Characterization of DNA methylation and transcriptomics during early pregnancy in dairy cows
DNA methylation is an essential epigenetic mechanism involved in cell differentiation, proliferation, apoptosis, and metabolism. However, the DNA methylation changes of related genes during early pregnancy in dairy cows remain unclear . The purpose of this study was to study DNA methylation-related changes in early pregnancy cows and construct gene pools associated with pregnancy. In this study, we used peripheral blood samples from 15 healthy, productive and similar cows at 30 days post-breeding (Pre30) and 10 non-pregnant cows (Ctrl) and association analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and Quantitative Real-time PCR (RT-qPCR) method are used to verify the Methylation differential genes. Our findings showed that DNA methylation majorly occurred in the cytosine-guanine (CG) range, and the amount of hypomethylation was higher than that of hypermethylation in 3Utrs, introns and promoters. A total of 76530 differentially methylated sites (DMCs) and 16411 differentially methylated regions (DMRs) were identified. RNA-seq identified 8858 differentially expressed genes (DEGs), with 4499 down-DEGs and 4359 up-DEGs. However, there were 44 significant differentially expressed genes, including 25 up-DEGs and 19 down-DEGs. DMC gene ontology (GO) analysis revealed significant enrichment in developmental process, cell differentitaion and nevous system development in biological processes (BP);Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer overview and cellular and Gastric cancer and Immune disease signaling pathways. DMRs GO analysis highlighted significant enrichment in the developmental process, cell differentiation and nervous system development; KEGG the results showed that significant enrichment in Signaling pathways regulating pluripotency of stem cells, Systemic lupus erythematosus,Transcriptional misregulation in cancer,Spliceosome,Herpes simplex virus 1 infection, Maturity onset diabetes of the young. DEG GO analysis results show that the main enrichment in BP differentiation of bone marrow cells, red blood cell differentiation and erythrocyte steady-state and anion during transportation and transshipment; KEGG analysis mainly concentrated in retinol metabolism, purine metabolism, cAMP signaling pathway and Gap junction pathway. Through a comprehensive analysis of RRBS and transcriptomics, 12 genes, including HBB, HBA 1, GPR 4, CPXM 2, SLC20A2, AFAP 1 and DOK 7, were different both in the methylome and the transcriptome. Early pregnancy changes in DNA methylation were significant and mainly down-regulated. These results can be the epigenetic changes in early pregnancy for cows provide new insights. Overall design: To investigate the changes in methylation associated with early pregnancy in cows, we used peripheral blood from 15 cows 30 days post-breeding (Pre30) and 10 non-pregnant cows (Ctrl).There were two groups, each with three biological replicates.