Szabó P, Mann J R
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010-0269.
Development. 1994 Jun;120(6):1651-60. doi: 10.1242/dev.120.6.1651.
Messenger RNA and methylation levels of four imprinted genes, H19, Igf2r, Igf-2 and Snrpn were examined by northern and Southern blotting in mouse parthenogenetic, androgenetic and normal or wild-type embryonic stem cell lines during their differentiation in vitro as embryoid bodies. In most instances, mRNA levels in parthenogenetic and androgenetic embryoid bodies differed from wild type as expected from previously determined patterns of monoallelic expression in midgestation embryos and at later stages of development. These findings implicate aberrant mRNA levels of these genes in the abnormal development of parthenogenetic and androgenetic embryos and chimeras. Whereas complete silence of one of the parental alleles has previously been observed in vivo, we detected some mRNA in the corresponding embryonic stem cell line. This 'leakage' phenomenon could be explained by partial erasure, bypass or override of imprints, or could represent the actual activity status at very early stages of development. The mRNA levels of H19, Igf2r and Igf-2 and the degree of methylation at specific associated sequences were correlated according to previous studies in embryos, and thereby are consistent with suggestions that the methylation might play a role in controlling transcription of these genes. Paternal-specific methylation of the H19 promoter region is absent in sperm, yet we observed its presence in undifferentiated androgenetic embryonic stem cells, or before the potential expression phase of this gene in embryoid bodies. As such methylation is likely to invoke a repressive effect, this finding raises the possibility that it is part of the imprinting mechanism of H19, taking the form of a secondary imprint or postfertilization epigenetic modification necessary for repression of the paternal allele.
通过Northern印迹和Southern印迹法，检测了小鼠孤雌生殖、雄核生殖以及正常或野生型胚胎干细胞系在体外分化形成类胚体过程中，四个印记基因H19、Igf2r、Igf-2和Snrpn的信使核糖核酸（mRNA）水平及甲基化水平。在大多数情况下，孤雌生殖和雄核生殖类胚体中的mRNA水平与野生型不同，这与先前在妊娠中期胚胎及发育后期确定的单等位基因表达模式预期一致。这些发现表明这些基因的异常mRNA水平与孤雌生殖和雄核生殖胚胎及嵌合体的异常发育有关。虽然之前在体内观察到亲本等位基因之一完全沉默，但我们在相应的胚胎干细胞系中检测到了一些mRNA。这种“渗漏”现象可以用印记的部分擦除、绕过或覆盖来解释，或者可能代表发育非常早期的实际活性状态。根据先前对胚胎的研究，H19、Igf2r和Igf-2的mRNA水平与特定相关序列的甲基化程度相关，因此与甲基化可能在控制这些基因转录中起作用的观点一致。精子中不存在H19启动子区域的父源特异性甲基化，但我们在未分化的雄核生殖胚胎干细胞中，或在类胚体中该基因的潜在表达阶段之前观察到了其存在。由于这种甲基化可能会产生抑制作用，这一发现增加了它是H19印记机制一部分的可能性，其形式为抑制父本等位基因所需的二级印记或受精后表观遗传修饰。
