Epigenetic Basis for the Establishment of Ruminal Tissue-Specific Functions (CUT&Tag-seq)

While DNA methylation in other tissues can be approximated through model species, the dynamic distribution and regulatory significance of DNA methylation in the rumen, a unique organ in ruminant, remain largely unknown. Here, we employed whole-genome bisulfite sequencing (WGBS), transcriptomics, and histone modification data to compare fetal and adult stages of bovine rumen with other tissues, including pluripotent stem cells (PSCs) approximating pre-implantation embryos. We found extensive methylation differences, including CG methylation (mCG) and non-CG methylation (mCH; H represents A, C and T) between the rumen at fetal and adult stages and other tissues and PSCs. These differentially methylated regions (DMRs) are closely associated with other epigenetic regulatory components, such as transcription factors (TFs) and histone modifications. These DMRs can also combine to form large hypo CG-DMRs to regulate a cluster of functionally related genes. We elucidated the reasons for morphological and functional differences between fetal and adult rumen at the epigenetic level and the interactions between epigenetic modifications and gene expression. This study highlights the differences in methylation patterns between the rumen and other tissues during development and the role of DNA methylation in controlling gene expression and establishing tissue-specific functions. Overall design: To assess the cytosine DNA methylation landscape during bovine development, we generated sequencing data for Holstein bovine fetal tissues (forebrain, hindbrain, testes, heart, liver, lung, kidney, muscle, rumen) and various bovine pluripotent stem cells (bESCs-F7, bESCs-F7-5, bESCs-F7-50, bEPSCs-AGS, bEPSCs-B18). Culture of bovine embryonic stem cell lines: The bovine embryonic stem cell line bESCs-F7 was established using the CTFR system. Two treatment methods were employed for bESCs-F7 using MM-102: one involved treating bESCs-F7 with a high concentration (50 µM) of MM-102 for 7 days, followed by normal culturing for 5 passages before characterization, labeled as bESCs-F7-102 (50). The other involved long-term treatment with a low concentration (5 µM) of MM-102, followed by characterization after 5 passages of culturing, labeled as bESCs-F7-102 (5). Bovine induced pluripotent stem cells and the bEPSCs-B18 cell line were established using the LCDM system. And bEPSCs-AGS was using a 500mL system, including 485mL of basic mTeSR1 medium, 5.0 mL of 100×Penicillin-Streptomycin Solution, 0.1 mM 2-mercaptoethanol, 1 µM GSK3ß inhibitor CHIR99021, 0.3 µM Lck/Src inhibitor WH-4-023, 5 µM Tankyrase inhibitor XAV939, 5 µM classic WNT signaling pathway inhibitor IWR-1, 50 µg/mL Vitamin C, 10 ng/mL Leukemia Inhibitory Factor (LIF), and 20.0 ng/mL Activin A for culturing bovine Embryonic Pluripotent Stem Cells.