MOLECULAR STUDIES OF GENOMIC IMPRINTING IN HUMANS

人类基因组印记的分子研究

• 批准号: 2204037
• 负责人: Robert D Nicholls
• 金额: $ 22.08万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2204037
• 关键词: DNA binding protein; DNA footprinting; DNA methylation; DNA replication; Prader Willi syndrome; RNase protection assay; embryonic stem cell; gene deletion mutation; gene expression; genetic disorder; genetic promoter element; genetic regulatory element; genetic transcription; genomic imprinting; human genetic material tag; hydatidiform mole; in situ hybridization; laboratory mouse; molecular cloning; northern blottings; nucleic acid sequence; ovary neoplasms; polymerase chain reaction; teratoma

The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting. The Angelman (AS) and Prader-Willi (PWS) syndromes are clinically distinct neurobehavioral disorders that represent the best example of this phenomenon in humans. They provide an ideal system in which to study the mechanisms of genomic imprinting. Loss of paternally active 15q11-q13 gene(s) results in PWS, whether by paternal deletion or maternal uniparental disomy, whereas loss of a maternally active 15q11-q13 gene(s) leads to AS, whether by maternal deletion, paternal uniparental disomy, or presumed point mutations. Our strategy to identify imprinted genes in 15q11-q13, which may thus be involved in AS and PWS, is to screen 15q11-q13 sequences for regions that display features of genomic imprinting. We have already identified one gene, ZNF127, that has a striking parental DNA methylation imprint. Two other loci in 15q11-q13 that may be imprinted have subsequently been identified by us and other workers. Furthermore, we have shown by replication timing studies that the entire 15q11-q13 region composes an imprinted domain. The three specific aims of our proposal are to: (1) Clone and characterize the complete ZNF127 gene (the model locus for our studies). We have isolated and sequenced a large part of the human and mouse ZNF127 genes, including the sites that show a methylation imprint in the CpG- island spanning the putative promotor, and will complete this work. The 5' start site of transcription will be determined by primer extension, 5' RACE and RNase protection. Promoter elements will be further defined by DNase I hypersensitivity, in vivo footprinting and deletion mapping. (2) Correlate the differential methylation patterns of ZNF127 with expression and function by examining expression via Northern analysis and RT-PCR on various tissues, including brain samples from AS and PWS patients, hydatidiform moles/ovarian teratomas, mouse uniparental disomies, and mouse parthenogenetic/androgenetic embryonic stem cells. Using the same strategies, we will also examine expression of another candidate human imprinted gene, SNRPN. (3) Assess replication timing in 15q11-q13 by FISH in unique AS and PWS patients with alterations in the DNA methylation imprint at ZNF127 as a consequence of distant chromosomal rearrangements. These studies will address the inter-relationships between DNA methylation, replication, chromatin structure, and DNA binding proteins in the mechanism of genomic imprinting.

某些哺乳动物基因表达的差异修饰 依赖父母的起源被称为基因组印迹。 这 Angelman（AS）和Prader-Willi（PWS）综合症在临床上是不同的 代表最佳例子的神经行为疾病 人类现象。 他们提供了一个理想的系统来研究 基因组印迹的机制。 丧失父亲活动15q11-Q13 基因导致PWS，无论是由父亲缺失还是母亲 单亲疾病，而母体活跃的15q11-Q13基因的丧失 导致AS，无论是通过母体删除，父亲单胎疾病， 或假定点突变。 我们识别印迹基因的策略 在15q11-Q13中，可能参与AS和PWS 15Q11-Q13序列用于显示基因组特征的区域 烙印。 我们已经确定了一个基因Znf127，该基因具有一个 醒目的父母DNA甲基化烙印。 15Q11-Q13中的另外两个基因座 随后，我们和其他 工人。 此外，我们通过复制时机研究表明 整个15q11-Q13区域组成一个印迹域。 我们建议的三个具体目的是：（1）克隆和 表征完整的ZNF127基因（我们研究的模型基因座）。 我们已经隔离并测序了人类和小鼠Znf127的大部分 基因，包括在CpG-中显示甲基化印记的位点 跨越推定的启动者的岛屿，并将完成这项工作。 这 5'转录的开始位点将由底漆扩展确定， 5'种族和RNase保护。 启动子元素将进一步定义 通过DNase I超敏反应，体内足迹和缺失映射。 （2）将Znf127的差分甲基化模式与 通过北方分析检查表达来表达和功能 各种组织上的RT-PCR，包括来自AS和PWS的脑样品 患者，氢摩尔/卵巢畸胎瘤，小鼠独立 疾病和小鼠孤pheneneod/雄激素发育胚胎干细胞。 使用相同的策略，我们还将检查另一种的表达 候选人类印迹基因，SNRPN。 （3）评估复制时间 15q11-Q13由鱼类在独特的AS和PWS患者中有改变 Znf127处的DNA甲基化烙印是由于远处的染色体 重排。 这些研究将解决DNA之间的关系 甲基化，复制，染色质结构和DNA结合蛋白 在基因组印迹的机制中。