Deciphering establishment of DNA methylation by transcription at the PWS-SRO

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。在第一项研究中，我们试图在体细胞系中建立人类体外系统，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。接下来，我们转向使用诱导多能干细胞（iPSC），因为有可能使这些细胞分化，并在此过程中监测转录通读的影响。将对Prader-Willi/Angelman综合征基因座gDMR处的卵母细胞特异性转录通读及其对DNA甲基化的影响进行建模。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。为了实现这一目标，首先对iPSC进行修饰以发挥诱导型细胞系统的功能，其次，将卵母细胞特异性AS-SRO启动子替换为诱导型启动子。通过诱导取代的AS-SRO，在其常规基因组背景下观察到跨gDMR的转录。接下来，转录，DNA甲基化和染色质修饰将通过高通量测序技术进行分析。在未来，转录依赖性DNA甲基化过程中对表观遗传修饰剂的直接需求可以通过补充或从细胞系统中删除它们来解决。有了这个，我们的研究将有助于理解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. In a first study we sought to establish a human in vitro system in a somatic cell line 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. Next, we shifted to the use of induced pluripotent stem cells (iPSCs), because of the possibility to differentiate these cells and to monitor the effect of transcriptional read-through during this process. Oocyte-specific transcriptional read-through at the gDMR of the Prader-Willi/Angelman syndrome locus and its effect on DNA methylation will be modelled. 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. To achieve this, the iPSCs were first modified to function as inducible cell system and second, the oocyte-specific AS-SRO promoter was replaced by an inducible promoter. By induction of the replaced AS-SRO, transcription was observed across the gDMR in its regular genomic context. Next, transcription, DNA methylation and chromatin modifications will be analyzed by high-throughput sequencing techniques. In the future, the direct 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.