高等真核生物 DNA N6-甲基腺嘌呤修饰分析与功 能研究
文献类型：学位论文
| 作者 | 黄华 |
| 学位类别 | 博士 |
| 答辩日期 | 2016-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 汪海林 |
| 关键词 | 6甲基腺嘌呤脱氧核苷酸，果蝇 6mA去甲基化酶，早期胚胎， 异染色质，白藜芦醇，5羟甲基胞嘧啶，α酮戊二酸 N6-methyladenine, DMAD, Early embryo, Heterochromatin, Resveratrol, 5hmC, αKG |
| 其他题名 | DNA N6-Methyladenine modification in high eukaryotes |
| 学位专业 | 环境科学 |
| 中文摘要 | 在哺乳动物体内，5-甲基胞嘧啶(5mC)被认为是唯一的DNA甲基化形式，可调控基因表达和多种生物学功能。5mC通过Fe(II)和 α-酮戊二酸(αKG)依赖的TET双加氧酶催化氧化，生成5-羟甲基胞嘧啶(5hmC)，并可进一步氧化形成5-醛基胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。5mC及其氧化产物与TET蛋白共同调节多种生理功能，如核重新编程、胚胎发育、细胞分化、疾病的发生发展等。与哺乳动物不同，原核生物存在三种 DNA甲基化形式，5mC、N4-甲基胞嘧啶(4mC)和N6-甲基腺嘌呤(6mA)。其中，6mA是主要的原核生物 DNA甲基化形式。对一些细菌而言，6mA缺乏将导致其死亡。一些低等单细胞真核生物也存在 6mA DNA修饰。令人惊奇的是：高等真核生物基因组中是否存在6mA修饰一直是一个悬而未决的科学难题。我们设想：1）过去所使用的分析技术和方法的检测灵敏度低，难以检测低丰度的 6mA；2）6mA可能分布于特定组织或特定发育阶段而难以探知；3）由于快速去甲基化， 6mA在体内高度动态变化而仅在短时间内出现。基于以上原因，人们难以确定高等真核生物中 6mA的存在。 为了回答上述科学难题，我们通过与中科院动物所陈大华研究组合作，以经典的模式生物果蝇作为研究对象考察 6mA的存在与可能的分布。在果蝇体内，5mC存在与否一直存在争议。我们设想，果蝇体内可能存在另外的DNA修饰，如6mA，并通过该修饰实现果蝇体内表观基因组调控。首先，我们发展了基于超高效液相色谱串联质谱（UHPLC-MS/MS）的高灵敏分析方法，可检测16mA/108 dA。利用该方法，我们发现 6mA修饰在果蝇体内多个组织基因组中广泛存在。液相色谱联用高分辨飞行时间质谱(LC-QTof)分析，显示可能的果蝇6mA组分的精确分子质量为 266.1250au，与 6mA标准品标准分子质量(266.1248au)偏差仅为 1.02ppm。另外，果蝇6mA组分分子峰碎裂模式和各个碎片(21碎片)准确质荷比，与标准 6mA化合物均一致，进一步确定了6mA的存在。 利用 UHPLC-MS/MS分析，发现果蝇在早期胚胎发育过程中6mA是高度动态变化的。0.75小时胚胎阶段，6mA修饰高达~0.07% 6mA/dA)；4-16h胚胎阶段，6mA的含量约下降至0.001% (6mA/dA)。这些结果显示随着胚胎发育的进行，6mA含量出现急剧下降，说明在胚胎发育后期 DNA 6mA去甲基化机制可能发挥作用。活性分析显示后期胚胎细胞核提取液的确存在显著的 DNA 6mA去甲基化活性，预示果蝇存在 DNA 6mA的去甲基化酶。 哺乳动物体内 TET蛋白家族在 DNA去甲基化过程中发挥重要作用。基因比对显示，果蝇体内存在一种 TET同原蛋白DMAD。DMAD在果蝇胚胎发育期的mRNA和蛋白质表达水平与6mA含量呈现反比关系。DMAD抗体处理的果蝇胚胎细胞核提取液 DNA去甲基化活性显著降低，同时，敲低果蝇胚胎早期DMAD表达会导致胚胎后期 6mA丰度明显提高。另外，敲除DMAD后，果蝇脑部和卵巢组织中6mA出现显著升高（增加10-70倍）。我们通过真核表达系统纯化了DMAD-CD和DMAD-CDmut蛋白，并建立DMAD的体外去甲基化反应体系。通过该体系，我们发现 DMAD-CD在体外具有明显6mA去甲基化活性，并具有明显剂量与效应关系，但DMAD-CDmut并没有显示去甲基化活性。因此我们确定DMAD是果蝇体内主要的 6mA去甲基化酶。 我们利用 6mA特异性抗体对野生型和 DMADdel果蝇基因组 DNA进行免疫沉淀，然后进行高通量二代测序分析。结果显示，在 DMADdel果蝇的卵巢中，6mA主要富集于转座子区域，表明在果蝇体内 6mA修饰可能与转座子表达调控有关。 在开展模式生物果蝇研究之前，我们就已着手研究哺乳动物基因组中的6mA修饰。我们发现，在成年小鼠的脑、肺、肾、脾等组织基因组 6mA修饰含量非常低，约 5-25 6mA/108 dA。通过与同济大学合作，我们进一步探讨小鼠生殖细胞和早期胚胎发育阶段基因组 6mA修饰。结果表明，在小鼠卵母细胞内存在较高丰度 6mA修饰(0.03%,6mA/dA)，是其它组织基因组的 1200-6000倍。令人奇怪的是，精子中却没有检测到 6mA修饰。体外培养受精卵，发现在胚胎二细胞时期 6mA的丰度达到最高(0.18%, 6mA/dA)。随着发育继续进行，6mA丰度出现急剧下降。至囊胚期时，6mA丰度为0.004% (6mA/dA)。免疫荧光成像分析表明，在小鼠生殖细胞和早期胚胎中，6mA主要分布于异染色质区域，暗示6mA在小鼠体内的存在可能与基因沉默有关。 与此同时，我们探讨了白藜芦醇对细胞 DNA羟甲基化的影响。已有研究表明，长期摄入一定剂量白藜芦醇对多种疾病具有预防作用，例如高血压和癌症。DNA羟甲基化是否有所贡献尚不清楚。利用小鼠胚胎干细胞(mESc)，白藜芦醇暴露可显著提升细胞内的 5hmC和5fC的含量，约为对照的 1.8-2.1倍。利用发展的 LC/MS/MS方法，分析细胞裂解液中α-酮戊二酸 (αKG)和二羟基戊二酸(2HG)的含量。结果显示，白藜芦醇处理可显著提升细胞αKG水平，约为对照组的 8倍，而 2HG的含量没有显著变化。该结果说明白藜芦醇可能通过提高细胞内 αKG含量来增强 Tet蛋白的活性，从而使 5hmC的含量增加。该过程表明白藜芦醇可能影响细胞内 DNA羟甲基化和能量代谢过程。 |
| 英文摘要 | 5-methylcytosine(5mC), which was thought to be the only form of DNA methylation in mammals, play critical roles in regulation of gene expression and modulation of diverse biological functions. Fe(II)- and α-ketoglutarate (α-KG)-dependent ten-eleven translocation(TET) family dioxygenases can catalyze the oxidation of 5mC tto form 5hmC, and iterately to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Tet proteins and their functions in oxidation of 5mC together display important biological process in mammals, including nuclear reprogramming, embryo development, cell differentiation and stemness, and diseases occurrence and development. Interestingly, three DNA methylations, 5mC, N4-methylcytosine (4mC) and N6-methyladenine (6mA), are found in prokaryote.Among these DNA methylations, 6mA is the predominant form and it is vital to some bacteria. 6mA is also found in some low unicellular eukaryotes. However, it remains unclear whether genomic 6mA exists in high eukaryotes. We reasoned this ambiguity: 1) Analytical technologies used for detection of genomic 6mA in the previous work are insensitive, so the trace amount of 6mA can’t be detected; 2) Genomic 6mA may only exist in certain tissues or at particular developmental stages: 3) Genomic 6mA is highly dynamic and hard to be captured due to rapid and one-step demehtylation involved in high eukaryotes. In colaboration with Chen Dahua’s Group, at the Institute of Zoology, CAS, We exploited Drosophila to investigate the 6mA existence and distribution in genome. Intriguingly, over a decade, it is controversial whether 5mC is present in Drosophila.We hypothesized that DNA methylation other than 5mC could occur, e.g., 6mA,which alternatively functions in epigenetics of Drosopholal. First, we developed a ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) analysis method, which can detect genomic6mA of 1 modification per 108 deoxyadenine dA). By taking advantage of this sensitive method, we can detect 6mA in Drosophila. By characterization of the peak fraction containing 6mA obtained from early embryo of drosophila usinga high-resolution mass spectrometry the drosophila 6mA display a mass/charge ratio of 266.1250 au, which match the theoretic mass of 6mA (266.1248 au) with a deviation of 1.02 ppm. Moreover, the measured mass/charge ratios of the obtained fragment ions (21 fragments) also match well with that of standard 6mA. All the results consistently support the presence of genomic 6mA in Drosophila. The quantitative UHPLC-MS/MS analysis of 6mA showed that 6mA was dynamic in Drosophila DNA during early embryonic development. The level of the 6mA appeared to be~0.07% (6mA/dA) at the ~0.75h stage but was dramatically reduced to a very low level (~0.001%, 6mA/dA) at the 4–16 h stages. The rapid decrease in genomic 6mA followed by development of the embryo indicated that the demethylation mechanism played a role at the late stage of embryo. Indeed, the nucleic extract of late stage embryo exhibited markedly demethylation activity, hinting the involvement ofa demethylase in embryonic development stage.TET proteins play a important role in the demethylation process in mammals. In Drosophila, there is a Tet homologous protei, which is named 6mA demethylase (DMAD), through sequence alignment. We observed that the DMAD expression was antagonistic to 6mA abundance and the demethylation activity of embryo nuclear extract show a significant decrease after DMAD depletion using anti-DMAD antibody.DMAD Knockout dramatically increase the level of 6mA for 70 folds in head and for 10 folds in ovary. The purified recombinant DMAD-CD protein showed demethylation activity in vitro 22-25%. In contrast, the DMAD-CDmut could not remove 6mA in the DNA substrates. All these results suggest that the DMAD is the Drosophila 6mA demethylase. We performed DNA-immunoprecipitation (DNA-IP) coupled with high-throughput DNA sequencing analysis and observed that more 6mA modification is present in the transposon regions in DMAD mutant ovaries than in wild-type ovaries.The result suggested that DMAD-mediated 6mA demethylation is correlated with transposon expression and this conclusion was confirmed by the RNA-seq data. Prior to the investigation of Drosophila, we set out to investigate the distribution of 6mA in mammals. We found that the 6mA abundance was extremely low (4-25 6mA/108dA) in gemomic DNA of tissues such as lung, heart, kidney and spleen,which were obtained from adult mice. In late cooperation with Tongji University, we carryed out some further research to evaluate 6mA in the germ cell and the early embryonic stages of mice. A higher abundance of 6mA was observed in oocyte (0.03%, 6mA/dA), about 1200-6000 fold compare to the other tissues. However, 6mA was not detected in sperm. The quantitative analysisof 6mA in the mouse early embryo cultured in vitro showed that 6mA abundance reached to a peak abundance at 2 cell stage (0.18%, 6mA/dA) and decreased dramatically following the embryo development to blastula (0.004%, 6mA/dA). Immunostaining assays using 6mA antibody showed that the 6mA was rich in the hereochromatinic regions both in the germ cell and early embryo stage. These results suggest that 6mA in genome may be related to gene silencing. We also conducted a preliminary investigation on the influence of resveratrol on DNA hydroxymethylation in mammalian cells. According to the literature,appropriate long-term intake of resveratrol is associated with a reduced risk for lifestyle-related diseases such as cardiovascular disease and cancer. However, it is not know whether DNA hydroxymethylation has a role in these protecting functions.Mass spectrometry analysis demonstrated that 5hmC and 5fC levels in genomic DNA both significantly increased (1.8- 2.1 folds) in wild type mouse embryonic stem cells (mESc) treated with 50μM resveratrol (24h). Moreover, we developed a LC/MS/MS analysis method for α-ketoglutarate (αKG) and 2-hydroxyglutaric (2HG) in the cell lysates. we observed a significant increase (more than 8 folds) of αKG in positive cells and no difference in the abundance of 2HG. The data demonstrated that resveratrol can enhanceTet oxidation activity by elevating αKG. These results indicated that the resveratrol could affect the DNA hydroxymethylation by impacting energy metabolism in cultured cells. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/36804]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 黄华. 高等真核生物 DNA N6-甲基腺嘌呤修饰分析与功 能研究[D]. 北京. 中国科学院研究生院. 2016. |
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来源：生态环境研究中心
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