The role of Tet1 in myofibroblast differentiation

项目概要： 肌成纤维细胞是分化的间充质细胞，其在发育、癌症、组织病理学和免疫组织化学中具有确定的作用。 修复/重塑。肌成纤维细胞分化的调节涉及多种信号通路， 各自的下游转录因子以及表观遗传因子的显著调节， DNA甲基化大量的研究集中在转录因子的鉴定及其调控作用上 α-平滑肌肌动蛋白（Acta 2）基因作为分化的标志物，但表观遗传的机制， 规则还不太清楚。Acta 2基因中的选择性CpG岛在小鼠中差异甲基化。 肌成纤维细胞与成纤维细胞和上皮细胞。DNA甲基化损伤促进分化 匡威则抑制分化。通过泰特（10 - 11易位）蛋白进行主动去甲基化， 是甲基胞嘧啶双加氧酶，与细胞分化有关。它们在细胞中的重要性 胚胎干细胞和其他干细胞的研究表明，分化，但它们在调节 肌成纤维细胞分化是未知的。初步数据显示Tet 1的选择性诱导，而不是另一个 2种亚型（Tet 2和Tet 3）在肌成纤维细胞分化中的作用，而Tet 1缺乏导致分化受损 无论是在体内还是体外。因此，Tet 1亚型的选择重要性，建议在分化。而 Tet 1并不通过去甲基化来全局调节所有基因，至少一个Tet 1调节靶点的身份 Acta 2基因，可以从初步数据推断。根据以前的调查结果和初步数据， 我们假设Tet 1通过调控基因的去甲基化来调节肌成纤维细胞分化， 选择分化过程所必需的靶基因中的DNA序列。选择招聘到 相关靶DNA序列由Tet 1结合蛋白/转录因子介导， 对这些DNA区域的特异性。为了检验这一假设，具体目标如下：1）筛选和 确定Tet 1在成纤维细胞中肌成纤维细胞分化中重要的靶基因，2）研究Tet 1的机制， Tet 1对肌成纤维细胞分化所必需的关键靶基因的调控，以及3）评估体内 Tet 1调控肌成纤维细胞分化意义这些研究将使用表观基因组方法， 评估差异甲基化基因和转基因小鼠，以评估Tet 1的生物学重要性， 在体内选择特定细胞类型中的分化相关基因。这些目标的实现将为我们提供新的视角， 与发育、癌症和组织相关的肌成纤维细胞分化的表观遗传调节 修复/重塑。

Project Summary: Myofibroblasts are differentiated mesenchymal cells with identified roles in development, cancer, tissue repair/remodeling. Regulation of myofibroblast differentiation involves multiple signaling pathways and respective downstream transcription factors as well as significant modulation by epigenetic factors such as DNA methylation. Abundant studies focused on identification of transcription factors and their role in regulation of the α-smooth muscle actin (Acta2) gene as a marker of differentiation, but the mechanism of epigenetic regulation is not as well understood. Select CpG islands in the Acta2 gene are differentially methylated in myofibroblasts vs. fibroblasts and epithelial cells. Impairment of DNA methylation enhances differentiation while the converse inhibits differentiation. Active demethylation by the Tet (Ten-eleven translocation) proteins, which are methylcytosine dioxygenases, is implicated in cell differentiation. Their importance in cell differentiation is suggested in studies of embryonal and other stem cells, but their significance in regulation of myofibroblast differentiation is unknown. Preliminary data showed selective induction of Tet1 and not the other 2 isoforms (Tet2 and Tet3) in myofibroblast differentiation, while Tet1 deficiency caused impaired differentiation both in vivo and in vitro. Thus the selective importance of the Tet1 isoform is suggested in differentiation. While Tet1 does not globally regulate all genes by demethylation, the identity of at least one Tet1 regulated target gene, Acta2 could be inferred from the preliminary data. Based on the previous findings and preliminary data we hypothesized that Tet1 importantly regulates myofibroblast differentiation by demethylation of regulatory DNA sequences in select target genes essential for the differentiation process. Select recruitment to the relevant target DNA sequences is mediated by Tet1 binding proteins/transcription factors with binding specificity for these DNA regions. To test this hypothesis the Specific Aims are as follows, 1) to screen for and identify Tet1 target genes important in myofibroblast differentiation in fibroblasts, 2) to study the mechanism of Tet1 regulation of key target genes essential for myofibroblast differentiation, and 3) to assess the in vivo significance of Tet1 regulation of myofibroblast differentiation. The studies will use epigenomic approaches to assess differentially methylated genes and transgenic mice to evaluate the biological importance of Tet1 and select differentiation relevant genes in specific cell types in vivo. Achievement of the goals will shed new light on the epigenetic regulation of myofibroblast differentiation of relevance to development, cancer and tissue repair/remodeling.