Imprint establishment at the PWS-SRO in a cellular model

• Applicant: Professorin Dr. Laura Steenpaß
• Subject Area: Human Genetics
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 285807807

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.

• DFG Programme: Research Grants