|Alternative Title||Identification and Analysis of DNA N6-methyladenine in Mammal|
|Place of Conferral||北京|
|Keyword||Dna 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)|
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。
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.
|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.|
|Files in This Item:|
|刘晓玲-哺乳动物DNA N6-甲基腺嘌呤（3814KB）||学位论文||开放获取||CC BY-NC-SA||Application Full Text|
|Recommend this item|
|Export to Endnote|
|Similar articles in Google Scholar|
|Similar articles in Baidu academic|
|Similar articles in Bing Scholar|
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.