基于ChIP-seq对山羊成纤维细胞转分化为乳腺上皮细胞的研究

Analysis of Goat(Capra hircus)Fibroblasts Transdifferentiation into Mammary Epithelial Cells Based on ChIP-seq

目前小分子化合物诱导山羊(Capra hircus)耳缘成纤维细胞(对照组)转分化为诱导乳腺上皮细胞(命名为CiMECs或5i8d,后文均以5i8d为准表明其处理条件)的技术平台已经被建立。本研究对转分化前后的细胞(分别命名为Control和5i8d)的两种组蛋白修饰,即组蛋白第三亚基4号赖氨酸的三甲基化(trimethylation of lysine 4 on histone H3 protein subunit,H3K4me3)和组蛋白第三亚基27号赖氨酸的三甲基化(trimethylation of lysine 27 on histone H3 protein subunit,H3K27me3)分别进行染色质免疫共沉淀实验(chromatin immunoprecipitation,ChIP),并进行测序分析,以探讨组蛋白修饰H3K4me3和H3K27me3在转分化前后细胞中表达模式的变化。GO富集显示H3K4me3中差异Peaks关联的基因富集在与乳腺发育分化相关的通路,包括上皮形态发生、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联调节、Wnt(Wingless-type MMTV integration site family)受体通路以及β-连环蛋白(β-catenin)受体通路,提示乳腺上皮细胞命运已经被激活;H3K27me3中差异Peaks关联的基因则富集在固有膜、固有质膜以及肌动蛋白丝等GO条目上,提示维持成纤维细胞膜及其骨架特性的通路被抑制,成纤维细胞的谱系被打破。KEGG富集显示H3K4me3中差异Peaks关联的基因富集在MAPK信号通路、胰岛素及分泌信号通路、磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-Akt)信号通路以及雌激素信号通路等,这些信号通路与乳腺发育分化密切相关,提示乳腺上皮细胞的谱系被触发;H3K27me3中差异Peaks关联的基因主要富集在黏着斑信号通路、细胞外基质(extracellular matrix,ECM)受体互作信号通路和细胞黏附因子等KEGG信号通路上,提示成纤维细胞间连接特性的有关通路被抑制。上述结果说明小分子化合物成功诱导山羊耳缘成纤维细胞转分化为具有泌乳功能的乳腺上皮细胞。该研究为乳腺上皮细胞命运决定、发育分化和泌乳的研究提供了一定的理论支持。

At present,a technical platform for inducing goat(Capra hircus)ear margin fibroblasts control group to transdifferentiate into mammary epithelial cells(named CiMECs or 5i8d,the latter will be subject to 5i8d to indicate the treatment conditions)has been established by small molecular compounds.In this study,the trimethylation of lysine 4 on histone H3 protein subunit(H3K4me3)and the trimethylation of lysine 27 on histone H3 protein subunit(H3K27me3)of chromatin immunoprecipitation(ChIP)were sequenced in cells before and after transdifferentiation(Control and 5i8d)to explore the changes of H3K4me3 and H3K27me3 expression patterns in cells before and after transdifferentiation.The differential Peaks-related genes of H3K4me3 were enriched in the pathways associated with mammary gland development and differentiation GO items,including epithelial morphogenesis,regulation of MAPK(Mitogen activated protein kinase)cascade,Wnt(Wingless type MMTV integration site family)receptor pathway andβ-catenin receptor pathway which indicated that the fate of mammary epithelial cells had been activated,the differential Peaks-related genes of H3K27me3 were enriched in Go items such as lamina propria,plasma propria and actin filaments,suggesting that the pathways maintained fibroblast membrane and its skeletal properties were inhibited,furthermore fibroblast lineage was broken.The differential Peaks-related genes of H3K4me3 were enriched in these KEGG pathway,such as MAPK signaling pathway,insulin and secretion signaling pathway,PI3K-Akt(Phosphatidylinositol 3-kinase protein kinase B)signaling pathway and estrogen signaling pathway.Moreover,these signaling pathways were closely related to mammary gland development and differentiation,showing that the lineage of mammary gland epithelial cells was triggered.The differential Peaks-related genes of H3K27me3 were mainly enriched in KEGG signaling pathways-adhesion plaque signaling pathway,ECM receptor interaction signaling pathway and cell adhesion factor,manifesting that the pathways related to the connectivity between fibroblasts were inhibited.Above all,it was inferred that the cell lineage of goat ear margin fibroblasts transformed from fibroblasts to mammary gland epithelial cells with lactation after eight days of this successful induction.The study provides some theoretical support for the study of fate determination,developmental differentiation and lactation.