He Hong, McHaney Mark, Hong James, Weiss Mark L
Department of Anatomy and Physiology and the Midwest Institute for Comparative Stem Cell Biology, Kansas State University, Manhattan, KS 66506, USA.
Open Stem Cell J. 2009 Jan 1;1(1):30-39. doi: 10.2174/1876893800901010030.
Here, the role of methylation in regulation of rat Oct4 gene was evaluated during embryonic development, in adult tissues and in embryo-derived cells. First, the region 3.1 kb upstream to the rat Oct4 ATG site was cloned and sequenced. The rat Oct4 upstream sequence was similar to that in bovine, mouse and human with two upstream elements: proximal (PE) and distal enhancers (DE) and four homology conserved regions (CR1-4). The conserved regions in the rat have 69% - 96% homology with bovine, human, mouse sequences. Next, the methylation pattern in the promoter was determined during embryonic development, in adult tissues, in rat embryonic stem cell (ESC)-like cells and umbilical cord-derived cells (the feeder for ESC-like cells) using the bisulfite method and DNA sequencing. The promoter was methylated in adult and fetal tissues, and in days post coitus (DPC) 10.5 and 12.5 embryos and hypomethylated in DPC4.5 embryos and in rat ESC-like cells. The expression of Oct4 was evaluated by qRT-PCR. DPC4.5 embryos and rat ESC-like cells had higher expression of the Oct4 gene compared to DPC10.5 and 12.5 embryos, adult tissues and embryoid bodies derived from rat ESC-like cells. Thus, the methylation status correlated with the qRT-PCR results. These results indicate that the rat Oct4 3.1kb promoter region is organized and contains transcription binding and regulatory sites similar to those described for bovine, mouse and human. The rat Oct4 promoter is methylated during embryonic development after 4.5 DPC and during differentiation of rat ESC-like cells to embryoid bodies.
在此，我们评估了甲基化在大鼠Oct4基因胚胎发育、成年组织及胚胎衍生细胞中的调控作用。首先，克隆并测序了大鼠Oct4基因ATG位点上游3.1 kb区域。大鼠Oct4上游序列与牛、小鼠和人类相似，具有两个上游元件：近端（PE）和远端增强子（DE）以及四个同源保守区域（CR1 - 4）。大鼠的保守区域与牛、人类、小鼠序列的同源性为69% - 96%。接下来，使用亚硫酸氢盐法和DNA测序确定了胚胎发育、成年组织、大鼠胚胎干细胞（ESC）样细胞和脐带衍生细胞（ESC样细胞的饲养层）中启动子的甲基化模式。启动子在成年和胎儿组织、交配后第10.5天和12.5天的胚胎中甲基化，在交配后第4.5天的胚胎和大鼠ESC样细胞中低甲基化。通过qRT - PCR评估Oct4的表达。与交配后第10.5天和12.5天的胚胎、成年组织以及源自大鼠ESC样细胞的胚状体相比，交配后第4.5天的胚胎和大鼠ESC样细胞中Oct4基因表达更高。因此，甲基化状态与qRT - PCR结果相关。这些结果表明，大鼠Oct4基因3.1 kb启动子区域结构有序，包含与牛、小鼠和人类中描述的类似的转录结合和调控位点。大鼠Oct4启动子在交配后4.5天之后的胚胎发育过程中以及大鼠ESC样细胞向胚状体分化过程中发生甲基化。
