DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.
DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.
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DOI：
10.1038/srep19134
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发表时间：
2016-01-11
影响因子：
4.6
通讯作者：
Takada T
中科院分区：
文献类型：
--
作者：
Okamoto Y;Yoshida N;Suzuki T;Shimozawa N;Asami M;Matsuda T;Kojima N;Perry AC;Takada T
哺乳动物受精后，父本基因组5-甲基-2 ′-脱氧胞苷（5 mC）含量被认为通过氧化为5-羟甲基-2 ′-脱氧胞苷（5 hmC）而降低。这种去甲基化和羟甲基化的相互作用模型是从间接的、非定量的方法推断出来的。我们在这里报告的小规模液相色谱串联质谱（SMM）直接定量基因组5 mC和5 hmC在小鼠胚胎。在体外受精（IVF）和卵胞浆内单精子注射（ICSI）产生的胚胎中，绝对5 mC水平的曲线几乎相同。受精后10小时，5 mC水平下降了约40%，与活跃的基因组DNA去甲基化一致。在雄基因（只包含一个父亲的基因组）和单性生殖（只包含一个母亲的基因组）的5 mC水平经历了积极的5 mC损失在第一个6小时，表明父母的基因组可以独立地进行去甲基化。我们没有发现受精后10 - 48小时5 mC净丢失的证据，这意味着DNA复制后的任何被动“去甲基化”都被主动5 mC维持甲基化所平衡。然而，在48小时后的发育过程中，5 mC水平下降，在囊胚（约96小时）中降至1%（作为G-残基的分数测量）。在正常二倍体胚胎的整个发育过程中，5 hmC水平始终较低（<0.2%的G-残基）。这项工作直接量化了小鼠植入前胚胎中全局基因组DNA修饰的动态，表明SMM将适用于样本量有限的其他生物医学情况。
Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.