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

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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.