Meg3 non-coding RNA expression controls imprinting by preventing transcriptional up-regulation in cis

Although many long non-coding RNAs (lncRNAs) are controlled by genomic imprinting, their roles often remain unknown. The Dlk1-Dio3 imprinted domain expresses the lncRNA Meg3 (also called Gtl2) and multiple microRNAs and snoRNAs from the maternal chromosome. This locus constitutes an epigenetic model for pluripotency and development, particularly for neurogenesis. The domain’s Dlk1 (Delta-like-1) gene encodes a ligand that inhibits Notch1 signalling and regulates diverse developmental processes. Using a hybrid embryonic stem (ES) cell system, we find that Dlk1 becomes imprinted during neural differentiation and that this involves chromatin activation and transcriptional up-regulation on the paternal chromosome. On the maternal chromosome, the Dlk1 gene remains poised. This allelic protection against gene activation is controlled in cis by Meg3 expression and also involves the H3-lysine-27 methyltransferase Ezh2. Maternal Meg3 expression additionally protects against de novo DNA methylation at its promoter. Concordantly, we find that the lncRNA Meg3 is nuclear, accumulates onto the imprinted locus and overlaps in cis with Dlk1 in embryonic cells. Our data evoke an imprinting model in which a mono-allelic lncRNA prevents chromatin and gene activation in cis during development. RNA-Seq profiles of NPCs generated from WT ES cells (line BJ-WT3) and 2 different Meg3 KO ES lines (ES lines promoter-/- and intron1-/- derived from ES BJ-WT3) using Illumina HiSeq 2500 as 2 technical replicates.

尽管诸多长链非编码RNA（long non-coding RNAs，lncRNAs）受基因组印记（genomic imprinting）调控，但其功能往往仍未明确。Dlk1-Dio3印记结构域（Dlk1-Dio3 imprinted domain）可从母源染色体（maternal chromosome）表达长链非编码RNA Meg3（亦称Gtl2）以及多种微小RNA（microRNAs）和核仁小RNA（snoRNAs），该位点是多能性与发育，尤其神经发生（neurogenesis）相关研究的经典表观遗传模型。此结构域中的Dlk1（Delta样蛋白1，Delta-like-1）基因编码一种可抑制Notch1信号通路（Notch1 signalling）、调控多种发育过程的配体。本研究借助胚胎干细胞（embryonic stem，ES）细胞杂交系统，发现Dlk1在神经分化（neural differentiation）过程中获得印记，该过程涉及父源染色体（paternal chromosome）的染色质激活（chromatin activation）与转录上调（transcriptional up-regulation）；而在母源染色体上，Dlk1基因始终处于表观遗传预激活状态。这种对基因激活的等位基因保护作用由Meg3的顺式（cis）表达调控，同时还依赖组蛋白H3赖氨酸27甲基转移酶Ezh2（H3-lysine-27 methyltransferase Ezh2）。母源Meg3的表达还可抑制其启动子（promoter）区域发生从头DNA甲基化（de novo DNA methylation）。与此一致，我们发现长链非编码RNA Meg3定位于细胞核内，并聚集于该印记结构域，且在胚胎细胞中与Dlk1存在顺式序列重叠。我们的研究数据提出了一种印记调控模型：单等位基因表达的长链非编码RNA可在发育过程中通过顺式作用阻止染色质与基因激活。本研究针对从野生型（wild type，WT）ES细胞系BJ-WT3，以及2株分别由BJ-WT3 ES细胞系衍生的Meg3敲除（knockout，KO）ES细胞系（启动子敲除型promoter-/-和内含子1敲除型intron1-/-）中诱导得到的神经前体细胞（neural progenitor cells，NPCs），采用Illumina HiSeq 2500测序平台进行测序并设置2次技术重复（technical replicates），获取其RNA测序（RNA-Seq）表达谱。