Tet2蛋白在成肌细胞体外分化的调控机制研究

发布时间：2018-06-16 20:48

  本文选题：Tet2 + 5hmC　； 参考：《中国农业大学》2016年博士论文

【摘要】：DNA甲基化是一种非常重要的表观遗传调控方式,参与细胞多种生理活动,在基因印迹、X染色体失活、染色质修饰及调控基因表达中发挥重要的作用。近年来发现的Pet (ten-eleven translocation)蛋白家族包括3个成员Tet1、Tet2和Tet3,均属于依赖α-酮戊二酸(a-KG)和Fe2+的DNA双加氧酶,能够催化5-甲基胞嘧啶(5-mC)转化为5-羟甲基胞嘧啶(5-hmC),是DNA去甲基化过程中一种至关重要的酶。由于Tet蛋白在DNA去甲基化过程中的关键性作用,近年来一直是研究的热点,特别是在胚胎干细胞分化、胚胎发生、发育及肿瘤的发生等过程。但是很少有报道涉及Tet蛋白在肌肉发育分化中的调控功能。然而,肌肉组织对机体是十分重要的,其中骨骼肌部分又是机体运动系统的动力部分,因此有必要对其发育调控进行深入和系统的研究。本课题的研究目的在于揭示Tet蛋白在骨骼肌细胞发育分化过程中的调控机制。首先,诱导小鼠成肌细胞系C2C12分化形成多核的肌管后,发现细胞内5hmC水平上升,同时5mC水平下降。而且,分化后Tet1和Tet2表达水平上升且一直维持较高的表达水平,并通过免疫荧光染色检测出Tet2主要定位在成肌细胞和肌管的细胞核中。于是,进一步研究了在成肌细胞中过表达或抑制Tetl和Tet2功能对肌特异基因表达的影响。首先构建了Tetl和Tet2催化活性域的真核表达载体,并瞬时转染至C2C12细胞中。qPCR结果表明在成肌细胞中,与分化相关的基因myogenin, Myf6和myomaker仅在Tet2过表达时其转录水平发生显著上升；而在Tetl过表达时,肌特异基因的表达水平并没有显著的变化。然后,利用siRNA分别抑制未分化的C2C12细胞中Tet1和Tet2的表达,发现同样仅当Tet2受抑制时,与分化相关的基因myogenin, Myf6和myomaker表达水平显著下调,说明是Tet2而非Tetl蛋白与成肌细胞的分化相关。接着,利用shRNA建立了稳定抑制Tet2表达的C2C12细胞系。研究发现Tet2抑制的C2C12细胞在诱导分化时,肌管形成率明显降低,同时与分化相关的基因表达水平在分化前和分化后的细胞中都发生了下调。亚硫酸氢盐测序的结果也表明Tet2抑制的细胞中,myogenin和myomaker启动子区的甲基化水平降低。以上结果证明,Tet2通过调节与分化相关基因启动子区的甲基化水平,增强基因的表达,从而促进成肌细胞的分化。另外,最近的研究表明维生素C能够增强Tet蛋白酶的催化活性。首先验证了维生素C在C2C12细胞中能够促进5hmC的生成,且具有剂量依赖效应。接着,维生素C对肌肉的分化具有促进作用。通过降低肌肉分化相关基因启动子区甲基化水平,促进相关基因的表达,显著提高了成肌细胞的分化效率。但是,在Tet2抑制的细胞中维生素C的促分化功能被显著的削弱了。主要表现为维生素C在Tet2抑制的成肌细胞和分化后的肌管中,均不能促进myogenin, Myf6和myomaker的表达,且5hmC的生成也减弱了,另外分化效率也受到了一定程度抑制。由此表明,维生素C是依赖于Tet2基因的调控通路参与调节成肌细胞的分化过程,同时也证明了Tet2是调控成肌细胞分化的关键因子。综上,Tet2在成肌细胞分化过程中高水平表达,并通过介导分化相关基因启动子区的DNA去甲基化促进基因的表达,从而调控骨骼肌细胞的分化过程。另外,维生素C是依赖于Tet2基因的调控作用促进成肌细胞分化的。
[Abstract]:DNA methylation is a very important epigenetic regulation. It plays an important role in many cell physiological activities. It plays an important role in gene imprinting, X chromosome inactivation, chromatin modification and regulation of gene expression. In recent years, the Pet (ten-eleven translocation) family, including 3 members, Tet1, Tet2 and Tet3, are all dependent on alpha. The DNA dioxygenase of ketopamyl diacid (a-KG) and Fe2+ can catalyze the conversion of 5- methylcytosine (5-mC) into 5- hydroxymethylcytosine (5-hmC), which is a vital enzyme in the process of DNA demethylation. Due to the key role of Tet protein in the process of demethylation of DNA, it has been a hot spot in recent years, especially in embryonic stem cells. Embryogenesis, development and the occurrence of tumor, but few reports involve the regulatory function of Tet protein in the development of muscle development. However, the muscle tissue is very important to the body, and the skeletal muscle part is the dynamic part of the body's motion system. Therefore, it is necessary to conduct in-depth and systematic Research on its development and regulation. The purpose of this study is to reveal the regulatory mechanism of Tet protein in the development and differentiation of skeletal muscle cells. First, after inducing the myoblast C2C12 to form a multicore myotube, the level of 5hmC in the cell is increased and the level of 5mC is decreased. Moreover, the expression level of Tet1 and Tet2 is increased and the high expression water has been maintained after the differentiation. Tet2 was detected mainly in the nuclei of myoblasts and myotubes by immunofluorescence staining. Thus, the effects of overexpression or inhibition of Tetl and Tet2 on the expression of specific genes in myoblasts were further studied. First, the eukaryotic expression vector of Tetl and Tet2 catalytic active domains was constructed and transfected to C2C12 fine. .qPCR results in the cell showed that in myoblasts, the transcriptional level of myogenin, Myf6 and myomaker was significantly increased when Tet2 was overexpressed, while the expression level of myo specific genes was not significantly changed when Tetl was overexpressed. Then, Tet1 and Tet2 in undifferentiated C2C12 cells were suppressed respectively by siRNA. It was found that when Tet2 was inhibited, the expression level of myogenin, Myf6 and myomaker associated with differentiation was significantly down, indicating that Tet2 but not Tetl protein was associated with the differentiation of myoblasts. Then, C2C12 cell lines were established with shRNA to stabilize the expression of Tet2. It was found that C2C12 cells inhibited by Tet2 were induced in differentiation, The formation rate of myotubes decreased significantly, and the gene expression levels associated with differentiation were downregulated before and after differentiation. The results of hydrogen sulfite sequencing also showed that the level of methylation in the myogenin and myomaker promoter regions decreased in Tet2 inhibited cells. The results showed that Tet2 regulated the genes associated with differentiation. The methylation level in the promoter region increases the expression of genes to promote the differentiation of myoblasts. In addition, recent studies have shown that vitamin C can enhance the catalytic activity of Tet protease. First, vitamin C can promote the production of 5hmC in C2C12 cells, and it has a dose dependent effect. Then, the differentiation of vitamin C to the muscles is made. Promoting the differentiation of myoblasts by reducing the level of methylation in the promoter region of the muscle differentiation related genes and promoting the expression of related genes, significantly improving the differentiation efficiency of myoblasts. However, the differentiation function of vitamin C in Tet2 inhibited cells is significantly weakened. It is mainly manifested in the myoblast and differentiation of vitamin C in Tet2. In myotubes, the expression of myogenin, Myf6 and myomaker can not be promoted, and the formation of 5hmC is also weakened, and the differentiation efficiency is also suppressed to a certain extent. Therefore, vitamin C is dependent on the regulatory pathway of the Tet2 gene to regulate the differentiation process of myoblast, and it is also proved that Tet2 is the key to modulate the differentiation of myocytes. Factors. In conclusion, Tet2 is expressed at a high level in the process of myoblast differentiation, and promotes the expression of gene by mediating DNA demethylation of differentiation related gene promoter region, thus regulating the differentiation process of skeletal muscle cells. In addition, vitamin C is dependent on the regulation of Tet2 gene to promote myoblast differentiation.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q254
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本文编号：2028058