Transcription-driven establishment of DNA methylation at the PWS/AS imprinted region
Genomic imprinting is regulated by allele-specific DNA methylation at imprinting control regions, ensuring parent-of-origin-specific gene expression. The human Prader-Willi/Angelman syndrome (PWS/AS) locus on chromosome 15q harbours a bipartite imprinting center consisting of the PWS-SRO, which acquires maternal-specific methylation, and the AS-SRO, an upstream promoter 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. Gain of DNA methylation up to 10% was observed only following directed differentiation into the endodermal lineage. 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.