Lee Jiyoung, Inoue Kimiko, Ono Ryuichi, Ogonuki Narumi, Kohda Takashi, Kaneko-Ishino Tomoko, Ogura Atsuo, Ishino Fumitoshi
Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Development. 2002 Apr;129(8):1807-17. doi: 10.1242/dev.129.8.1807.
Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.
基因组印记是一种表观遗传机制，它导致父本和母本基因组之间的功能差异，并在哺乳动物发育中发挥重要作用。印记基因DNA甲基化模式的阶段特异性变化表明，它们的印记在原始生殖细胞（PGC）阶段的某个时间被消除，然后在配子发生过程中根据个体性别重新建立其配子模式。为了确定消除过程的确切时间和模式，我们分析了印记基因的亲本来源特异性表达以及通过核移植从单个第11.5天到第13.5天的PGC衍生的胚胎中差异甲基化区域（DMR）的DNA甲基化模式。从第12.5天到第13.5天的PGC产生的克隆胚胎在第9.5天左右出现生长迟缓并早期胚胎致死。印记基因完全失去了它们的亲本来源特异性表达模式，变成了双等位基因表达或沉默。我们证实，来自雄性和雌性PGC的克隆都给出了相同的结果，这表明雄性和雌性生殖系存在共同的基因组印记默认状态。当我们从第11.5天的PGC产生克隆胚胎时，它们的发育得到了显著改善，使它们能够存活到至少第11.5天的胚胎阶段。有趣的是，在这些胚胎中观察到了体细胞状态和默认状态之间的几种基因组印记中间状态。印记基因单等位基因表达的丧失以逐步方式进行，每个印记基因都有特定的协调。还观察到印记基因DMR的DNA去甲基化与它们印记单等位基因表达的丧失完全一致。对第10.5天到第12.5天的PGC中的DNA甲基化分析表明，PGC克隆很好地代表了供体PGC的DNA甲基化状态。这些发现提供了强有力的证据，表明基因组印记记忆的消除过程在第10.5天到第11.5天的PGC中进行，每个印记基因的时间都受到精确控制。核移植技术使我们能够分析每个PGC的印记状态，并清楚地证明了表达和DNA甲基化模式与印记基因支持发育能力之间的密切关系。
