Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cell lines.
Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cell lines.
复制标题
作者：
P. Szabó;J. Mann
采用北方和南方杂交技术检测了小鼠孤雌生殖、雄核发育和正常或野生型胚胎干细胞系在体外向胚状体分化过程中H19、Igf 2 r、Igf-2和Snrpn 4个印迹基因的mRNA和甲基化水平。在大多数情况下，mRNA水平在孤雌生殖和雄核发育胚状体不同于野生型预期从先前确定的模式，在妊娠中期胚胎和发育后期的单等位基因表达。这些发现暗示这些基因的异常mRNA水平在孤雌生殖和雄核发育胚胎和嵌合体的异常发育中。而完全沉默的父母等位基因之一，以前已经在体内观察到，我们检测到一些mRNA在相应的胚胎干细胞系。这种“泄漏”现象可以用部分擦除、绕过或覆盖印记来解释，或者可以代表开发早期阶段的实际活动状态。H19、Igf 2 r和Igf-2的mRNA水平与特定相关序列的甲基化程度相关，这与先前在胚胎中的研究一致，因此与甲基化可能在控制这些基因的转录中发挥作用的建议一致。父亲特异性甲基化的H19启动子区域是不存在的精子，但我们观察到它的存在，在未分化的雄激素胚胎干细胞，或之前的潜在表达阶段，该基因在胚状体。由于这种甲基化可能引起抑制效应，这一发现提出了它是H19印迹机制的一部分的可能性，其形式为抑制父本等位基因所必需的次级印迹或受精后表观遗传修饰。
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.