Imprint establishment at the PWS-SRO in a cellular model

转录在印迹基因gDMR的DNA甲基化和基因表达调控中的作用已被广泛接受。然而，转录过程如何与DNA甲基转移酶的募集相联系尚不完全清楚。我们试图建立一个人类体外系统，以确定所需的机制和因素。虽然我们可以观察到通过在几个启动子处诱导转录通读来下调基因表达，但在任何产生的细胞系中都没有获得DNA甲基化。补充所涉及的表观遗传因子，如DNMT 3A、DNMT 3L和ZFP 57，不足以在诱导转录通读后引起DNA甲基化。我们的结论是，基因组环境可能是必不可少的，或者细胞需要处于允许状态才能对转录通读的DNA甲基化做出反应。在这项提案中，我们希望通过在人类iPSCs中Prader-Willi/Angelman综合征基因座的gDMR处模拟卵母细胞特异性转录通读来解决这两种可能性。为了实现这一点，卵母细胞特异性AS-SRO启动子将被诱导型启动子取代。在Angelman综合征患者中，AS-SRO（Angelman综合征最短重叠区）的缺失与gDMR处缺乏DNA甲基化相关。因此，我们假设起始于AS-SRO并贯穿gDMR的转录负责在gDMR处建立DNA甲基化。通过诱导取代的AS-SRO，可以在常规基因组背景下测试该假设。此外，通过使用iPSC，我们有可能将这些细胞分化为三个胚层的衍生物，并在此过程中监测转录通读的效果。在未来的实验中，可以使用相同的策略替换其他印记基因的调控元件。此外，转录依赖性DNA甲基化过程中对表观遗传修饰剂的需求可以通过从细胞系统中补充或删除它们来解决。有了这个，我们的研究将有助于了解DNA甲基化，转录和细胞潜力的依赖性。

The role of transcription in establishment of DNA methylation at gDMRs of imprinted genes and in the regulation of gene expression is widely accepted. However, how the process of transcription is linked to recruitment of DNA methyltransferases is not entirely clear. We sought to establish a human in vitro system to identify the involved mechanisms and factors needed. Although we could observe downregulation of gene expression by induction of transcriptional read-through at several promoters, DNA methylation was not acquired in any of the generated cell lines. Supplementation of epigenetic factors involved, like DNMT3A, DNMT3L and ZFP57, was not sufficient to provoke DNA methylation upon induction of transcriptional read-through. We concluded that either genomic context could be essential or that cells need to be in a permissive state to be able to respond with DNA methylation to transcriptional read-through. In this proposal we want to address these two possibilities by modelling oocyte-specific transcriptional read-through at the gDMR of the Prader-Willi/Angelman syndrome locus in human iPSCs. To achieve this, the oocyte-specific AS-SRO promoter will be replaced by an inducible promoter. In patients with Angelman syndrome, deletions at the AS-SRO (Angelman syndrome shortest region of overlap) are associated with lack of DNA methylation at the gDMR. We therefore hypothesize that transcription initiating at the AS-SRO and running through the gDMR is responsible for establishment of DNA methylation at the gDMR. By induction of the replaced AS-SRO, this hypothesis can be tested in the regular genomic context. In addition, with the use of iPSCs we have the possibility to differentiate these cells into derivatives of the three germ layers and to monitor the effect of transcriptional read-through during this process. In future experiments, regulatory elements at other imprinted genes could be replaced using the same strategy. Also, the need of epigenetic modifiers in the process of transcription-dependent DNA methylation could be addressed by either supplementing or deleting them from the cell system. With this, our study will contribute to understand the dependencies of DNA methylation, transcription and cellular potential.