Kawasumi Miyuri, Unno Yuichi, Matsuoka Toshiki, Nishiwaki Megumi, Anzai Masayuki, Amano Tomoko, Mitani Tasuku, Kato Hiromi, Saeki Kazuhiro, Hosoi Yoshihiko, Iritani Akira, Kishigami Satoshi, Matsumoto Kazuya
Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kinki University, Wakayama, Japan.
Mol Reprod Dev. 2009 Apr;76(4):342-50. doi: 10.1002/mrd.20966.
Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos--the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.
八聚体结合转录因子4（Oct-4）对于正常胚胎发育至关重要，克隆胚胎中Oct-4表达异常导致克隆效率低下。然而，克隆胚胎中Oct-4表达异常的原因尚不清楚。由于已知调控区域的DNA甲基化可控制转录活性，我们研究了克隆小鼠胚胎中Oct-4基因的三个转录调控区域——远端增强子（DE）、近端增强子（PE）和启动子区域的甲基化状态。我们还研究了克隆胚胎中Oct-4基因的表达水平。免疫化学分析显示，85%的克隆囊胚在滋养外胚层和内细胞团细胞中均表达Oct-4。DNA甲基化分析显示，克隆桑葚胚中PE区域的甲基化程度高于正常桑葚胚。然而在克隆囊胚中，该区域的甲基化程度低于正常囊胚。我们发现克隆囊胚中从头甲基转移酶3b表达异常。这些结果表明，克隆胚胎在Oct-4基因PE区域的CpG位点存在异常DNA甲基化，这可能直接导致该基因在克隆胚胎中表达异常。
