Genomic imprinting is an epigenetic process regulated by DNA methylation and resulting in parent-of-origin-specific gene expression. DNA methylation is established in only one of the parental germ lines, either the maternal or the paternal one. Once established, the DNA methylation imprint is stable throughout lifetime and differentiation. The Prader-Willi / Angelman syndrome (PWS/AS) locus on human chromosome 15 is controlled by DNA methylation at the PWS-SRO (SRO: shortest region of overlap) encompassing the promoter of the gene SNRPN. The project explores if and how transcription from an upstream, oocyte-specific promoter, called the AS-SRO, regulates acquisition of DNA methylation at the PWS-SRO. In order to do so, the AS-SRO was replaced by a doxycycline-inducible promoter in induced pluripotent stem cells derived from a patient with Angelman syndrome. Induction of transcription across the unmethylated PWS-SRO and its consequences regarding changes in DNA methylation and associated histone modifications will be investigated in undifferentiated and differentiated iPSCs.

The project is funded by DFG grants STE1987/5-2 and STE1987/11-1.

In humans, biallelic inactivation of RB1 results in the development of retinoblastoma. This is a tumor of the developing neural retina occurring in children under the age of five. RB1 is also an imprinted gene, with DNA methylation on the maternal allele resulting in an expression bias in favor of the maternal allele. Retionblatoma cannot be modeled in the mouse because of differences in gene expression and retina development. To enable studies on retinoblastoma in a human model, we established in vitro differentiation of human pluripotent stem cells into neural retina in 3D organoids. To mimic the RB1 genomic status in heterozygous carriers and the tumor, we introduced RB1 mutations in human embryonic stem cells (hESCs) by CRISPR/Cas9. Comparative differentiation revealed no difference in retinal organoid development between wildtype and heterozygous mutant hESCs. However, in homozygous mutant hESCs retinal differentiation was disturbed: the structure of retinal layering was destroyed and certain retinal cell types did not differentiate, like amacrine cells and rod photoreceptors. In contrast, immature cone photoreceptors were over-represented and showed persistent proliferation. This model will now enable further studies on the development and spreading of retinoblastoma.

The project was funded by DFG grant STE1987/6-1.

This project is conducted in cooperation with Prof. Christel Depienne, Institute of Human Genetics, University Hospital Essen. Mutations in the gene RBMX are related to the development of X-linked intellectual disability in, predominantly, male patients. However, the exact role of RBMX and the genotype-phenotype correlations are unclear. To enable the study of neuronal cells, stem cells that can be differentiated along the neuronal lineage are needed. Fibroblasts of three patients were reprogrammed to induced pluripotent stem cells (iPSCs) and characterized. Currently, these iPSCs are differentiated into cortical neurons to enable further analyses.

This project is conducted in cooperation with Dr. Ilaria Parenti, Institute of Human Genetics, University Hospital Essen. Cohesinopathies are characterized by mutations affecting proteins that are part of the mulit-subunit complex cohesin or take part in its regulation. Defects affect chromosome distribution in mitosis and gene expression. We employ CRISPR/Cas9 base editing to generate various iPSC lines carrying specific point mutations. Such lines will be useful in studying the molecular basis of the associated diseases.