Dissecting genetic determinants of epigenetic instability in pluripotent stem cells

项目摘要 多能干细胞(PSCs)产生大量功能齐全的体细胞的非凡能力 在体外培养中，由于DNA甲基化标记的异常获得或丢失，类型会逐渐受损 在必要的发育基因座上。这种普遍存在的表观遗传不稳定性是 使用PSCs的生物医学应用，但潜在的分子原因几乎完全未知。一个 PSCs表观遗传不稳定性的重要例子是基因组印迹的丢失，这是一种重要的基因调控 哺乳动物的机制。通过将印记稳定性的转基因报告等位基因与小鼠遗传学相结合，我们 发现PSCs中的印记失调是由影响反式作用的可识别的遗传变异引起的 染色质调节剂。这项提议的目标是发展一个全面的分子理解 特定的遗传变异如何影响PSCs中重要表观遗传标记的稳定性。为了实现这一目标， 我们将首先从功能上剖析我们发现的基因组易感区域内的因果变异 并导致特定于基因座的DNA高甲基化和发育潜力的丧失。这将使我们能够 精确定位易使印迹基因和其他基因获得DNA异常的基因调控特征 甲基化(目标1)。然后我们将在杂交PSCs中使用遗传图谱来发现数量性状基因座 可防止有害的DNA低甲基化，并验证相关基因产物的功能 小鼠和人类多能细胞(目标2)。最后，我们将对印记稳定性的遗传学进行建模 一组具有高度多样化但特征良好的基因组的PSCs。这将使我们能够剖析分子 在与人类群体相似的复杂遗传背景中调节表观遗传稳定性并帮助我们 建立新的生物标志物，以确定保持发展潜力的培养条件 遗传背景未知的PSCs(目标3)。我们的工作将建立新的概念和机制 多能细胞的有害表观遗传脆弱性框架，这将有助于安全和可靠地 利用这些细胞研究发育和疾病过程。此外，我们的发现将具有相关性 用于多种以表观遗传不稳定为特征的人类疾病和发育综合征。

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.