Sasaki H, Ferguson-Smith A C, Shum A S, Barton S C, Surani M A
Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, UK.
Development. 1995 Dec;121(12):4195-202. doi: 10.1242/dev.121.12.4195.
The mouse H19 gene is imprinted so that the paternal copy is both methylated and repressed during fetal development. However, the CpG-rich promoter region encompassing the transcription start is not methylated in sperm; this region must therefore become methylated postzygotically. We first examined the timing of DNA methylation of this region and the corresponding expression of H19. Both parental copies are initially undermethylated in blastocysts and the paternal copy then becomes fully methylated in the embryo around implantation; this methylation is more protracted in the extraembryonic lineages, especially in the trophoblast. By contrast to the lineage-dependent methylation, we observed exclusive expression of the maternal copy in preimplantation embryos and in all the lineages of early postimplantation embryos although variability may exist in cultured embryos. This indicates that methylation of the CpG-rich promoter is not a prerequisite for the paternal repression. We then examined whether methylation and expression occurs appropriately in the absence of a maternal or a paternal genome. Both H19 copies in androgenetic embryos are fully methylated while they are unmethylated in parthenogenetic embryos. This correlates with the lack of expression in androgenetic embryos but expression in parthenogenetic embryos. However, the androgenetic trophoblast was exceptional as it shows reduced methylation and expresses H19. These results suggest that promoter methylation is not the primary inactivation mechanism but is a stabilizing factor. Differential methylation in the more upstream region, which is established in the gametes, is a likely candidate for the gametic signal and may directly control H19 activity.
小鼠H19基因是印记基因，在胎儿发育过程中，父本拷贝会发生甲基化并受到抑制。然而，包含转录起始位点的富含CpG的启动子区域在精子中并未发生甲基化；因此，该区域必定在合子形成后才会发生甲基化。我们首先研究了该区域DNA甲基化的时间以及H19的相应表达情况。在囊胚中，双亲拷贝最初均处于低甲基化状态，随后父本拷贝在着床前后的胚胎中完全甲基化；这种甲基化在胚外谱系中持续时间更长，尤其是在滋养层中。与谱系依赖性甲基化不同，我们观察到在植入前胚胎以及植入后早期胚胎的所有谱系中，母本拷贝均有特异性表达，尽管在培养的胚胎中可能存在变异性。这表明富含CpG的启动子甲基化并非父本抑制的先决条件。然后，我们研究了在缺失母本或父本基因组的情况下，甲基化和表达是否会正常发生。孤雄胚胎中的两个H19拷贝均完全甲基化，而孤雌胚胎中的H19拷贝则未甲基化。这与孤雄胚胎中缺乏表达但孤雌胚胎中有表达的情况相关。然而，孤雄滋养层是个例外，因为它的甲基化程度降低且表达H19。这些结果表明，启动子甲基化并非主要的失活机制，而是一个稳定因子。在配子中建立的更上游区域的差异甲基化可能是配子信号的候选因素，并且可能直接控制H19的活性。
