The role of Tet1 in myofibroblast differentiation

项目总结： 肌成纤维细胞是一种分化的间充质细胞，在发育、癌症、组织发育中起重要作用。 修理/改建。肌成纤维细胞分化的调控涉及多条信号通路和 各自的下游转录因子以及表观遗传因子的显著调控，如 DNA甲基化。大量的研究集中在转录因子的识别及其在调控中的作用 α-平滑肌肌动蛋白(Acta2)基因作为分化的标志，但表观遗传学的机制 人们对监管的理解还不够深入。Acta2基因中的部分CpG岛存在差异甲基化 肌成纤维细胞与成纤维细胞和上皮细胞。DNA甲基化障碍促进分化 反之则抑制分化。Tet(Ten-11易位)蛋白的活性去甲基化， 它们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 分化是在胚胎干细胞和其他干细胞的研究中提出的，但它们在调节 肌成纤维细胞的分化尚不清楚。初步数据显示，有选择性地诱导了Tet1而不是另一种 肌成纤维细胞分化中的2种亚型(TET2和Tet3)，而Tet1缺乏则导致分化障碍 无论是在体内还是体外。因此，Tet1亚型在分化过程中具有选择性的重要性。而当 Tet1不是通过去甲基化来全局调节所有基因，去甲基化是至少一个Tet1调节的靶标 从初步数据中可以推断出Acta2基因。根据之前的调查结果和初步数据 我们推测，Tet1通过去甲基化调节基因对肌成纤维细胞分化起重要调节作用。 选择对分化过程至关重要的靶基因中的DNA序列。选择招聘到 相关的靶DNA序列由Tet1结合蛋白/转录因子结合介导 这些DNA区域的特异性。为了检验这一假设，具体目的如下：1)筛选和 筛选成纤维细胞中重要的肌成纤维细胞分化相关基因Tet1，2)研究Tet1的作用机制 Tet1对肌成纤维细胞分化所必需的关键靶基因的调控；3)体内评估 Tet1调控肌成纤维细胞分化的意义。这些研究将使用表观基因组学方法来 评估差异甲基化基因和转基因小鼠以评估Tet1和Tet1的生物学重要性 在体内筛选特定细胞类型的分化相关基因。实现这些目标将带来新的曙光 肌成纤维细胞分化与发育、肿瘤和组织的表观遗传调控 修理/改建。

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.