Imprinting defects leading to Angelman and Prader Willi syndromes

描述（由申请人提供）：本申请旨在了解Angelman和Prader-Willi综合征（两种终身神经行为障碍）的遗传印记机制。拟议的研究将确定该位点被印迹的机制，测试AS印迹缺陷中涉及的DNA元件的分子作用，并创建AS印迹缺陷动物模型。与其他印记基因簇相似，印记中心（IC）负责PWS-AS位点的单等位基因表达。在PWS-AS位点的IC是二分的，由PWS-IC和AS-IC组成。PWS-IC在体细胞中起作用，以激活来自父本等位基因的基因表达。AS-IC在卵母细胞中的功能是在表观遗传学上抑制PWS-IC，从而使未来的母系等位基因上的父系表达基因沉默。因此，AS-IC在该区域中启动压印。小鼠AS-IC已经逃避检测，排除了对AS-IC功能的机制研究。然而，我们最近证明，通过卵母细胞中的PWS-IC转录转运是必要的，足以正确地印记来自该位点的转基因。我们还发现卵母细胞活性转录启动子对于观察可靠的转基因印记是必要的。总之，这些结果表明跨PWS-IC的转录转运包括AS-IC活性。在第一个具体目标中，我们将修改内源性PWS-AS基因座以检验整个内源性PWS-AS基因座由类似的基于转录的机制印迹的假设。目标2将确定建立印记的发展窗口。目的3将提供AS-IC在人类卵母细胞中的第一个功能表征，该组织在其中起作用。在这些研究的结论，我们将有一个机械的理解印记是如何建立在PWS-AS基因座，并将其特征的发展阶段和细胞类型中的印记发生。我们还将了解是否有类似的机制在人类中起作用，以及DNA序列在缺失时导致AS的分子作用。这些结果将告知未来的研究的AS印记缺陷的原因，自发出现的，作为一个删除的AS-IC的结果，或在辅助生殖技术后频率增加。

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.