MOLECULAR STUDIES OF GENOMIC IMPRINTING IN HUMANS

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印迹被称为基因组印迹。这个 Angelman(AS)和Prader-Willi(PWS)综合征在临床上是不同的 神经行为障碍就是最好的例子 人类的一种现象。它们提供了一个理想的研究系统 基因组印迹的机制。失去父系活动15q11-q13 基因(S)导致PWS，无论是父亲的缺失还是母亲的缺失 单亲二体，而母亲活跃的15q11-q13基因缺失(S) 导致AS，无论是由于母亲的缺失，父亲的单亲二体， 或推定的点突变。我们识别印记基因的策略 在15q11-q13，可能涉及AS和PWS，将进行筛查 显示基因组特征的区域的15q11-q13序列 印记。我们已经鉴定出一种名为ZNF127的基因，它具有 父母DNA甲基化的显著印记。15q11-q13中的另外两个基因座 后来被我们和其他人确认了 工人们。此外，我们已经通过复制计时研究表明 整个15q11-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 鉴定完整的ZNF127基因(我们研究的模式基因座)。 我们已经分离并测定了人类和小鼠ZNF127的大部分序列 基因，包括在CpG中显示甲基化印记的位置- 横跨推定推定推动者的岛屿，并将完成这项工作。这个 转录的5‘起始点将通过引物延伸来确定， 5‘种族和核糖核酸酶保护。将进一步定义启动子元素 通过DNase I超敏反应、体内足迹和缺失作图。 (2)ZNF127的差异甲基化模式与 通过Northern分析检测表达和功能 RT-PCR检测各种组织，包括AS和PWS的脑组织样本 患者，葡萄胎/卵巢畸胎瘤，小鼠单亲 二体和小鼠孤雌生殖/雄生殖胚胎干细胞。 使用相同的策略，我们还将检查另一个 候选人类印记基因Snrpn。(3)评估中的复制时间 15q11-q13在独特的AS和PWS患者中的FISH检测 远缘染色体导致ZNF127的DNA甲基化 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 在基因组印记的机制中。

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.