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题名: 镍化合物抑制DNA羟甲基化效应与机理研究
作者: 尹瑞川
学位类别: 博士后
答辩日期: 2016-01
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 汪海林 ; 刘思金
关键词: DNA甲基化、DNA去甲基化、Tet双加氧酶、5’-甲基胞嘧啶氧化产物、镍化合物、尿液5hmC分析、碳酸氢铵增强效应 ; DNA methylation, DNA demethylation, Tet dioxygenases, nickel compounds, 5’- methylcytosine oxidation products, urinary 5hmC analysis, ammonium bicarbonate enhanced effects
其他题名: Studies of the mechanisms of nickel inhibition on Tet-mediated DNA demethylation
学位专业: 生物学
中文摘要:      Tet(Tet-eleven translocation)双加氧酶能够氧化5-甲基胞嘧啶(5mC),生成三种新的DNA修饰—5-羟甲基胞嘧啶(5hmC)、5-醛甲基胞嘧啶(5fC)和5-羧甲基胞嘧啶(5caC)。该家族在哺乳动物DNA去甲基化过程中发挥着关键的作用:通过生成5hmC,Tet蛋白质促使哺乳动物发生依赖于DNA复制的被动去甲基化过程;通过5fC和5caC,Tet蛋白质还能介导快速的、主动的DNA去甲基化。Tet蛋白质氧化功能的异常与癌症密切相关。Tet2基因突变体普遍存在于恶性骨髓肿瘤和淋巴肿瘤中,并在这些癌组织基因组中5hmC含量显著降低。除了Tet基因改变以外,我们课题组发现小分子物质(如维生素C和卤代苯醌)同样能影响到Tet蛋白质的功能,进而改变DNA甲基化状态。因而,研究其它小分子物质(如镍化合物)对Tet蛋白质功能的影响具有重要的意义。
     镍化合物是各种环境介质中广泛存在的一类物质,包括大气、水体、土壤和植物等。除了自然来源外,镍金属及其化合物被大量用于工业。全球约有几百万工人暴露在含有高浓度镍的颗粒、飞尘和烟雾之中。镍化合物已经被国际癌症组织归类为已知的致癌物质。镍化合物不容易引起哺乳动物细胞内发生基因突变,而且其DNA损伤能力较弱。因而,镍化合物可能通过调控表观遗传修饰,特别是改变基因甲基化状态,进而引起癌症的形成。镍化合物能够引起特定基因高度甲基化,降低了这些基因的表达,其中包含抑癌基因(p16、RAR‐β2和CDKN2A)和DNA损伤修复基因(MGMT)。这些基因启动子高度甲基化的现象经常出现在各种癌症组织中。
     然而,至今不能解释镍化合物引起DNA高度甲基化的机理。我们猜想镍化合物通过抑制Tet蛋白质氧化5mC的能力,进而阻碍Tet介导的DNA去甲基化,导致基因高甲基化。为了证明猜想,我们考查了镍离子对体细胞、Tet转染细胞和小鼠胚胎干细胞5mC及其氧化产物的效应。在体细胞(293T和MRC5)内,镍离子能显著抑制生成5hmC(8.6 - 64 %),但由于这些细胞内Tet蛋白质表达低,镍离子的抑制作用还不足于改变整体5mC水平。以Tet1CD过表达的细胞为模型,证实了镍离子不仅能抑制Tet介导的5mC氧化,还能阻碍Tet引导的DNA去甲基化过程。镍离子(500 μM)处理转染细胞后,绝大多数5hmC和5fC(78 - 95%)的生成已经被抑制,与之对应整体5mC水平上升了30 %。此外,镍离子对小鼠胚胎干细胞5hmC和5fC具有类似的抑制效应,而且还能阻碍维生素C的DNA去甲基化作用。这暗示着镍离子对Tet酶的抑制作用具有重要的生物学意义。我们进一步探讨了镍离子抑制Tet双加氧酶的机理。体外氧化实验揭示了镍离子直接阻碍Tet酶的催化活性,并且其抑制效应极其显著。2 μM镍离子能引起5hmC和5fC的生成量下降84 - 96%,10 μM镍离子完全阻碍了5fC的形成。这表明Ni2+很可能与Fe2+竞争结合Tet1CD蛋白质中HxD区域,当Ni2+占据HxD区域后,将阻碍Fe2+与该区域结合,因而导致Tet1CD丧失催化活性。亚铁离子竞争实验和体积排阻色谱-ICPMS数据为这个猜想提供了直接的证据。同样,在动物水平上我们考查了镍离子对新生及成年小鼠的效应,然而没有足够的数据来表明镍离子能够影响小鼠体内5hmC和5mC的水平。
     此外,研究表明5hmC可能作为一种新型的可用于疾病诊断的生物标志物。在患有癌症、骨髓增生异常综合症、神经性舞蹈病、阿尔兹海默病或精神病等病人体内普遍发现5hmC含量异常。由于具有无菌、样品量大和取样无损等优点,因而人体尿液5hmC应该是疾病诊断、治疗及预后诊断的首选标志物。然而,还不清楚尿液中是否含有游离状态的5hmC。为了解决这个问题,我们发展了一种离线固相萃取-稳定同位素稀释-高效液相色谱-串联质谱方法,用于定量人体尿液中5mC、5hmC及其他氧化产物。在尿样分析前,采用固相萃取柱去除尿液中高浓度的盐分,以降低基质效应。该方法使用碳酸氢铵溶液和甲醇作为流动相。碳酸氢铵能显著提高5mC、5hmC和5fC的检测灵敏度(1.8 -14.3倍)。该方法具有良好的准确度和精密度,5hmC和5mC加标回收率在70 -100%之间,其日内测量偏差约为1.4 - 7.7%,日间测量偏差约为2.9 -10.6%。该方法被用于分析13个健康人体尿液样品。我们首次证实了人体尿液中含有高丰度的5hmC,在13个中样品5hmC平均水平为22.6 ± 13.7 nM,与其前体5mC含量(52.4 ± 50.2 nM)相当。考虑到基因组DNA内5hmC水平远低于5mC(约两个数量级),这表明人体基因组中5hmC代谢速率远高于其前体5mC。
英文摘要:     Tet (Ten-eleven translocation) dioxygenases can oxidize 5’- methylcytosine (5mC) to produce 5’- hydroxylmethylcytosine (5hmC), 5’-formylmethylcytosine (5fC), and 5’-carboxylmethylcytosine (5caC) in genomic DNA. Tet proteins play critical roles in mammalian DNA demethylation. They can promote replication-dependent passive DNA demethylation by the formation of 5hmC, and also have the potential to mediate rapid and active DNA demethylation by 5fC and 5caC that is independent of DNA replication. Impaired function of Tet proteins is frequently found in various cancers and other diseases. Mutations of Tet2 gene are frequently observed in diverse myeloid and lymphoid malignancies accompanied by reduced levels of genomic 5hmC. Our previous data illustrated that some small molecular compounds (ascorbic acid and redox-active quinones) alter the distribution of 5hmC in genome via Tet dioxygenases. Therefore, it is urgent to study the effects of other compounds (e.g. nickel compounds) on the function of Tet proteins.
     Nickel compounds are widely distributed in air, water, animals, plants, soil, and sediments. In the world, millions of workers are being occupationally exposed to air particles with high levels of nickel compounds (about 7.6 - 452 μg/m3), mainly from industries regarding mining, refining, alloy production, electroplating, and welding. Nickel compounds can depress the expression of a set of genes through hypermethylation of these genes, including p16, RAR‐β2, CDKN2A, and MGMT, which are frequently depressed in various cancers. However, the mechanism of nickel-induced DNA methylation is unclear. Here, we hypothesize that nickel (II) ion hinders Tet-mediated DNA demethylation through the inhibition of 5mC oxidation. In this study, we tested epigenetic effects of nickel (II) ion in somatic cells, Tet-transfected cells, and mouse ES cells. In somatic cells (HEK 293T and MRC5 cells), nickel (II) ion significantly inhibited the generation of 5hmC (8.6 – 64 %). There was no significant increase in global 5mC levels of HEK 293T and MRC5 cells after NiCl2 treatment due to low expression of Tet proteins. We further demonstrated that nickel (II) ion hindered Tet-mediated DNA demethylation through the inhibition of 5mC oxidation in Tet1CD transfected cells. For 500 μM NiCl2 treatment, all of 5hmC and 5fC (about 78 – 95%) were almost inhibited in Tet1CD-transfected cells.
     Correspondingly, nickel treatment caused a significant increase in 5mC level by 30% compared with transfected cells without nickel treatment. The similar inhibitions of 5hmC and 5fC (about 70 - 90%) were observed in nickel-treated ES cells. Although global level of 5mC did not change in nickel-treated ES cells, nickel treatment can diminish ascorbic acid (AA)-induced DNA demethylation in ES cells, possibly through depletion of intracellular AA. Besides, we demonstrated nickel (II) ion directly inhibited the in-vitro catalytic activity of Tet dioxygenases for the oxidation of 5mC. In particular, only 2 μM NiCl2 can almost prevent all of 5hmC and 5fC (84 - 96%) being generated at the presence of 10 μM FeCl2, suggesting Tet dioxygenases are highly sensitive to nickel inhibition. Together, these data demonstrate that nickel (II) ion blocks Tet-mediated DNA demethylation in mammalian cells through direct inhibition of 5mC oxidation.
     In addition, abnormal levels of 5hmC have been widely found in various tumors and neurological diseases. Due to its advantages of being sterile, easily accessible in large volumes, and noninvasive to patients, urinary 5hmC analysis should be a first choice for clinical diagnosis, disease treatment, and prognosis. However, the concentrations of 5hmC in human urine are not clear. We developed a stable isotope dilution-HPLC-MS /MS method for accurate quantification of 5mC and its oxidation products in human urine. The urinary samples were desalted using offline solid phase extraction followed by HPLC-MS/MS analysis for urinary 5hmC and 5mC. Ammonium bicarbonate enhances the detection of 5mC, 5hmC, and 5fC by 1.8-14.3 times. The precision and accuracy of this method were excellent. The recovery for 5mC and 5hmC ranged from 70 -100 %. The relative standard deviations of the interday precision and the intraday precision were about 2.9 −10.6% and 1.4−7.7%, respectively. By the analysis of 13 volunteers using the developed method, we for the first time demonstrate the presence of 5hmC in human urine. Unexpectedly, we observed that the level of 5hmC (22.6 ± 13.7 nmol/L) is comparable to that of its precursor 5mC (52.4 ± 50.2 nmol/L) in human urine.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/37038
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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尹瑞川. 镍化合物抑制DNA羟甲基化效应与机理研究[D]. 北京. 中国科学院研究生院. 2016.
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