Induction of DNA methylation by transcription in an in vitro iPS cell model

Genomic imprinting is regulated by allele-specific DNA methylation at imprinting control regions (ICRs), ensuring parent-of-origin-specific gene expression. The human Prader-Willi/Angelman syndrome (PWS/AS) locus on chromosome 15q11–q13 harbors a bipartite imprinting center comprising the PWS-SRO, which acquires maternal-specific methylation, and the upstream AS-SRO, a promoter ~35 kb upstream that initiates transcription in oocytes. To directly test whether transcription across the PWS-SRO is sufficient to trigger de novo DNA methylation, a human iPSC-based in vitro system was established in which the endogenous AS-SRO was replaced by a doxycycline-inducible promoter driving transcription across the PWS-SRO. Inducible transcription was robustly activated upon doxycycline treatment and confirmed by splicing to SNRPN exon 2, mirroring the structure of oocyte-derived transcripts. However, in undifferentiated iPSCs and after embryoid body formation, this transcriptional activation did not lead to DNA methylation at the PWS-SRO, as measured by targeted bisulfite sequencing. Strikingly, DNA methylation gains of up to ~10% were observed only following directed differentiation into the endodermal lineage, suggesting that transcription alone is insufficient, and that a permissive differentiation context is required for methylation establishment. In contrast, mesodermal and ectodermal derivatives showed little to no methylation gains despite transcriptional induction. The presence of endogenous SNRPN expression, which is absent in oocytes but active in all differentiated lineages examined, may contribute to transcriptional interference, limiting the efficiency of read-through across the PWS-SRO. Notably, SNRPN expression is lowest in liver tissue. To explore whether extended differentiation and reduced SNRPN activity further facilitate methylation, the system was applied to hepatic differentiation. Although SNRPN expression was downregulated in hepatic-like cells, concurrent silencing of the inducible transactivator prevented further conclusions regarding methylation acquisition in this context. In summary, this study provides the first direct evidence in a human model that transcription across the PWS-SRO is necessary but not sufficient for de novo DNA methylation. Instead, differentiation-dependent cues, potentially resembling aspects of the oocyte environment, are required. The established in vitro system enables controlled dissection of the molecular factors and chromatin dynamics underlying imprint establishment at the PWS/AS locus.