Wu F-R, Zhang Y, Ding B, Lei X-H, Huang J-C, Wang C-H, Liu Y, Wang R, Li W-Y
School of Biological Science and Food Engineering, Fuyang Teachers College, Key Laboratory of Embryo Development and Reproductive Regulation, Anhui Province, Fuyang, China.
School of Biological Science and Food Engineering, Fuyang Teachers College, Key Laboratory of Embryo Development and Reproductive Regulation, Anhui Province, Fuyang, China
Genet Mol Res. 2014 Dec 4;13(4):10121-9. doi: 10.4238/2014.December.4.6.
As a core member of polycomb repressive complex 2, the transcription and enzyme activity of enhancer of zeste homolog 2 (Ezh2) is directly involved in the trimethylation of lysine 27 on histone H3. In this study, the fluorescence intensity of H3K27me3 in mouse in vivo morulae and blastocysts was compared by indirect immunofluorescence staining. We found that demethylation of H3K27me3 occurred during the blastocyst stage. Real-time polymerase chain reaction was performed to investigate Ezh2 expression in oocytes and in preimplantation embryos. Ezh2 expression peaked during the zygote stage and gradually decreased from the 2-cell stage, exhibiting an inverse pattern when compared with Oct4 and Sox2 mRNA in mouse preimplantation embryos. To understand the role of development-related genes on the transcription of mouse Ezh2, a promoter assay was performed in NIH/3T3 cells. Ezh2 expression was markedly suppressed by Oct4 and Sox2 alone in a dose-dependent manner, while Ezh2 promoter activity in co-transfection with Nanog, Klf-4, and c-Myc groups showed no significant change as compared with the control. Our data suggest that the demethylation of H3K27me3 is caused by the degressive expression and activity of Ezh2 in blastocysts, leading to increased expression of developmentally important transcription factors. We also observed negative effects of Oct4 and Sox2 on the transcription of Ezh2 and identified Oct4 and Sox2 as novel negative regulators of Ezh2 at the post-translation level in a mouse preimplantation embryo.
作为多梳抑制复合物2的核心成员，zeste同源物2（Ezh2）的转录和酶活性直接参与组蛋白H3赖氨酸27的三甲基化。在本研究中，通过间接免疫荧光染色比较了小鼠体内桑椹胚和囊胚中H3K27me3的荧光强度。我们发现H3K27me3的去甲基化发生在囊胚阶段。进行实时聚合酶链反应以研究Ezh2在卵母细胞和植入前胚胎中的表达。Ezh2表达在合子期达到峰值，并从2细胞期开始逐渐下降，与小鼠植入前胚胎中的Oct4和Sox2 mRNA相比呈现相反的模式。为了了解发育相关基因对小鼠Ezh2转录的作用，在NIH/3T3细胞中进行了启动子分析。单独的Oct4和Sox2以剂量依赖性方式显著抑制Ezh2表达，而与Nanog、Klf-4和c-Myc组共转染时Ezh2启动子活性与对照组相比无显著变化。我们的数据表明，H3K27me3的去甲基化是由囊胚中Ezh2表达和活性的下降引起的，导致发育重要转录因子的表达增加。我们还观察到Oct4和Sox2对Ezh2转录的负面影响，并确定Oct4和Sox2是小鼠植入前胚胎翻译后水平上Ezh2的新型负调节因子。
