Department of Obstetrics, University of Western Ontario, Schulich School of Medicine and Dentistry, London, ON N6A 5W9, Canada.
Development. 2011 Sep;138(17):3667-78. doi: 10.1242/dev.057778. Epub 2011 Jul 20.
To understand the complex regulation of genomic imprinting it is important to determine how early embryos establish imprinted gene expression across large chromosomal domains. Long non-coding RNAs (ncRNAs) have been associated with the regulation of imprinting domains, yet their function remains undefined. Here, we investigated the mouse Kcnq1ot1 ncRNA and its role in imprinted gene regulation during preimplantation development by utilizing mouse embryonic and extra-embryonic stem cell models. Our findings demonstrate that the Kcnq1ot1 ncRNA extends 471 kb from the transcription start site. This is significant as it raises the possibility that transcription through downstream genes might play a role in their silencing, including Th, which we demonstrate possesses maternal-specific expression during early development. To distinguish between a functional role for the transcript and properties inherent to transcription of long ncRNAs, we employed RNA interference-based technology to deplete Kcnq1ot1 transcripts. We hypothesized that post-transcriptional depletion of Kcnq1ot1 ncRNA would lead to activation of normally maternal-specific protein-coding genes on the paternal chromosome. Post-transcriptional short hairpin RNA-mediated depletion in embryonic stem, trophoblast stem and extra-embryonic endoderm stem cells had no observable effect on the imprinted expression of genes within the domain, or on Kcnq1ot1 imprinting center DNA methylation, although a significant decrease in Kcnq1ot1 RNA signal volume in the nucleus was observed. These data support the argument that it is the act of transcription that plays a role in imprint maintenance during early development rather than a post-transcriptional role for the RNA itself.
为了理解基因组印记的复杂调控，重要的是要确定早期胚胎如何在大染色体区域建立印记基因表达。长非编码 RNA（ncRNA）与印迹域的调控有关，但它们的功能仍然未知。在这里，我们利用小鼠胚胎和胚胎外干细胞模型研究了小鼠 Kcnq1ot1 ncRNA 及其在植入前发育过程中对印迹基因调控的作用。我们的研究结果表明，Kcnq1ot1 ncRNA 从转录起始位点延伸 471 kb。这很重要，因为它提出了通过下游基因转录可能在其沉默中发挥作用的可能性，包括我们证明在早期发育过程中具有母系特异性表达的 Th。为了区分转录本的功能作用和长 ncRNA 转录固有的特性，我们采用了基于 RNA 干扰的技术来耗尽 Kcnq1ot1 转录本。我们假设 Kcnq1ot1 ncRNA 的转录后耗竭会导致父系染色体上正常母系特异性蛋白编码基因的激活。在胚胎干细胞、滋养层干细胞和胚胎外内胚层干细胞中，转录后短发夹 RNA 介导的耗尽对该域内印迹基因的表达或 Kcnq1ot1 印迹中心 DNA 甲基化没有明显影响，尽管核内 Kcnq1ot1 RNA 信号体积明显减少。这些数据支持这样一种观点，即在早期发育过程中维持印迹的是转录本身的作用，而不是 RNA 本身的转录后作用。
