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）是罕见的发育障碍，具有父母来源效应，在美国发生在1/15,000活产婴儿中。这些疾病的分子基础是11 p15和15q11上的印迹基因簇的失调，主要是由印迹控制中心（ICC）的缺失或ICC的表观遗传DMA甲基化变化引起的。AS和BWS的风险增加与辅助生殖技术有关，这些技术被认为会在体外胚胎培养过程中诱导印迹的表观遗传丧失。这一建议验证了整体假设，即单独的表观遗传改变可以导致发育病理，这些发育病理是由于整体印迹丧失（LOI）造成的，并进一步验证了纠正LOI事件的治疗方法。为了确定全局LOI的累积发育效应，我们建立了一个新的小鼠模型，通过对胚胎干细胞中必需的DMA甲基转移酶Dnmtl的顺序基因失活和再激活，来实现全基因组表观遗传失调。在哺乳动物中，DMA甲基化是印迹位点以及整个基因组中表观遗传沉默稳定传播的主要机制。使用此意向书模型，我们将：(1)利用转录谱分析和DNA甲基化分析确定所有DMA甲基化依赖的印迹基因；(2)确定全基因组印迹缺失的发育表型影响，特别强调胎儿生长和在胰腺、肾脏和心脏等关键器官中观察到的BWS、PWS和AS的特征性症状；(3)利用一种新的转基因RNAi策略，选择性地纠正个体基因表达异常，以评估单个基因对复杂表型的贡献。该项目将定义在胚胎着床前发育过程中对DNA甲基化状态变化做出反应的一组基因，当表观遗传失调时，这些基因会导致随后的发育异常。本研究将通过前瞻性地将表观遗传改变与器官发生过程中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，从而填补目前的知识空白。

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.