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哺乳动物DNA N6-甲基腺嘌呤的分析与鉴定
Alternative TitleIdentification and Analysis of DNA N6-methyladenine in Mammal
刘晓玲
Subtype博士
Thesis Advisor汪海林
2018-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline分析化学
KeywordDna N6-甲基腺嘌呤(6ma),小鼠胚胎干细胞(Mes),慢病毒,Hiv-1假病毒,多重免疫沉淀(Mrip) Dna N6-methyladenine (6ma), Mouse Embryonic Stem Cells (Mes), Lentivirus, Hiv-1 Pseudovirus, Multiple Immunoprecipitation (Mrip)
Abstract

       2015年,我们在高等真核生物果蝇中发现  DNA  N6-甲基腺嘌呤(6mA)新修饰,同时人们在秀丽隐杆线虫基因组中也检测到 6mA。此时研究也发现果蝇、哈德莱茵衣藻和秀丽隐杆线虫基因组DNA 6mA具有激活基因表达或转座子表达的功能,表明 DNA 6mA是真核生物中潜在的表观遗传标记。随后,在斑马鱼和非洲爪蟾中也检测到 6mA修饰。虽然有报道猪胚胎细胞和小鼠胚胎干细胞中存在 DNA 6mA,但目前人们对于哺乳动物是否存在 DNA 6mA这一科学问题仍未得出结论。
      我们以小鼠胚胎干细胞(mESCs)为模型,利用本研究组发展的一种检测6mA的高灵敏度 UHPLC-MS/MS方法,寻找哺乳动物DNA6mA。在mES细胞中,我们可检测到 6mA,其含量约2.0-6.0 6mA/107dA。利用细胞周期同步化,发现 G1期   6mA水平升高,可达  1.0 – 2.6 6mA/106 dA。我们在人胚胎干细胞中也检测到 DNA 6mA。由此证明了胚胎干细胞中存在 6mA。
      利用稳定同位素标记的脱氧腺嘌呤核苷([15N5]-dA),在 mES细胞中没有检测到新的含有同位素标记的 6mA,暗示  mES细胞中的  6mA可能不是由甲基转移酶甲基化产生的;利用[13CD3] L-甲硫氨酸示踪,结果显示>50% 5mdC被标记成[13CD3]-5mC,但仍未检测到[13CD3]-6mA,再次表明 mES细胞中的6mA可能不是由甲基转移酶甲基化产生的。
      由以上结果,我们提出一个关键科学问题,小鼠胚胎干细胞中 6mA是如何产生的?为此,我们将细胞暴露于RNA m6A核苷、DNA   6mA脱氧核苷,均可检测到暴露基因组中6mA的增加。暴露于稳定同位素标记的6mA脱氧核苷([15N5]-6mA),可在基因组 DNA中检测[  15N5]-6mA的出现。暴露于含6mA的脱氧寡核苷酸链或含 RNAm6A寡核苷酸链,均检测到基因组中6mA的增加。这些结果表明,含N6-甲基腺嘌呤碱基的外源核苷/(脱氧)寡核苷酸在 mES细胞中经过一系列的核苷代谢途径可掺入到基因组中。在正常培养的 mESC中,我们并未检测到 N6-甲基腺嘌呤碱基相关的外源物质。mESC中6mA的产生仍是一个谜,有待进一步深入研究。
      由于在原核生物中 DNA  6mA的一个主要功能与宿主-病原体的相互作用有关,我们设想病毒的侵入是否可引起哺乳动物基因组 6mA的出现。我们以人类免疫缺陷病毒 1型(HIV-1)的假病毒和常见的慢病毒为模型。HIV-1假病毒基因组 gRNA在感染细胞内逆转录成   cDNA,然后整合进细胞的基因组DNA中。我们猜想 HIV-1假病毒感染细胞的整个过程是否改变宿主DNA甲基化水平,如DNA 6mA修饰。结果显示  HIV-1假病毒感染  Hela细胞后,未检测到DNA 6mA水平的变化;慢病毒感染  Hela细胞后,6mA呈现从无到有的增加。结果表明,病毒感染有可能产生 DNA 6mA,但具有病毒种类的依赖性。
      为了理解哺乳动物 DNA 6mA的来源及其分布与功能,我们需要发展相应的测序技术与方法。利用免疫沉淀法富集含 6mA的   DNA片段是一个极为关键的步骤。我们分析了五种 6mA抗体对  Dam-/-  lambda  DNA的富集效果,富集倍数为 5.3-37倍,然而我们发现在最终富集的   DNA中含有大量的不含  6mA修饰的DNA(73.5-94.6%)。理论上,残存的不含  6mA的片段以序列无关的方式随机地与抗体结合,不会被序列分析算法判为 6mA峰。实际上,大量不含  6mA片段的存在严重干扰 6mA峰的判定。为此,发展出一种多重免疫沉淀(MrIP)富集方法。利用这一方法,对 Dam-/- lambda DNA中的  6mA片段富集。经过两轮富集,每条  DNA片段含有一个或两个   6mA修饰。对于含有极低   6mA丰度的基因组DNA (0.44/106 nt)。经过三轮富集后,6mA水平升高约 9100倍,可达  0.4% (6mA/nt),也就是说最终富集的 DNA中有  60%的 DNA片段中含有  6mA修饰,并且富集的DNA质量约  13.7ng,已满足 DNA文库构建和基因组  DNA测序分析的需要。所发展的这种生物化学方法将在很大程度上减少 6mA峰有关的偏差,并且有利于6mA相关数据的挖掘,将极大地促进基因组  6mA测序分析。类似地,MrIP方法可用于各种 DNA修饰的基因组测序。

Other Abstract

      In 2015, we discovered DNA N6-methyladenine (6mA) in Drosophila melanogaster, representing a breakthrough in epigenetics. Meanwhile, 6mA was also detected in the genome Caenorhabditis elegans. At that time, the functional studies from Drosophila melanogaster, Chlamydomonas algae demonstrated that DNA 6mA might activate gene expression or stimulate transposon expression. Therefore, DNA 6mA is a potential epigenetic marker in eukaryotes. Subsequently, 6mA modification was also detected in zebrafish and Xenopus Laevis. Although it reported that DNA 6mA was present in pig embryos and mouse embryonic stem cells, it remains elusive whether DNA 6mA is present in mammals.

       We used mouse embryonic stem cells (mESCs) as a model, and explored highly sensitive UHPLC-MS/MS method to search for 6mA in mammalian DNA. In mES cells, we could detect 6mA and its level was about 2.0 – 6.0 6mA/107 dA. By cynchronizing cell cycle, it was found that the level of 6mA in G1 phase increased and reached 1.0 – 2.6 6mA/106 dA. We also detected DNA 6mA in human embryonic stem cells. These results clearly proves the presence of 6mA in embryonic stem cells.

      We further traced 6mA formation using stable isotope labeled deoxyadenosine ([15N5]-dA), and did not detect any stable isotope-labeled 6mA in mES cells, suggesting that dA in the genome of mES cells might not be methylated by certain methyltransferase. Then, we utilized [13CD3] L-methionine as a stable isotope tracer to explore the 6mA formation. Although >50% of 5mC could be labeled as [13CD3]-5mC, it failed to detect [13CD3]-6mA. Again, this stable isotope tracing study confirmed that dA in the genome of mES cells might not be methylated by methyltransferase.

      Now we asked a critical question how DNA 6mA formed in mouse embryonic stem cells. To this end, we treated mES cells with N6-methyl-adenosine (RNA m6A nucleoside) or N6-methyl-2’deoxyadenosine (DNA 6mA deoxynucleoside). Surprisingly, we observed that the level of 6mA elevated in genomic DNA. Treated with a stable isotope-labeled 6mA deoxyribonucleoside ([15N5]-6mA), we could detect [15N5]-6mA in genomic DNA. Treated with a 6mA-containing DNA oligo or m6A-containing RNA oligo, we also detected an increase of 6mA in the genome. These results indicated that exogenous nucleoside/(deoxy)oligonucleotides containing N6-methyladenine bases could be incorporated into the genome of mES cells. In normal cultured mESCs, we did not detect N6-methyladenine base-related exogenous substances. The production of 6mA in mESC is still a mystery and needs to further study.

      Recalling that a major function of DNA 6mA in prokaryotes is related to host-pathogen interactions, we envisaged whether the virus infection could cause the emergence of mammalian genome 6mA. We used one pseudoviruse similar to human immunodeficiency virus type 1 (HIV-1) and a common lentiviruse as two models. The HIV-1 pseudovirus RNA genome is reversely transcribed into cDNA in the infected cells and then integrated into the genomic DNA of host cells. We hypothesized whether the entire process of HIV-1 pseudovirus infection of cells altered host DNA methylation levels, but the level of DNA 6mA was not changed. However, the infection of HeLa cells with the lentivirus, 6mA showed an increase from none. The results showed that the virus infection might produce DNA 6mA, but it was dependent on the virus species.

      As described above, we identified two possible ways to induce DNA 6mA in genome of mammals. To explore the distribution and functions of 6mA generated in mammalian genomes, it is of paramount option to use immunoprecipitation to select 6mA-containing DNA fragments for genome-wide sequencing. Presumably, most of the 6mA-free fragments are removed, and the co-pulling down of the residual is stochastic and sequence-independent and thus they should not be called as peaks by computation. Surprisingly, we observed the predominance of 6mA-free fragments in the pulled-down fractions. By taking advantage of the submicromolar affinity of the antibodies, we further developed an elegant, multiple-round immunoprecipitation (MrIP) approach and showed that 6mA-containing fragments could be enriched over 9100-fold and dominated in the final pulled-down fractions. This biochemical approach would greatly reduce the peak calling bias, which is caused by handling of dominated 6mA-free DNA fragments with an assumption-based algorithm computation and facilitates 6mA-pertinent data mining. The MrIP concept is extendable for the genome-wide sequencing of diverse DNA modifications.

Pages88
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/41494
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
刘晓玲. 哺乳动物DNA N6-甲基腺嘌呤的分析与鉴定[D]. 北京. 中国科学院生态环境研究中心,2018.
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