IMPRINTING IN THE AS/PWS REGION IN HUMAN GAMETOGENESIS

印记是一种表观遗传现象，其中基因在亲本配子中被独特地“标记”，并且在受精后差异表达，这取决于亲本的性别。因此，每个印记基因具有活性和非活性等位基因，其转录状态取决于亲本来源。一些遗传性疾病和癌症是由于基因表达异常而引起的。Angelman（AS）和Prader-Willi（PWS）综合征是临床上不同的神经行为障碍，代表了人类这种现象的最佳例子。父系活性15 q11-q13基因的缺失导致PWS，而母系活性15 q11-q13基因的缺失导致AS。印迹基因的个体等位基因根据其亲本标记的分子机制尚不清楚，尽管已经为已经充分表征的每个印迹基因鉴定了不同的DNA甲基化印迹。据推测，印记必须首先发生在生殖细胞中，从消除父母印记并根据个人的性别建立新印记开始。关于哺乳动物配子发生的过程，特别是人类的配子发生过程，我们知之甚少。因此，本项目的目标是确定人类配子发生中的印记机制和时间，特别是在AS/PWS区域的15 q11-q13。这将通过首先分离雄性和雌性配子发生的阶段来实现，然后：1）评估配子发生各个阶段细胞中印记基因的表达模式; 2）识别差异甲基化位点并检查它们在建立印记中的作用; 3）识别和表征与印记基因表达调节相关的序列特异性DNA-蛋白质相互作用;和4）评估减数分裂中跨越15 q11-q13区域的组蛋白乙酰化状态，以鉴定母本和父本印记结构域之间的边界。这些研究将使我们能够直接解决有关建立和维持印迹基因表达的分子机制。

Imprinting is an epigenetic phenomenon in which genes are uniquely "marked" in the parental gametes and differentially expressed post- fertilization, dependent upon the sex of the parent. Thus each imprinted genes has an active and inactive allele with transcription status dependent upon parent-of-origin. Several genetic diseases and cancers arise due to abnormal gene expression. The Angelman (AS) and Prader-Willi (PWS) syndromes are clinically distinct neurobehavioral disorders that represent the best example of this phenomenon in humans. Loss of paternally active 15qll-q13 genes results in PWS, whereas loss of a maternally active 15q11-q13 gene leads to AS. The molecular mechanisms by which individual alleles of imprinted genes are marked according to their parent- of-origin is not known, although distinct DNA methylation imprints have been identified for every imprinted gene that has been well characterized. It has been postulated that imprinting must first occur in the germ cells, beginning with erasure of the parental imprint and establishment of a new imprint depending upon the sex of the individual. Very little is known about how this process occurs in mammalian gametogenesis, particularly in humans. Therefore, the goals of this project are to determine the mechanisms and timing of imprinting in human gametogenesis specifically in the AS/PWS region of 15qll-q13. This will be accomplished by first separating the stages of male and female gametogenesis, and then: 1) assessing the expression pattern of imprinted genes in cells from various stages of gametogenesis; 2) identifying sites of differential methylation and examining their role in establishing imprinting; 3) identifying and characterizing sequence-specific DNA- protein interactions associated with the regulation of imprinted gene expression; and 4) assessing the state of histone acetylation across the 15q11-q13 region in meiosis in order to identify boundaries between maternally and paternally imprinted domains. These studies will allow us to directly address molecular mechanisms regarding the establishment and maintenance of imprinted gene expression.