Dissecting genetic determinants of epigenetic instability in pluripotent stem cells

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Project Summary The remarkable ability of pluripotent stem cells (PSCs) to give rise to a plethora of fully functional somatic cell types becomes progressively impaired upon ex vivo culture by aberrant gain or loss of DNA methylation marks at essential developmental gene loci. This pervasive epigenetic instability represents a significant hurdle for biomedical applications using PSCs, but the underlying molecular reasons remain almost entirely unknown. An important example for epigenetic instability in PSCs is loss of genomic imprinting, an essential gene regulatory mechanism in mammals. By combining transgenic reporter alleles for imprint stability with mouse genetics, we found that imprint dysregulation in PSCs is caused by identifiable genetic variants that affect trans acting chromatin regulators. The goal of this proposal is to develop a comprehensive molecular understanding on how specific genetic variants affect the stability of vital epigenetic marks in PSCs. To work towards this goal, we will first functionally dissect the causal variants within a genomic susceptibility region that we discovered and which drives locus-specific DNA hypermethylation and loss of developmental potential. This will allow us to pinpoint gene regulatory features that predispose imprinted and other genes to aberrant gain of DNA methylation (Aim 1). We will then use genetic mapping in hybrid PSCs to discover quantitative trait loci that can protect against detrimental DNA hypomethylation and validate the function of associated gene products in both mouse and human pluripotent cells (Aim 2). Finally, we will model the genetics of imprint stability in a panel of PSCs with highly diverse yet well-characterized genomes. This will enable us to dissect the molecular regulation of epigenetic stability in complex genetic backgrounds resembling the human population and help us to establish new biomarkers for the identification of culture conditions that preserve developmental potential in PSCs of unknown genetic background (Aim 3). Our work will establish novel conceptual and mechanistic frameworks for the detrimental epigenetic vulnerability of pluripotent cells, which will aid the safe and reliable use of these cells to study developmental and disease processes. In addition, our findings will have relevance for a wide range of human diseases and developmental syndromes characterized by epigenetic instability.