IMPRINTING IN THE AS/PWS REGION IN HUMAN GAMETOGENESIS

人类配子发生中 AS/PWS 区域的印记

• 批准号: 6053848
• 负责人: Daniel J Driscoll
• 金额: $ 22.57万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6053848
• 关键词: DNA binding protein; DNA footprinting; DNA methylation; Prader Willi syndrome; acetylation; alleles; chromosome aberrations; clinical research; developmental genetics; flow cytometry; fluorescent in situ hybridization; gametogenesis; gene expression; gene mutation; genomic imprinting; happy puppet syndrome; histones; human genetic material tag; human subject; meiosis; molecular cloning; nucleic acid sequence; nucleic acid structure; polymerase chain reaction; protein protein interaction; southern blotting

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.

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，每个印记基因都有一个活性和非活性等位基因，其转录状态取决于亲本。一些遗传疾病和癌症是由于基因表达异常而产生的。 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 15qll-q13 基因的缺失会导致 PWS，而母系活性 15q11-q13 基因的缺失会导致 AS。尽管已为每个已充分表征的印记基因鉴定出不同的DNA甲基化印记，但印记基因的各个等位基因根据其亲本来源进行标记的分子机制尚不清楚。据推测，印记必须首先发生在生殖细胞中，首先是消除父母印记并根据个体的性别建立新的印记。人们对哺乳动物配子发生过程中如何发生这一过程知之甚少，尤其是在人类中。因此，该项目的目标是确定人类配子发生中印记的机制和时间，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阶段来实现，然后：1）评估配子发生各个阶段的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； 4) 评估减数分裂中 15q11-q13 区域的组蛋白乙酰化状态，以确定母系和父系印记域之间的界限。这些研究将使我们能够直接解决有关印记基因表达的建立和维持的分子机制。