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.

DNA甲基化（DNA methylation）是一类关键的表观遗传调控机制，参与细胞分化、增殖、凋亡及代谢过程。然而，奶牛早期妊娠过程中相关基因的DNA甲基化变化仍有待阐明。本研究旨在探究奶牛早期妊娠中的DNA甲基化相关调控变化，并构建与妊娠相关的基因池。本研究采集了15头配种后30天的健康高产同龄奶牛（Pre30组）与10头未妊娠奶牛（Ctrl组）的外周血样本，采用简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）与转录组测序技术开展关联分析；并通过靶向亚硫酸氢盐测序（Targeted bisulfite sequencing, TBS）与实时定量聚合酶链式反应（Quantitative Real-time PCR, RT-qPCR）对差异甲基化基因进行验证。本研究结果显示，DNA甲基化主要发生于胞嘧啶-鸟嘌呤（Cytosine-Guanine, CG）位点区域；在3'非翻译区（3'UTR）、内含子及启动子区域中，低甲基化位点数量高于高甲基化位点。共计鉴定得到76530个差异甲基化位点（Differentially Methylated Sites, DMCs）与16411个差异甲基化区域（Differentially Methylated Regions, DMRs）。转录组测序（RNA-seq）共鉴定得到8858个差异表达基因（Differentially Expressed Genes, DEGs），其中4499个为下调差异表达基因（down-DEGs），4359个为上调差异表达基因（up-DEGs）；但其中仅44个为显著差异表达基因，包含25个上调基因与19个下调基因。差异甲基化位点的基因本体论（Gene Ontology, GO）富集分析显示，其在生物过程（Biological Process, BP）中显著富集于发育过程、细胞分化及神经系统发育等条目；京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析则显示，其显著富集于癌症概览、细胞通路、胃癌及免疫疾病相关信号通路。差异甲基化区域的GO富集分析同样显示其显著富集于发育过程、细胞分化及神经系统发育；KEGG富集分析结果表明，其显著富集于干细胞多能性调控通路、系统性红斑狼疮、癌症转录失调、剪接体、单纯疱疹病毒1型感染及青少年发病的成年型糖尿病相关通路。差异表达基因的GO富集分析结果显示，其主要富集于骨髓细胞分化、红细胞分化、红细胞稳态及阴离子转运等生物过程条目；KEGG富集分析则主要集中于视黄醇代谢、嘌呤代谢、cAMP信号通路及间隙连接通路。通过对RRBS数据与转录组数据的联合分析，共筛选得到12个在甲基化组与转录组中均存在显著差异的基因，包括HBB、HBA1、GPR4、CPXM2、SLC20A2、AFAP1及DOK7等。奶牛早期妊娠过程中的DNA甲基化变化显著，且整体以下调为主。本研究结果可为奶牛早期妊娠的表观遗传调控机制研究提供全新视角。实验整体设计：为探究奶牛早期妊娠相关的甲基化变化，本研究采集了15头配种后30天的奶牛（Pre30组）与10头未妊娠奶牛（Ctrl组）的外周血样本，分为两组，每组设置3个生物学重复。