BACKGROUND Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and microRNA (miRNA)-mediated mRNA degradation of KPNA7 were examined. RESULTS Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza-CdR showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR (RT-qPCR) analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. CONCLUSIONS These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.

中文翻译：

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DNA甲基化和miRNA-1296协同作用，介导牛卵母细胞和早期胚胎发育过程中KPNA7的时空表达。

背景技术卵母细胞特异性母体因子的表观遗传学调节对于卵母细胞和早期胚胎发育是必不可少的。KPNA7是卵母细胞特异性的母体因子，它控制着对早期胚胎发育很重要的核蛋白的转运。为了阐明参与KPNA7受控表达的表观遗传机制，研究了DNA甲基化相关的转录沉默和microRNA（miRNA）介导的KPNA7的降解。结果比较了在卵母细胞和6个不同的体细胞组织之间KPNA7基因近端启动子的DNA甲基化谱，鉴定出3个卵母细胞特异性差异甲基化CpG位点。用甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-氮杂-CdR）处理后，KPNA7 mRNA的表达重新引入牛肾源CCL2细胞。分析用5-Aza-CdR处理的CCL2细胞中KPNA7基因的启动子区域，显示所有CpG位点的甲基化率均较低。生物信息学分析预测KPNA7 mRNA编码区中有4个miRNA-1296结合位点。miRNA-1296和KPNA7在HEK293细胞中异位共表达导致KPNA7蛋白表达降低。实时荧光定量PCR（RT-qPCR）分析显示，miRNA-1296在卵母细胞和早期胚胎中表达，并且在8细胞阶段胚胎中表达达到峰值，这与胚胎基因组激活时间和启动时间一致。 KPNA7表达下降。结论这些结果表明，DNA甲基化可能解释了KPNA7的卵母细胞特异性表达，