Erzeugung der genomischen Prägung am PWS-SRO in einem zellulären Modell
Zusammenfassung der Projektergebnisse
The Prader-Willi / Angelman syndrome locus on human chromsome 15 shows parent-of-origin dependent expression of several genes, which is regulated by DNA methylation at the PWS-SRO element. The PWS-SRO resides in the CpG island promoter of the SNURF-SNRPN gene. It was shown that integrity of the element AS-SRO, located 35 kb upstream to the PWS-SRO, is essential for proper establishment of DNA methylation at the PWS-SRO. In addition, the AS-SRO acts as promoter for non-coding transcripts apparently only in the oocyte. We hypothesized that transcription initiating the the AS-SRO and traversing the PWS-SRO is needed for recruitment of DNA methylation to the PWS-SRO. Since human oocytes are not available and not suited for experimental research we aimed to establish an in vitro system in human induced pluripotent stem cells (iPSCs) to model the genomic landscape and the transcription processes at the PWS / AS locus. Using CRISPR/Cas9 genome editing, iPSC lines were generated that stably express the reverse tetracycline transactivator (rtTA) protein and can be utilized in inducible systems. Then the AS-SRO element was replaced by an inducible promoter in iPSC line AGI-0_rtTA (DSMZi017-A-1), preserving the genomic spacing between the upstream promoter and the PWS-SRO. Of note, the parental cell line AGI-0 was established from a patient with Angelman syndrome due to a large deletion on the maternal chromosome, thus carrying only the paternal, not methylated copy of the PWS-SRO. Upon addition of doxycycline, the promoter was induced, transcription initiated and transcripts spliced to exon 2 of the SNRPN gene, as described in the literature. Measured by qRT-PCR induction of upstream transcription was 100- to 1000-fold and was stably achieved in all experiments. In the undifferentiated modified iPSC line AGI-0_rtTA_AS-SRO expression of SNRPN was reduced upon induction of upstream transcription, but was not accompanied by DNA methylation at the PWS-SRO as analyzed by amplicon bisulfite sequencing. We hypothesized that an additional stimulus is needed to result in DNA methylation. Such a stimulus could be set by differentiation, involving remodeling of the epigenetic landscape in iPSCs by, among other processes, DNA methylation. However, no substantial gain of DNA methylation could be achieved upon undirected embryoid body differentiation, probably due to heterogenous cell populations. Directed trilineage differentiation into derivatives of the three germ layers, endoderm, mesoderm and ectoderm resulted in induction of upstream transcription in all layers, a reduction of SNRPN expression and acquisition of DNA methylation up to a level of 10% in endoderm only. We conclude that the in vitro system is suitable to study the effect on upstream transcription on establishment of DNA methylation at the PWS-SRO. However, optimization of endoderm differentiation is needed to reach at least 50% of DNA methylation to be able to conduct further experiments like chromatin analyses. In addition, it appears that expression of SNRPN hinders acquisition of DNA methylation. Interestingly, SNRPN is not expressed in human oocytes at the time when the upstream transcripts can be detected, but it is expressed in undifferentiated and differentiated iPSCs. This might indicate that transcription interference occured at the PWS-SRO in the iPSC model and could result in low efficiency of DNA methylation. Further experiments will adress this point to fully understand the mechanisms of DNA methylation at the PWS-SRO in the oocyte.
Projektbezogene Publikationen (Auswahl)
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Generation of three iPSC lines with inducible systems to be used in Angelman syndrome research. Stem Cell Research, 78, 103454.
Haake, Josephine; Kaufmann, Maren & Steenpass, Laura
