Genomic Imprinting and Embryonic Development

描述(由申请人提供)：Beckwith-Wiedeman综合征(BWS)和Prader-Willi/Angelman综合征(PWS/AS)是一种罕见的发育障碍，具有父母起源的影响，在美国每15,000名活产儿中就有1人发生。这些疾病的分子基础是11p15和15q11上印记基因簇的解除调控，主要是由于印记控制中心(ICC)内的缺失或由ICC中表观遗传的DMA甲基化变化引起的。AS和BWS风险的增加与辅助生殖技术有关，辅助生殖技术被认为在体外胚胎培养过程中导致表观遗传印记丢失。这项建议测试了表观遗传改变本身就可以导致发育病理的总体假设，该病理模型将这些综合征建模为全球印记丢失(LOI)的结果，并进一步测试了纠正LOI事件的治疗方法。作为一种确定全球LOI累积发育效应的手段，我们建立了一个新的小鼠模型，通过对胚胎干细胞中必要的DNA甲基转移酶Dnmtl进行顺序基因失活和重新激活来实现全基因组表观遗传去调控。在哺乳动物中，DNA甲基化是表观遗传沉默在印迹基因座以及整个基因组中稳定传播的主要机制。使用这个LOI模型，我们将：(1)使用转录图谱和DNA甲基化分析来确定所有依赖于DMA甲基化的印记基因；(2)确定全基因组印记丢失的发育表型效应，特别强调胎儿的生长和来自BWS、PWS以及包括胰腺、肾脏和心脏在内的关键器官的特征症状；(3)选择性地纠正单个基因表达异常，以使用新的转基因RNAi策略评估单个基因对复杂表型的贡献。这个项目将定义一组基因，这些基因对植入前发育过程中DNA甲基化状态的变化做出反应，当表观遗传错误时，这些变化会导致随后的发育异常。这项研究将填补目前的知识空白，前瞻性地将表观遗传变化与器官发生过程中由此产生的发育缺陷联系起来，并进一步建立在成人中观察到的后续病理。

DESCRIPTION (provided by applicant): Beckwith-Wiedeman Syndrome (BWS), and Prader-Willi/Angelman Syndromes (PWS/AS) are rare developmental disorders with parent-of-origin effects that occur in 1/15,000 live births in the US. The molecular basis for these diseases is deregulation of imprinted gene clusters on 11 p15 and 15q11 caused predominantly by either deletions within imprinting control centers (ICC) or by epigenetic DMA methylation changes in the ICC. Elevated risk for AS and BWS has been associated with assisted reproductive technologies, which are thought to induce epigenetic loss of imprinting during in vitro embryo culture. This proposal tests the overall hypothesis that epigenetic alterations alone can cause developmental pathologies that model these syndromes as a result of global loss of imprinting (LOI), and further tests a therapeutic approach for correcting LOI events. As a means to determine the cumulative developmental effects of global LOI, we have established a new mouse model for genome-wide epigenetic deregulation using sequential gene inactivation and reactivation for the essential DMA methyltransferase Dnmtl in embryonic stem cells. DMA methylation is the primary mechanism in mammals for stable propagation of epigenetic silencing at imprinted loci as well as across the entire genome. Using this LOI model we will: (1) identify all DMA methylation-dependent imprinted genes using transcriptional profiling and DNA methylation analyses, (2) determine developmental phenotypic effects of genome-wide loss of imprinting with particular emphasis on fetal growth and characteristic symptoms from BWS, PWS, and AS observed in critical organs including pancreas, kidney and heart, (3) selectively correct individual gene expression abnormalities to assess the contribution of single genes to the complex phenotype using a novel transgenic RNAi strategy. This project will define the set of genes responsive to changes in DNA methylation state during preimplantation development that cause subsequent developmental anomalies when epigenetically misregulated. This study will fill a current gap in knowledge by prospectively connecting epigenetic alterations to resultant developmental defects during organogenesis, and further establishing the subsequent pathologies that are observed in the adult.