deciphering the molecular mechanisms underlying DON toxic effects on the intestinal cells,and revealed the changes in gene expression patterns at genom-wide.Our findings may advance the identification of biomarkers and drug targets for predicting and controlling the toxic effects of this common mycotoxin.