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综合征和Prader-Willi综合征这两种终身神经行为障碍的遗传印记机制。拟议的研究将确定基因座被印迹的机制，测试涉及AS印迹缺陷的DNA元件的分子作用，并创建AS印迹缺陷动物模型。与其他印迹基因簇类似，印迹中心（IC）负责PWS-AS位点的单等位基因表达。PWS-AS位点IC由PWS-IC和AS-IC组成。PWS-IC在体细胞中起作用，激活来自父系等位基因的基因表达。AS-IC在卵母细胞中起表观遗传失活PWS-IC的作用，从而沉默未来母体等位基因上父系表达的基因。AS-IC因此在该区域启动印记。小鼠AS-IC逃避了检测，妨碍了对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转录转移是正确标记来自该位点的转基因的必要和充分条件。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。总之，这些结果表明，通过PWS-IC传递的转录包括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，以验证整个内源性PWS-AS基因座被类似的基于转录的机制所印记的假设。目标2将确定建立印记的发育窗口期。目的3将提供人类卵母细胞中AS-IC的第一个功能表征，其功能所在的组织。在这些研究的结论中，我们将对印记如何在PWS-AS位点建立的机制有一个了解，并将表征印迹发生的发育阶段和细胞类型。我们还将了解类似的机制是否在人类中起作用，以及当删除导致AS的DNA序列的分子作用。这些结果将为未来研究AS印迹缺陷的原因提供信息，这些缺陷是由于AS- ic缺失或辅助生殖技术后频率增加而自发产生的。

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.