Hou Jian, Liu Lei, Zhang Jing, Cui Xiu-Hong, Yan Feng-Xiang, Guan Hong, Chen Yong-Fu, An Xiao-Rong
State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100094, PR China.
BMC Dev Biol. 2008 May 29;8:60. doi: 10.1186/1471-213X-8-60.
Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation.
By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels.
Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.
先前的研究表明，与小鼠受精卵不同，绵羊受精卵缺乏父本DNA去甲基化事件。另一种表观遗传标记，即组蛋白修饰，尤其是组蛋白3赖氨酸9（H3K9）处的修饰，已被认为与DNA甲基化存在机械联系。在小鼠受精卵中，父本原核中缺乏甲基化的H3K9被认为归因于父本DNA去甲基化。
通过免疫荧光染色方法，我们发现，尽管在DNA甲基化方面存在差异，但绵羊和小鼠受精卵中H3K9的修饰是相似的。在这两个物种中，相对于母本原核，父本原核中的H3K9均发生了高乙酰化或低甲基化。事实上，绵羊受精卵也能发生父本DNA去甲基化，尽管程度比小鼠小。通过双重免疫染色对单个受精卵的进一步检查显示，父本DNA甲基化水平与H3K9乙酰化或三甲基化水平并无密切关联。用5-氮杂胞苷或曲古抑菌素A处理均未导致父本DNA甲基化水平显著降低。
我们的结果表明，绵羊父本基因组较低的DNA去甲基化并非由于H3K9修饰，并且父本原核中持续存在的甲基化DNA并非来自DNA从头甲基化。
