Doherty A S, Mann M R, Tremblay K D, Bartolomei M S, Schultz R M
Department of Biology and Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Biol Reprod. 2000 Jun;62(6):1526-35. doi: 10.1095/biolreprod62.6.1526.
The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.
H19基因呈现印记状态，母本等位基因优先表达。该基因座的假定印记控制区域在被抑制的父本等位基因上高度甲基化。尽管在体内发育的小鼠囊胚中观察到H19的母本特异性表达，但在体外培养条件下实验操作的胚胎和胚胎干细胞中已记录到双等位基因表达。在本研究中，确定了培养对印记H19表达和甲基化的影响。将2细胞胚胎在惠顿氏培养基中培养至囊胚阶段后，通常沉默的父本H19等位基因异常表达，而在含有氨基酸的KSOM（KSOM+AA）中培养后，几乎未观察到父本表达。对位于上游印记控制区域的一个CpG二核苷酸的甲基化状态分析显示，在惠顿氏培养基中培养的胚胎中甲基化缺失，而在KSOM+AA中培养的胚胎中未出现这种情况。因此，惠顿氏培养基中的培养对H19表达和甲基化产生不利影响，而H19对KSOM+AA培养的反应更接近体内情况。在惠顿氏培养基中培养后H19的双等位基因表达不太可能是较低甲基化水平的普遍效应，因为体内来源和培养的胚胎中DNA甲基转移酶活性的量和Dnmt1蛋白的空间分布相似。此外，在任何一种培养基中培养后，Snrpn的印记表达均得以维持，这表明并非所有印记基因都受到相同严格的印记控制。培养条件可显著但选择性地影响印记基因表达这一发现为进一步研究DNA甲基化与基因表达之间的联系提供了一个模型系统。
