Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the DNA (de)methylation machinery.

中文翻译：

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具有等位基因特异功能的双分子元件可保护Dlk1-Dio3基因座上的DNA甲基化印记

印迹基因位点的失调也称为印迹丢失（LOI），可能导致严重的发育缺陷和其他疾病，但是确保印迹稳定性的分子机制仍然不完全清楚。在这里，我们剖析了基本Dlk1-Dio3基因座（称为IG-DMR）的印迹控制区域的功能组件，以及它们确保印迹维护的机制。使用携带等位基因特异性报告系统的多能干细胞，我们证明了IG-DMR由两个拮抗性调控元件组成：一个阻止Tet双加氧酶活性的父本甲基化CpG-岛和一个母本未甲基化的调控元件，它充当了非典型增强子，并通过排除从头DNA甲基转移酶功能来维持母体Gtl2 lncRNA的表达。这些元件的定向遗传或表观遗传编辑会导致LOI具有双亲或双亲表达模式，以及整个Dlk1-Dio3基因座的DNA甲基化和3D染色质拓扑的等位基因变化。尽管对IG-DMR或Gtl2启动子的靶向抑制足以引起LOI，但LOI表型的稳定性取决于IG-DMR状态，提示功能层次。这些发现将IG-DMR确立为一种新型的两部分控制元件，并通过等位基因特异性限制DNA（去）甲基化机制为Dlk1-Dio3印迹控制提供了机械学见解。