Optimization of epigenomic modification with regulatory RNAs considering the presence of endogenous non-coding RNAs

近年来越来越多的证据表明，多种非编码rna （ncRNAs）参与了区域特异性表观遗传调控。由于许多内源性ncrna表现出组织/细胞类型依赖性表达模式，因此揭示组织/细胞类型依赖性表达与表观遗传调控之间的关系非常重要。我们的数据表明，许多基因在组织/细胞类型特异性基因的启动子区域内含有ncrna，这些基因的DNA甲基化状态因缺乏ncrna相关酶而改变。Sall4基因是胚胎干细胞发育的关键基因，其内源性反义ncRNA（ASncRNA）与编码Sall4蛋白的启动子区域重叠。Sall4 ASncRNA独立于Dicer双链RNA合成调节Sall4启动子的DNA甲基化状态。ASncRNA在成纤维细胞中的异位过表达导致Sall4启动子区域的DNA去甲基化。通过敲低内源性ASncRNA， Sall4 ASncRNA被证明是维持ES细胞非甲基化和激活状态不可或缺的。我们的数据还表明，卵母细胞特异性组蛋白和环境化学物质参与表观遗传调控。总之，我们可以鉴定出许多以靶特异性方式调节DNA甲基化状态的ASncRNAs，这些ASncRNAs被认为可用于表观基因组修饰，以建立表观基因组相关疾病模型动物和细胞。

Recent accumulating evidences have indicated that various non-coding RNAs(ncRNAs) are involved in region-specific epigenetic regulations. Since many endogenous ncRNAs show tissue/cell-type-dependent expression patterns, it is important to reveal the relationships between tissue/cell-type-dependent expression and epigenetic regulations. Our data indicated that many genes had ncRNAs within promoter regions of tissu/cell-type-specific genes whose DNA methylation status changed by deficiency of ncRNA-related enzymes. The Sall4 gene, which is crucial for stemness of embryonic stem(ES) cells, had an endogenous antisense ncRNA(ASncRNA) overlapping with the promoter region of the protein-coding Sall4 gene. The Sall4 ASncRNA regulated DNA methylation status of the Sall4 promoter independent of double-strand RNA synthesis by Dicer. An ectopic overexpression of the ASncRNA in fibroblast cells resulted DNA demethylation of the Sall4 promoter region. The Sall4 ASncRNA was proven indispensable for maintenance of unmethylated and activated status in ES cells by shRNA knockdown of the endogenous ASncRNA. Our data also indicated that oocyte-specific histone and environmental chemicals were involved in epigenetic regulations. In conclusion, we could identify many ASncRNAs that regulate DNA methylation status in a target-specific manner, and these ASncRNAs are thought as useful for epigenome modification to establish epigenome-related disease model animals and cells.