INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France.
PLoS One. 2012;7(5):e38156. doi: 10.1371/journal.pone.0038156. Epub 2012 May 31.
In the mouse zygote, DNA methylation patterns are heavily modified, and differ between the maternal and paternal pronucleus. Demethylation of the paternal genome has been described as an active and replication-independent process, although the mechanisms responsible for it remain elusive. Recently, 5-hydroxymethylcytosine has been suggested as an intermediate in this demethylation.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, we quantified DNA methylation and hydroxymethylation in both pronuclei of the mouse zygote during the replication period and we examined their patterns on the pericentric heterochromatin using 3D immuno-FISH. Our results demonstrate that 5-methylcytosine and 5-hydroxymethylcytosine localizations on the pericentric sequences are not complementary; indeed we observe no enrichment of either marks on some regions and an enrichment of both on others. In addition, we show that DNA demethylation continues during DNA replication, and is inhibited by aphidicolin. Finally, we observe notable differences in the kinetics of demethylation and hydroxymethylation; in particular, a peak of 5-hydroxymethylcytosine, unrelated to any change in 5-methylcytosine level, is observed after completion of replication.
CONCLUSIONS/SIGNIFICANCE: Together our results support the already proposed hypothesis that 5-hydroxymethylcytosine is not a simple intermediate in an active demethylation process and could play a role of its own during early development.
在小鼠受精卵中，DNA 甲基化模式被大量修饰，并且在母源和父源原核之间存在差异。已经描述了父本基因组的去甲基化是一个主动的、与复制无关的过程，尽管负责该过程的机制仍不清楚。最近，5-羟甲基胞嘧啶被认为是这种去甲基化的中间产物。
方法/主要发现：在这项研究中，我们在复制期定量测定了小鼠受精卵中两个原核的 DNA 甲基化和羟甲基化，并使用 3D 免疫荧光原位杂交（immuno-FISH）检查了它们在着丝粒异染色质上的模式。我们的结果表明，着丝粒序列上的 5-甲基胞嘧啶和 5-羟甲基胞嘧啶定位并不互补；实际上，我们在一些区域没有观察到任何一种标记的富集，而在另一些区域则同时富集了这两种标记。此外，我们表明 DNA 去甲基化在 DNA 复制过程中持续进行，并被阿霉素抑制。最后，我们观察到去甲基化和羟甲基化的动力学有显著差异；特别是，在复制完成后，观察到 5-羟甲基胞嘧啶的峰值，与 5-甲基胞嘧啶水平的任何变化无关。
结论/意义：综上所述，我们的结果支持了已经提出的假说，即 5-羟甲基胞嘧啶不是主动去甲基化过程中的简单中间产物，并且在早期发育过程中可能发挥自身作用。
