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 依赖于亲本来源的基因组印记。 的 Angelman（AS）和Prader-Willi（PWS）综合征在临床上是不同的 神经行为障碍是最好的例子 人类的现象。 他们提供了一个理想的系统，在其中研究 基因组印记的机制 父亲活动丧失15 q11-q13 一个或多个基因导致PWS，无论是通过父系缺失还是母系缺失 单亲二体性，而缺失母系活性15 q11-q13基因 导致AS，无论是通过母系缺失，父系单亲二体性， 或推测的点突变。 我们识别印记基因的策略 在15 q11-q13，这可能因此参与AS和PWS，是筛选 显示基因组特征的区域的15 q11-q13序列 印记 我们已经确定了一个基因，ZNF 127，它具有一个 父母DNA甲基化印记。 15 q11-q13中的另外两个位点 我们和其他人随后发现了可能被印记的东西， 工人 此外，我们通过复制时间研究表明， 整个15 q11-q13区域构成印迹结构域。 我们建议的三个具体目标是：（1）克隆和 描述完整的ZNF 127基因（我们研究的模型基因座）。 我们已经分离并测序了人类和小鼠ZNF 127的大部分基因， 基因，包括CpG中显示甲基化印记的位点， 岛跨越假定的启动子，并将完成这项工作。 的 转录的5 ′起始位点将通过引物延伸来确定， 5' RACE和RNase保护。 启动子元件将被进一步定义 通过DNA酶I超敏反应、体内足迹和缺失图谱。 (2)将ZNF 127的差异甲基化模式与 通过北方分析检测表达， 各种组织的RT-PCR，包括来自AS和PWS的脑样品 患者，葡萄胎/卵巢畸胎瘤，小鼠单亲 双体和小鼠孤雌生殖/雄核发育胚胎干细胞。 使用相同的策略，我们还将研究另一个 候选人印迹基因SNRPN。 (3)评估复制时间 FISH检测AS和PWS患者的15 q11-q13， ZNF 127的DNA甲基化印记是染色体远距离甲基化的结果 重新安排 这些研究将解决DNA之间的相互关系， 甲基化、复制、染色质结构和DNA结合蛋白 基因组印记的机制。