Imprinted gene expression at the Dlk1-Dio3 cluster is controlled by both maternal and paternal IG-DMRs in a tissue-specific fashion.

Imprinting at the Dlk1-Dio3 cluster is controlled by the IG-DMR, an imprinting control region differentially methylated between maternal and paternal chromosomes. The maternal IG-DMR is essential for imprinting control, functioning as a cis enhancer element. Meanwhile, DNA methylation at the paternal IG-DMR is thought to prevent enhancer activity. To explore whether suppression of enhancer activity at the methylated IG-DMR requires the transcriptional repressor TRIM28, we analyzed Trim28chatwo embryos and performed epistatic experiments with IG-DMR deletion mutants. We found that while TRIM28 regulates the enhancer properties of the paternal IG-DMR, it also controls imprinting through other mechanisms. Additionally, we found that the paternal IG-DMR, previously deemed dispensable for imprinting, is required in certain tissues, demonstrating that imprinting is regulated in a tissue-specific manner. Using ChRO-seq to analyze nascent transcription, we show that different tissues have a distinctive regulatory landscape at the Dlk1-Dio3 cluster, providing insight into potential mechanisms of tissue-specific imprinting control. ChRO-seq identified 30 novel transcribed regulatory elements, including a candidate regulatory region that depends on the paternal IG-DMR. Together, our findings challenge the model that Dlk1-Dio3 imprinting is regulated through a single mechanism and demonstrate that different tissues use distinct strategies for imprinting control. Duplicates of PRO-seq in E14.5 liver and yolk sac samples obtained from mating C57BL/6J females with males of a mixed genetic background (C57BL/6J and CAST/EiJ). Males in this cross were selected to exclusively carry CAST/EiJ polymophisms at the Dlk1/Gtl2 locus on chromosome 12Dlk1-Dio3基因簇的印记调控由IG-DMR（印记控制区域）介导，该区域在母本与父本染色体间存在差异甲基化。母本IG-DMR是印记调控的核心元件，可作为顺式增强子发挥功能。与此同时，父本IG-DMR上的DNA甲基化被认为会抑制其增强子活性。为探究甲基化IG-DMR的增强子活性抑制是否依赖转录抑制因子TRIM28，本研究分析了Trim28chatwo胚胎，并结合IG-DMR缺失突变体开展上位性实验。研究发现，TRIM28不仅调控父本IG-DMR的增强子特性，还可通过其他机制实现印记调控。此外，本研究还发现，此前被认为对印记调控非必需的父本IG-DMR，在特定组织中却是必需的，这表明印记调控具有组织特异性。利用ChRO-seq分析新生转录，我们证实不同组织在Dlk1-Dio3基因簇上具有独特的调控图谱，为解析组织特异性印记调控的潜在机制提供了新视角。ChRO-seq共鉴定出30个新的转录调控元件，其中包括一个依赖父本IG-DMR的候选调控区域。综上，本研究的发现挑战了"Dlk1-Dio3印记调控仅通过单一机制实现"的传统模型，并证实不同组织采用不同策略进行印记调控。本研究还获取了C57BL/6J雌性小鼠与混合遗传背景（C57BL/6J与CAST/EiJ）雄性小鼠交配得到的E14.5胎肝及卵黄囊样本，并开展了PRO-seq重复实验。本次交配所用的雄性小鼠经筛选，仅在12号染色体上的Dlk1/Gtl2位点携带CAST/EiJ多态性。