IMPRINTING IN THE AS/PWS REGION IN HUMAN GAMETOGENESIS

印记是一种表观遗传现象，基因在双亲配子中被唯一地“标记”，并在受精后有不同的表达，这取决于双亲的性别。因此，每个印记基因都有一个活跃和不活跃的等位基因，其转录状态取决于父母的起源。一些遗传性疾病和癌症是由于基因表达异常引起的。Angelman(AS)和Prader-Willi(PWS)综合征是临床上截然不同的神经行为障碍，代表了人类这种现象的最好例子。丢失父系活性的15q11-q13基因会导致PWS，而丢失母系活性的15q11-q13基因会导致AS。印记基因的个体等位基因根据其亲本来源进行标记的分子机制尚不清楚，尽管已经为每个已被很好表征的印记基因识别出不同的DNA甲基化印记。据推测，印记必须首先在生殖细胞中发生，首先是擦除父母的印记，然后根据个体的性别建立新的印记。关于这一过程在哺乳动物配子发生中是如何发生的，尤其是在人类中，人们知之甚少。因此，本项目的目标是确定人类配子发生中印记的机制和时机，特别是在15qll-q13的AS/PWS区域。这将通过首先分离雄性和雌性配子发生的阶段，然后：1)从配子发生的不同阶段评估印记基因在细胞中的表达模式；2)识别差异甲基化的位置并研究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控相关的序列特异性DNA-蛋白质相互作用；以及4)评估组蛋白乙酰化在减数分裂中的状态，以便确定母系和父系印记结构域之间的界限。这些研究将使我们能够直接解决与印记基因表达的建立和维持有关的分子机制。

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.