Epigenetic Basis for the Establishment of Ruminal Tissue-Specific Functions (ChIP-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. 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 GSK3β 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.

尽管其他组织的DNA甲基化可通过模式生物进行推测，但反刍动物特有器官瘤胃的DNA甲基化动态分布及其调控意义，在很大程度上仍未明确。本研究采用全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing, WGBS）、转录组学（transcriptomics）及组蛋白修饰（histone modification）数据，对牛瘤胃的胎儿期与成年期样本，以及包括模拟植入前胚胎的多能干细胞（pluripotent stem cells, PSCs）在内的其他组织进行了比较分析。我们发现，胎儿期与成年期瘤胃及其他组织、PSCs之间存在广泛的甲基化差异，涵盖CG甲基化（CG methylation, mCG）与非CG甲基化（non-CG methylation, mCH；其中H代表A、C、T）。此类差异甲基化区域（differentially methylated regions, DMRs）与转录因子（transcription factors, TFs）、组蛋白修饰等其他表观遗传调控元件密切相关。此类DMRs还可协同形成大片段低甲基化CG-DMR，调控一组功能相关的基因。本研究从表观遗传层面阐明了胎儿期与成年期瘤胃在形态与功能上产生差异的原因，以及表观遗传修饰与基因表达之间的互作机制。本研究凸显了发育过程中瘤胃与其他组织的甲基化模式差异，以及DNA甲基化在调控基因表达、建立组织特异性功能中的作用。为评估牛发育过程中的胞嘧啶DNA甲基化图谱，我们生成了荷斯坦奶牛胎儿组织（前脑、后脑、睾丸、心脏、肝脏、肺脏、肾脏、肌肉、瘤胃）及多种牛多能干细胞（bESCs-F7、bESCs-F7-5、bESCs-F7-50、bEPSCs-AGS、bEPSCs-B18）的测序数据。牛胚胎干细胞系的培养：牛胚胎干细胞系bESCs-F7采用CTFR系统构建。针对bESCs-F7，我们采用两种MM-102处理方案：其一为用50μM高浓度MM-102处理7天，随后正常培养5代后进行鉴定，标记为bESCs-F7-102 (50)；其二为用5μM低浓度MM-102进行长期处理，培养5代后完成鉴定，标记为bESCs-F7-102 (5)。牛诱导多能干细胞及bEPSCs-B18细胞系采用LCDM系统构建。而bEPSCs-AGS则采用500mL培养体系，其培养基成分为：485mL基础mTeSR1培养基、5.0mL 100×青霉素-链霉素溶液、0.1mM 2-巯基乙醇、1μM GSK3β抑制剂CHIR99021、0.3μM Lck/Src抑制剂WH-4-023、5μM 端锚聚合酶抑制剂XAV939、5μM 经典WNT信号通路抑制剂IWR-1、50μg/mL 维生素C、10ng/mL 白血病抑制因子（Leukemia Inhibitory Factor, LIF）以及20.0ng/mL 激活素A（Activin A），用于培养牛胚胎多能干细胞。