Institut für Humangenetik, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany.
PLoS One. 2013 Sep 3;8(9):e74159. doi: 10.1371/journal.pone.0074159. eCollection 2013.
The human retinoblastoma gene (RB1) is imprinted; the mouse Rb1 gene is not. Imprinted expression of RB1 is due to differential methylation of a CpG island (CpG85), which is located in the pseudogene PPP1R26P1 in intron 2 of RB1. CpG85 serves as promoter for an alternative RB1 transcript, which is expressed from the unmethylated paternal allele only and is thought to suppress expression of the full-length RB1 transcript in cis. PPP1R26P1 contains another CpG island (CpG42), which is biallelically methylated. To determine the influence of PPP1R26P1 on RB1 expression, we generated an in vitro murine embryonic stem cell model by introducing human PPP1R26P1 into mouse Rb1. Next generation bisulfite sequencing of CpG85 and CpG42 revealed differences in their susceptibility to DNA methylation, gaining methylation at a median level of 4% and 18%, respectively. We showed binding of RNA polymerase II at and transcription from the unmethylated CpG85 in PPP1R26P1 and observed reduced expression of full-length Rb1 from the targeted allele. Our results identify human PPP1R26P1 as a cis-repressive element and support a connection between retrotransposition of PPP1R26P1 into human RB1 and the reduced expression of RB1 on the paternal allele.
人类视网膜母细胞瘤基因（RB1）是印记基因；而小鼠的 Rb1 基因则不是。RB1 的印记表达是由于 CpG 岛（CpG85）的差异甲基化所致，该 CpG 岛位于 RB1 内含子 2 中的假基因 PPP1R26P1 中。CpG85 作为一个替代 RB1 转录本的启动子，仅从未甲基化的父系等位基因表达，据认为该转录本在顺式中抑制全长 RB1 转录本的表达。PPP1R26P1 含有另一个 CpG 岛（CpG42），其为双等位基因甲基化。为了确定 PPP1R26P1 对 RB1 表达的影响，我们通过将人 PPP1R26P1 引入小鼠 Rb1 中，生成了一种体外鼠胚胎干细胞模型。对 CpG85 和 CpG42 的下一代亚硫酸氢盐测序显示，它们对 DNA 甲基化的敏感性存在差异，分别获得了中位数为 4%和 18%的甲基化。我们在 PPP1R26P1 中发现了 RNA 聚合酶 II 在未甲基化的 CpG85 处的结合和转录，并观察到靶向等位基因全长 Rb1 的表达减少。我们的结果将人 PPP1R26P1 鉴定为一个顺式抑制元件，并支持 PPP1R26P1 反向转位进入人类 RB1 与父系等位基因 RB1 表达减少之间的联系。
