Imprint establishment at the PWS-SRO in a cellular model

在 PWS-SRO 的细胞模型中建立印记

• 批准号: 285807807
• 负责人: Professorin Dr. Laura Steenpaß
• 金额: --
• 依托单位: Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/285807807?language=en
• 关键词: Imprint; establishment; PWS; SRO; cellular

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.

转录在印记基因的gDMR上建立DNA甲基化和在基因表达调控中的作用被广泛接受。然而，转录过程如何与DNA甲基转移酶的招募有关还不完全清楚。我们试图建立一个人类体外系统，以确定所需的相关机制和因素。虽然我们可以观察到通过在几个启动子上诱导转录通读而下调基因表达，但在任何产生的细胞系中都没有获得DNA甲基化。补充表观遗传因子，如DNMT3A、DNMT3L和ZFP57，不足以在诱导转录通读时引发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的状态，才能对转录通读做出DNA甲基化反应。在这项建议中，我们希望通过在人类ipscs中Prader-Willi/Angelman综合征基因座的gDMR处建立卵母细胞特异性转录通读模型来解决这两种可能性。为了实现这一点，卵母细胞特异性的AS-SRO启动子将被可诱导的启动子取代。在Angelman综合征患者中，AS-SRO(Angelman综合征最短重叠区域)缺失与gDMR缺乏DNA甲基化有关。因此，我们假设，在AS-SRO启动并贯穿gDMR的转录负责在gDMR建立DNA甲基化。通过诱导被替换的AS-SRO，这一假说可以在常规基因组环境中得到验证。此外，通过IPSCs的使用，我们有可能将这些细胞分化为三个胚层的衍生物，并在这一过程中监测转录通读的效果。在未来的实验中，其他印迹基因上的调控元件可以用同样的策略来取代。此外，在转录依赖的DNA甲基化过程中对表观遗传修饰物的需求可以通过在细胞系统中补充或删除它们来解决。有了这一点，我们的研究将有助于了解DNA甲基化、转录和细胞潜力的相关性。