Imprinting defects leading to Angelman and Prader Willi syndromes

描述(由申请者提供)：本申请旨在了解Angelman和Prader-Willi综合征这两种终生神经行为障碍的遗传印记机制。这项拟议的研究将确定该基因座被印记的机制，测试与AS印记缺陷有关的DNA元件的分子作用，并创建AS印记缺陷动物模型。与其他印迹基因簇相似，印迹中心(IC)负责PWS-AS基因座的单等位基因表达。在PWS-AS位点上的IC是两部分的，由PWS-IC和AS-IC组成。PWS-IC在体细胞中工作，激活父系等位基因的基因表达。AS-IC在卵母细胞中发挥表观遗传功能，使PWS-IC失活，从而使父系表达的基因在未来的母系等位基因上沉默。因此，AS-IC在该区域启动印记。小鼠的AS-IC逃避了检测，排除了对AS-IC功能的机械性研究。然而，我们最近证明，在卵母细胞中，通过PWS-IC的转录过渡是正确印记来自该基因座的转基因的必要条件和充分条件。我们还发现，卵母细胞活性转录启动子对于观察忠实的转基因印迹是必要的。综上所述，这些结果表明，跨越PWS-IC的转录包括AS-IC活性。在第一个特定的目标中，我们将修改内源PWS-AS基因座来检验这一假设，整个内源PWS-AS基因座是由类似的基于转录的机制印记的。目标2将确定印记形成的发育窗口。目标3将提供人类卵母细胞中AS-IC的第一个功能特征，即它在其中发挥作用的组织。在这些研究的结论中，我们将对印记是如何在PWS-AS位点上建立的有一个机械性的理解，并将表征发生印记的发育阶段和细胞类型。我们还将了解人类是否存在类似的机制，以及当DNA序列缺失时会导致AS的分子作用。这些结果将为未来的研究提供关于AS印记缺陷的原因的信息，这些缺陷是由于AS-IC的缺失而自发产生的，或者是在辅助生殖技术之后频率增加的结果。

DESCRIPTION (provided by applicant): This application seeks to understand genetic imprinting mechanisms underlying Angelman and Prader-Willi syndromes, two lifelong neurobehavioral disorders. The proposed studies will determine the mechanisms by which the locus is imprinted, test the molecular role of a DNA element implicated in AS imprinting defects, and create an AS imprinting defect animal model. Similar to other imprinted gene clusters, an imprinting center (IC) is responsible for monoallelic gene expression at the PWS-AS locus. The IC at the PWS-AS locus is bipartite, consisting of the PWS-IC and the AS-IC. The PWS-IC works in somatic cells to activate gene expression from the paternal allele. The AS-IC functions in oocytes to epigenetically inactivate the PWS-IC, and thereby silence paternally-expressed genes on the future maternal allele. The AS-IC thus initiates imprinting in the region. The murine AS-IC has evaded detection, precluding mechanistic investigations into AS-IC function. However we recently demonstrated that transcription transiting through the PWS-IC in oocytes is necessary and sufficient to correctly imprint transgenes derived from the locus. We also found that oocyte-active transcriptional promoters were necessary to observe faithful transgene imprinting. Together, these results indicate that transcription transiting across the PWS-IC comprises AS-IC activity. In the first specific aim we will modify the endogenous PWS-AS locus to test the hypothesis the entire endogenous PWS-AS locus is imprinted by a similar transcription-based mechanism. Aim 2 will determine the developmental window in which imprints are established. Aim 3 will provide the first functional characterization of the AS-IC in human oocytes, the tissue in which it functions. At the conclusion of these studies, we will have a mechanistic understanding of how imprints are established at the PWS-AS locus and will have characterized the developmental stage and cell type in which imprinting occurs. We will also understand whether a similar mechanism operates in humans, and the molecular role of DNA sequences that when deleted result in AS. These results will inform future studies of the causes of AS imprinting defects that arise spontaneously, as a result of a deletion of the AS-IC, or at increased frequency following assisted reproductive technologies.