Our recent research focuses on the development of cellular models to understand human imprinting diseases and the development of tumors of the eye, retinoblastoma and uveal melanoma. We use human cell lines and human pluripotent stem cells (induced and embryonic) to establish such models and we employ differentiation of these cells and molecular biology techniques, like genome editing and next generation sequencing for analysis.
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 Angelman syndrome gene, UBE3A, is active on both chromosomes in most tissues, except brain. In neurons, only the maternally inherited chromosome expresses UBE3A, whereas an epigenetic mechanism silences UBE3A on the paternal copy. If the maternal chromosome carries a genetic defect, expression of UBE3A is lost completely and the symptoms of Angelman syndrome develop.
To understand the switch from biallelic UBE3A expression in stem cells to maternal-only expression in neurons of the human brain, we reprogrammed fibroblasts from a patient with Angelman syndrome into induced pluripotent stem cells (iPSCs). We differentiated these cells, together with two other iPSC lines with different molecular causes of Angelman syndrome, into neurons. Indeed, we observed and confirmed induction of silencing of paternal UBE3A expression upon neuronal differentiation. UBE3A locates to the synapse and the nucleus. Studying if loss of UBE3A changes gene expression on transcriptome level, we could not observe substantial differences between neurons derived from healthy control iPSCs and those derived from patients with Angelman syndrome. The function of UBE3A in the nucleus is therefore still unclear.
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. For an imprinted gene, we would expect parent-specific effects on tumor development. Indeed, some rare mutations in RB1 are associated with a higher risk for tumor development when inherited from the father. To enable studies on parent-specific effects in retinoblastoma, 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 by CRISPR/Cas9. Currently we analyze the effects of RB1 loss on neural retina differentiation and cell type composition in the organoids.
Uveal melanoma is a tumor of the eye in elderly patients. Prognosis in uveal melanoma is related to chromosome 3 status: tumors with two copies have good prognosis, whereas tumors with only one copy often metastasize. Monosomy of chromosome 3 goes along with presence mutations in the BAP1 gene, whereas tumors with disomy of chromosome 3 acquire mutations in other genes, eg EIF1AX. We started to modify these two genes in uveal melanoma cell lines using CRISPR/Cas9 and analyze the effects on DNA methylation pattern and gene expression.