本文選題：Tet2 + 5hmC�。� 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：DNA甲基化是一種非常重要的表觀遺傳調(diào)控方式,參與細(xì)胞多種生理活動(dòng),在基因印跡、X染色體失活、染色質(zhì)修飾及調(diào)控基因表達(dá)中發(fā)揮重要的作用。近年來(lái)發(fā)現(xiàn)的Pet (ten-eleven translocation)蛋白家族包括3個(gè)成員Tet1、Tet2和Tet3,均屬于依賴α-酮戊二酸(a-KG)和Fe2+的DNA雙加氧酶,能夠催化5-甲基胞嘧啶(5-mC)轉(zhuǎn)化為5-羥甲基胞嘧啶(5-hmC),是DNA去甲基化過(guò)程中一種至關(guān)重要的酶。由于Tet蛋白在DNA去甲基化過(guò)程中的關(guān)鍵性作用,近年來(lái)一直是研究的熱點(diǎn),特別是在胚胎干細(xì)胞分化、胚胎發(fā)生、發(fā)育及腫瘤的發(fā)生等過(guò)程。但是很少有報(bào)道涉及Tet蛋白在肌肉發(fā)育分化中的調(diào)控功能。然而,肌肉組織對(duì)機(jī)體是十分重要的,其中骨骼肌部分又是機(jī)體運(yùn)動(dòng)系統(tǒng)的動(dòng)力部分,因此有必要對(duì)其發(fā)育調(diào)控進(jìn)行深入和系統(tǒng)的研究。本課題的研究目的在于揭示Tet蛋白在骨骼肌細(xì)胞發(fā)育分化過(guò)程中的調(diào)控機(jī)制。首先,誘導(dǎo)小鼠成肌細(xì)胞系C2C12分化形成多核的肌管后,發(fā)現(xiàn)細(xì)胞內(nèi)5hmC水平上升,同時(shí)5mC水平下降。而且,分化后Tet1和Tet2表達(dá)水平上升且一直維持較高的表達(dá)水平,并通過(guò)免疫熒光染色檢測(cè)出Tet2主要定位在成肌細(xì)胞和肌管的細(xì)胞核中。于是,進(jìn)一步研究了在成肌細(xì)胞中過(guò)表達(dá)或抑制Tetl和Tet2功能對(duì)肌特異基因表達(dá)的影響。首先構(gòu)建了Tetl和Tet2催化活性域的真核表達(dá)載體,并瞬時(shí)轉(zhuǎn)染至C2C12細(xì)胞中。qPCR結(jié)果表明在成肌細(xì)胞中,與分化相關(guān)的基因myogenin, Myf6和myomaker僅在Tet2過(guò)表達(dá)時(shí)其轉(zhuǎn)錄水平發(fā)生顯著上升；而在Tetl過(guò)表達(dá)時(shí),肌特異基因的表達(dá)水平并沒(méi)有顯著的變化。然后,利用siRNA分別抑制未分化的C2C12細(xì)胞中Tet1和Tet2的表達(dá),發(fā)現(xiàn)同樣僅當(dāng)Tet2受抑制時(shí),與分化相關(guān)的基因myogenin, Myf6和myomaker表達(dá)水平顯著下調(diào),說(shuō)明是Tet2而非Tetl蛋白與成肌細(xì)胞的分化相關(guān)。接著,利用shRNA建立了穩(wěn)定抑制Tet2表達(dá)的C2C12細(xì)胞系。研究發(fā)現(xiàn)Tet2抑制的C2C12細(xì)胞在誘導(dǎo)分化時(shí),肌管形成率明顯降低,同時(shí)與分化相關(guān)的基因表達(dá)水平在分化前和分化后的細(xì)胞中都發(fā)生了下調(diào)。亞硫酸氫鹽測(cè)序的結(jié)果也表明Tet2抑制的細(xì)胞中,myogenin和myomaker啟動(dòng)子區(qū)的甲基化水平降低。以上結(jié)果證明,Tet2通過(guò)調(diào)節(jié)與分化相關(guān)基因啟動(dòng)子區(qū)的甲基化水平,增強(qiáng)基因的表達(dá),從而促進(jìn)成肌細(xì)胞的分化。另外,最近的研究表明維生素C能夠增強(qiáng)Tet蛋白酶的催化活性。首先驗(yàn)證了維生素C在C2C12細(xì)胞中能夠促進(jìn)5hmC的生成,且具有劑量依賴效應(yīng)。接著,維生素C對(duì)肌肉的分化具有促進(jìn)作用。通過(guò)降低肌肉分化相關(guān)基因啟動(dòng)子區(qū)甲基化水平,促進(jìn)相關(guān)基因的表達(dá),顯著提高了成肌細(xì)胞的分化效率。但是,在Tet2抑制的細(xì)胞中維生素C的促分化功能被顯著的削弱了。主要表現(xiàn)為維生素C在Tet2抑制的成肌細(xì)胞和分化后的肌管中,均不能促進(jìn)myogenin, Myf6和myomaker的表達(dá),且5hmC的生成也減弱了,另外分化效率也受到了一定程度抑制。由此表明,維生素C是依賴于Tet2基因的調(diào)控通路參與調(diào)節(jié)成肌細(xì)胞的分化過(guò)程,同時(shí)也證明了Tet2是調(diào)控成肌細(xì)胞分化的關(guān)鍵因子。綜上,Tet2在成肌細(xì)胞分化過(guò)程中高水平表達(dá),并通過(guò)介導(dǎo)分化相關(guān)基因啟動(dòng)子區(qū)的DNA去甲基化促進(jìn)基因的表達(dá),從而調(diào)控骨骼肌細(xì)胞的分化過(guò)程。另外,維生素C是依賴于Tet2基因的調(diào)控作用促進(jìn)成肌細(xì)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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q254
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本文編號(hào)：2028058