Silencing of maternally expressed RNAs in Dlk1-Dio3 domain is critical for embryonic development

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternal RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3polyA termination sequence in Gtl2 locus. By analyzing mouse embryos RNA-Seq data combined with histological analysis, we found that silence of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of fetal liver, resulting hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of the maternally expressed RNAs and activation of the paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the above phenotypes. In conclusion, these findings illuminate a novel mechanism by which silencing of the maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through activation of the apoptosis. We used Easi-CRISPR technology to insert three polyadenylation cassettes into the promoter of Gtl2 in the Dlk1-Dio3 imprinted domain to generate Gtl2 polyA knock-in mouse model. At embryonic day 12.5 (E12.5), the embryos of paternally Gtl2 polyA knock-in (PKI), maternally Gtl2 polyA knock-in (MKI), homozygous (HOMO) and wild-type (WT) mice were selected to do the RNA-sequencing.哺乳动物印记基因Dlk1-Dio3结构域包含多种长链非编码RNA（lncRNAs）、信使RNA（mRNAs）、基因组中规模最大的微小RNA（miRNA）簇以及4个差异甲基化区域（DMRs）；该位点内母源RNA的缺失会引发胚胎致死，但具体的分子机制尚未明确。本研究优化了该结构域内母源表达RNA的转录终止模型，发现胚胎致死的核心诱因是胚胎（尤以肝脏组织最为显著）发生细胞凋亡。本研究通过在Gtl2位点插入3聚腺苷酸（3polyA）终止序列，成功构建了Dlk1-Dio3结构域内母源表达RNA沉默的小鼠模型。结合小鼠胚胎RNA测序（RNA-Seq）数据与组织学分析，本研究发现该结构域内母源表达RNA的沉默会激活细胞凋亡，引发胎肝血管破裂，进而导致出血与组织损伤。从分子机制来看，Gtl2转录的终止会导致该区间内母源表达RNA的沉默以及父源表达基因的激活，而引发上述表型的核心因素是基因本身，而非IG-DMR与Gtl2-DMR。综上，本研究揭示了一种全新的分子机制：Dlk1-Dio3结构域内母源表达RNA的沉默通过激活细胞凋亡，引发肝出血并最终导致胚胎致死。本研究利用Easi-CRISPR技术，在Dlk1-Dio3印记结构域的Gtl2启动子区域插入3个聚腺苷酸（polyA）表达盒，成功构建了Gtl2 polyA敲入（knock-in）小鼠模型。本研究选取胚胎发育第12.5天（E12.5）的父系Gtl2 polyA敲入（PKI）、母系Gtl2 polyA敲入（MKI）、纯合子（HOMO）以及野生型（WT）小鼠胚胎进行RNA测序。