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题名: 代谢依赖的稳定同位素标记脱氧核苷代码与 DNA N6-甲基腺嘌呤溯源
作者: 刘保东1
学位类别: 博士
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 汪海林
关键词: [15N5]-dA示踪,腺苷脱氨酶,N6-甲基-2’-腺嘌呤脱氧核苷,[15N3]-dC,细胞增殖 ; [15N5]-dA Tracing, Ada, 6mA, [15N3]-dC, Cell Proliferation
其他题名: Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-methyladenine in Human Cells
学位专业: 环境科学
中文摘要: 2015年,我们课题组和中国科学院动物研究所陈大华组合作,证实果蝇基 因组 DNA上含有大量的 6mA(6mA/dA,0.001~0.07%),并且其紧密调控果蝇 早期胚胎发育和转座子表达。这与 6mA在秀丽隐杆线虫以及哈德莱茵衣藻中的 研究一起,共同表明 6mA是一种新的真核基因组表观遗传印记。随后,在小鼠、 斑马鱼、猪和人等高等动物基因组 DNA中也检测到 6mA的存在。现有少数几 种检测 6mA的分析方法,如超高效液相色谱串联质谱(UHPLC-MS/MS)、抗体 依赖的免疫分析和 DNA测序等。然而,LC-MS和抗体依赖的实验面临细菌污染 的难题。DNA测序技术虽然能够通过测序结果与实验物种的基因组序列比对从 而扣除来自细菌的 6mA干扰,但它可能同样需要抗体富集含有 6mA的 DNA片 段以用来测序。为此,我们开发了一种基于稳定同位素标记的脱氧腺嘌呤核苷 ([15N5]-dA)示踪, UHPLC-MS/MS检测的方法,来精确和快速地检测细胞基 因组真实的 6mA水平。 当 HEK293T细胞暴露[15N5]-dA时,仅以[15N4]-dA的形式掺入基因组 DNA。 Q-TOF-MS的 Target MS/MS模式的分析结果表明,[15N4]-dA的环外氨基上为普 通的 14N原子。因而,我们推测在细胞内 [15N5]-dA→[15N4]-dA的生物过程可能 是腺苷脱氨酶 Ada介导的。 为了证实这一猜测,我们采用 siRNA敲低 Ada的表达,和阴性对照组相比 [15N4]-dA/dA和[15N4]-dG/dG分别下降了 37.2%和 31.8%,表明在这一过程中 Ada 确实发挥了作用,但未检测到[15N5]-dA。为了证实以上结果,我们采用不同浓度 的 Ada抑制剂(EHNA)抑制 Ada活性,发现和对照组相比 [15N4]-dA/dA和 [15N4]-dG/dG分别下降了 75%−96.4%和 75.4−96.8%,同时检测到了[15N5]-dA,但 是[15N5]-dA /dA不超过 5%。以上结果表明,Ada介导的嘌呤补救合成途径的确 是[15N5]-dA掺入 DNA的主要途径。 共生于 293T细胞的支原体,既能够摄取培养基中 [15N5]-dA,也能够摄取宿 主核酸池中的 [15N4]-dA和[15N4]-dG。然而, 293T细胞主要摄取核酸池中的 [15N4]-dA和[15N4]-dG。利用支原体 DNA富有 [15N5]-dA和[15N5]-6mA而人细胞 富有[15N4]-dA和潜在的[15N4]-6mA的特点,该方法能够区分细胞是否发生支原因为扩增自细菌的质粒无 15N标记,所以质粒携带的 6mA也无 15N标记,因而可以采用[15N5]-dA示踪的方法将质粒 DNA 6mA和细胞 DNA [15N4]-6mA区 分开来。 利用上述原理,我们评估了细菌 DNA N6-腺嘌呤甲基转移酶(Dam)在细胞 内的活性。突变体DamD181N拥有较野生型Dam低约100倍的甲基转移酶活性, 但采用 [15N5]-dA示踪, UHPLC-MS/MS检测的方法仍然可以检测到 2.8个 [15N4]-6mA/106 dA,即使此时存在高达≈500个 6mA/106 dA的背景干扰。而作 为阴性对照,EGFP和 DamP183R过表达均未显示甲基转移酶活性。以上结果表 明,[15N5]-dA示踪能够准确地、灵敏地评价外源蛋白在细胞内 DNA N6-腺嘌呤 甲基转移酶活性。 鉴于一些细胞不能利用胸腺嘧啶脱氧核苷来评价细胞增殖,我们试图建立 [15N3]-dC标记 DNA合成来评价细胞增殖的方法。 首先,我们证实 [15N3]-dC掺入小鼠胚胎干细胞基因组 DNA的形式为 [15N3]-dC和[15N2]-dT。当细胞暴露 10 µM [15N3]-dC时,[15N2]-dT/dT和细胞增殖 呈正相关(0−24 h),是较[15N3]-dC/dC更为理想的评价细胞增殖的指标。而在撤 掉示踪剂[15N3]-dC后,[15N3]-dC/dC和[15N2]-dT/dT的对数和细胞数目的对数呈 负相关,二者均是评价细胞增殖的理想指标。另外,不同 [15N3]-dC的剂量和不 同的细胞铺板密度影响[15N3]-dC/dC和[15N2]-dT/dT的标记比例。以上结果表明, [15N3]-dC在一定的条件下,可以用来评价细胞增殖。采用[15N3]-dC示踪的方法, 我们发现 Tet1-/ Tet2-/ Tet3-三敲的 mES细胞拥有较野生型更快的细胞增殖速度。
英文摘要: In 2015, we and our co-worker showed that 6mA is abundantly present in genome of Drosophila melanogaster and tightly regulates early embryonic development and control transposon’s expression. Together with the study of Caenorhabditis elegans and Chlamydomonas algae, it showed that 6mA is a new epigenetic mark in eukaryotic genome. Late works suggested that DNA 6mA was detectable in mice, zebrafish, pig, and human. A few of methods have been developed for detection of 6mA in eukaryotic genome, including liquid chromatography-tandem mass spectrometry (LC-MS/MS), immunoassays, and DNA sequencing. Both LC-MS and antibody-based assays confronted bacterial contamination. DNA sequencing-based assays might be used to discriminate endogenous DNA 6mA from that caused by bacterial contamination through genome-wide sequence mapping. However, it may also require anti-6mA antibody to enrich 6mA-carrying DNA fragments for sequencing. In this study, we reported the development and application of stable isotope-labeled deoxynucleoside [15N5]-deoxyadenosine ([15N5]-dA) as a tracer, combined with UHPLC-MS/MS analysis, for accurate and rapid identification and detection of authentic DNA 6mA. Our data demonstated [15N5]-dA incorporated into genomic DNA of human 293T cells only in the form of [15N4]-dA. Target MS/MS mode of Q-TOF-MS was used to identify which 15N of [15N4]-dA was lost. All the fragmentation analysis consistently supports a loss of 15N occurs at the position of exocyclic N6-amino-15N by the conversion of [15N5]-dA into [15N4]-dA in the treated cells. To investigate whether Ada involves with the exocyclic deamination of [15N5]-dA, we further knocked down Ada gene expression using siRNA. Interestingly, we observed that the signals of [15N4]-dA and [15N4]-dG in genomic DNA decreased by 37.2% and 31.8%, respectively, confirming the involvement of Ada. Of note, despite the decrease in both [15N4]-dA and [15N4]-dG, we did not observe any [15N5]-dA incorporated into genomic DNA. To corroborate this conclusion, we next examined the DNA incorporation of [15N5]-dA using the Ada inhibitor. By treating 293T cells with an Ada inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), the signals of [15N4]-dA and [15N4]-dG in genomic DNA decreased by 75.0%−96.4% and 75.4%−96.8%, respectively. Meanwhile, we observed the presence of [15N5]-dA in genomic DNA, but the signal of [15N5]-dA is less than 5% of that for dA. All these data consistently support that the DNA incorporation of [15N5]-dA predominantly involves with adenine deamination-dominated purine salvage pathway. Mycoplasma co-inhabited in the cultured human cells can uptake [15N5]-dA in the medium accompanying with [15N4]-dA and [15N4]-dG in the nucleoside pool of the host cells, however, the cultured human 293T cells (host cells) mainly uptake [15N4]-dA and [15N4]-dG in the nucleoside pool. By taking advantage of this metabolic difference in deamination of dA between the mycoplasmas and the host human cells, we could discriminate the mycoplasma DNA and the genomic DNA of host cells. Since bacteria-amplified plasmid is not labeled by stable 15N isotope-involved metabolism, the 6mA carried by the plasmid would not have 15N isotope and thus be discriminated using [15N5]-dA tracing. For example, by transfection with N6-methyltransferase Dam and its mutants, we could sensitively measure their methyltransferases activity. Even for the mutant of DamD181N with 100-fold lower methyltransferases activity, by the use of stable isotope-labeled [15N5]-dA tracing, we could detect its weak activity, which only generates 2.8 6mA per million dA in cultured 293T cells among abundant interfering unlabeled 6mA (about 500 6mA per million dA). As negative controls, neither EGFP nor Dam P183R overexpression showed adenine methylation activity in vivo. This presents a proof-of-principle for monitoring DNA adenine N6-methyltransferases activity (writing), and it is also possible to apply for the assay to accurately evaluate other DNA 6mA-related gene activities. Because of lacking ability of uptaking thymidine for some cell types, [15N3]-deoxycytidine ([15N3]-dC) utilizing pyrimidine salvage pathway was investigated as a tracer. When mouse embryonic stem cells (mESCs) treated by [15N3]-dC, there were [15N3]-dC and [15N2]-thymidine ([15N2]-dT) in genomic DNA of mESCs detected by UHPLC-MS/MS. When logarithmic growth mESCs treated by 10 µM [15N3]-dC, the ratio of dT ([15N2]-dT/dT) was linearly increased in 0−24 h, but not labeling ratio of dC ([15N3]-dC/dC). These results indicate [15N3]-dC as a tracer is also able to to measure cell proliferation by index labeling of dT (0−24 h). Washout of [15N3]-dC in genomic DNA from mESCs, labeling ratio of dC or dT were negative correlation with cell number. All the results supported [15N3]-dC was competent for accurate measurement of cell proliferation. Also, dose of [15N3]-dC and cell density affected the labeling ratio of dC or dT was studied. Further, we used the developed [15N3]-dC tracing method to measure the difference of proliferation rate between Tet1-/Tet2-/Tet3- triple-knock out (TKO) mESCs and wild type (Wt), and the results indicated the Tet TKO mESCs have higher proliferation rate in our cell culture condition.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38661
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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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
刘保东. 代谢依赖的稳定同位素标记脱氧核苷代码与 DNA N6-甲基腺嘌呤溯源[D]. 北京. 中国科学院大学. 2017.
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