Induction of site-specific methylation in induced pluripotent stem cells of a patient with Angelman syndrome

In this study, an iPSC model of Angelman syndrome was developed. iPSCs were generated from an Angelman syndrome patient harbouring a three-base pair deletion in the maternal UBE3A allele and from a healthy control person. The maternal UBE3A allele is expressed but the protein UBE3A is not functional. Therefore, the cells exhibit normal expression of all genes at the PWS/AS locus. In comparison with all iPSC lines generated as a model of Angelman syndrome so far, this is a significant advantage because all observed effects may be attributed directly and solely to the inactive UBE3A protein. Using lentiviral transduction, we reprogrammed fibroblasts from the patient and from the healthy control person, and isolated several clones. Their thorough characterisation followed confirming their pluripotency, identity and integrity by various methods. Based on the results, clones most suitable for further analyses were selected. The stability of the imprint at the PWS/AS locus is a critical prerequisite for the faithful analysis of imprinted expression and for the induction of DNA methylation on the paternal allele. Therefore, the methylation status at six imprinted loci and two pluripotent loci was determined in the iPSCs generated at the beginning of this project, their parental fibroblasts, in hESCs H1 and H9, and in published cell lines AGI-0 and MCH2-10 that represented iPSC controls. Most importantly, I observed an exceptional stability of the methylation imprint at PWS-SRO. Three imprinted loci showed a tendency towards a loss of methylation and two exhibited susceptibility to acquiring methylation. Both pluripotent loci displayed a higher level of methylation in differentiated cells than in pluripotent cells. My results on the stability of the assayed loci upon reprogramming are in agreement with previously reported observations. In cooperation with Anika Neureiter from the Institute for Transfusion Medicine, iPSCs were differentiated into neurons. We determined by single-nucleotide primer extension assay that silencing of the paternal UBE3A allele by SNHG14 occurs late during the process of neuronal differentiation. These findings are also in line with previously published results. I developed a quantitative qRT-PCR analysis to follow the expression dynamics of the maternal and the paternal UBE3A allele separately in the generated iPSCs during differentiation. The method allows for a much more precise evaluation of the respective alleles’ expression than single-nucleotide primer extension assay. In the second part of the study, I aimed to induce methylation at the paternal PWS-SRO in the cell line AGI-0, which only harbours the unmethylated paternal allele. I observed a transient increase of methylation at the paternal PWS-SRO to 38.6 % upon transfecting the cells with methylated DNA fragments. By employing the modified CRISPR/Cas9 system fused with DNMT3A and DNMT3L, I was able to induce methylation at the paternal PWS-SRO in AGI-0 to 30.0 %. Not only was the methylation stable at the locus over the course of two weeks but its level also increased during the time. The result is especially valuable because the approach does not require mitosis for its functioning and could therefore potentially be used even in non-dividing cells such as neurons. It would certainly be worthwhile to pursue the stability of the induced methylation in a longer time-course experiment. Also, the investigation of a direct biological impact in the form of a qRT-PCR assay of SNURF-SNRPN expression would be of importance. The effect of the methylation on the expression of SNHG14 in neurons is even more relevant for my purposes and so is the subsequent impact on the expression of UBE3A. Should the methylation lead to abolishment of SNHG14 expression and thereby prevent silencing of the paternal UBE3A allele in neurons, it would be a significant step towards possible compensation for the loss of maternal UBE3A by the intact paternal allele. Therefore, further optimisation of the method and its application in iPSCs generated in this study ought to be performed.