H19--A MODEL FOR PARENTAL IMPRINTING IN THE MOUSE

H19——小鼠父母印记模型

• 批准号: 2190003
• 负责人: SHIRLEY M. TILGHMAN
• 金额: $ 26.03万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190003
• 关键词: Wilms' tumor; alleles; artificial chromosomes; biochemical evolution; biological models; embryonic stem cell; gene dosage; gene expression; gene mutation; genetic disorder; genetic enhancer element; genetic models; genetic recombination; genetic regulatory element; genetic transcription; genomic imprinting; hepatocellular carcinoma; laboratory mouse; molecular cloning; protein structure function; rhabdomyosarcoma; transfection

The purpose of this application is to investigate the function and mechanisms of parental imprinting in mammals, using as a model system the murine Igf2 and H19 genes. Igf2 encodes the fetal-specific growth factor insulin-like growth factor II, and is expressed throughout development primarily from the paternally inherited chromosome. The closely linked H19 gene is expressed in a very similar pattern to Igf2, but exclusively from the maternal chromosome. The imprinting and linkage of these genes is conserved in humans, where disruptions in their gene dosage have been implicated in Beckwith-Wiedeman syndrome, Wilm's tumor and susceptibility to childhood tumors such as rhabdomyosarcomas and hepatocellular carcinomas. A series of mutations will be generated in mice to test the role of these genes in these disorders. The first specific aim is to test a model to explain the reciprocal imprinting of Igf2 and H19. The model proposes that the genes compete in cis for common regulatory elements. The competition is biased differently on the two chromosomes by the presence of paternal-specific DNA methylation, which acts to silence the H19 promoter and facilitate Igf2 transcription. Experiments are proposed to test the role of specific enhancers which lie 3' to the H19 gene by mutating them via homologous recombination in embryonic stem cells. The effects of both maternal and paternal inheritance of the deletion of the enhancers on Igf2 and H19 will be examined. The basis for the preferential transcription of H19 on the apparently unmarked maternal chromosome will be investigated by transfecting both wild type and mutant yeast artificial chromosomes bearing the genes into tissue culture cells. The relaxation of imprinting of Igf2 in vivo will be studied to understand how imprinting can be erased in somatic cells. The second specific aim will focus on the function of the H19 gene, and its possible involvement in the mechanism of imprinting. A series of mutations in the gene and its transcriptional control regions will be generated through homologous recombination in embryonic stem cells. The phenotype of mice that have inherited either maternal and paternal null mutations will be determined. The basis for the late fetal lethality caused by extra copies of H19 will be studied by introducing both wild type and mutant copies of the gene into embryonic stem cells, in order to establish lines of mice which transmit extra copies of the gene. The nature of the proteins complexed in the H19 particle will be determined by purifying the particle and cloning the proteins contained within it. The third specific aim is to elucidate the function of imprinting in eutherian mammals by identifying how it evolved. The Igf2 and H19 genes will be identified and cloned from marsupials and monotremes, and tested for allele-specific expression. The likelihood that the H19 gene arose during mammalian speciation as a duplication of XIST, a gene which maps to the X chromosome inactivation center and is expressed exclusively from the inactive X chromosome, will be tested.

此应用程序的目的是调查功能和 哺乳动物中父母印记的机制，以系统为模型 小鼠免疫球蛋白2和H19基因。IGF2编码胎儿特异性生长因子 胰岛素样生长因子II，在发育过程中表达 主要来自父系遗传的染色体。紧密相连的H19 基因的表达模式与Igf2非常相似，但完全来自 母体染色体。这些基因的印记和连锁是 在人类中保守，在那里他们的基因剂量的中断 与Beckwith-Wiedeman综合征、Wilm瘤和易感性有关 儿童肿瘤，如横纹肌肉瘤和肝细胞瘤 癌症。将在小鼠身上产生一系列突变来测试 这些基因在这些疾病中的作用。第一个具体目标是测试 一个解释Igf2和H19互易印记的模型。模型 提出基因在顺式结构中竞争共同的调控元件。这个 竞争在两条染色体上是不同的，因为存在 父亲特有的DNA甲基化，它可以沉默H19 启动子和促进Igf2转录。建议进行实验，以 检测位于H19基因3‘端的特异性增强子的作用 通过胚胎干细胞中的同源重组使它们发生突变。这个 基因缺失对父母亲遗传的影响 将研究免疫球蛋白Igf2和H19的增强剂。的基础是 H19在明显未标记的母体上的优先转录 染色体将通过同时导入野生型和突变型进行研究 将携带基因的酵母人工染色体送入组织培养细胞。 体内Igf2印迹的松弛将被研究以了解 如何在体细胞中消除印记。第二个具体目标 将重点放在H19基因的功能及其可能的参与 在印记的机制中。该基因及其基因的一系列突变 转录控制区将通过同源基因产生 胚胎干细胞中的重组。小鼠的表型 将确定遗传的母系和父系零突变。 H19基因额外拷贝导致晚期胎儿死亡的基础 通过引入基因的野生型和突变型拷贝进行研究 转化为胚胎干细胞，以便建立小鼠品系 传递额外的基因拷贝。结合在一起的蛋白质的性质 H19颗粒将通过提纯和克隆来确定 它所含的蛋白质。第三个具体目标是澄清 印记在真兽类哺乳动物中的作用 进化了。Igf2和H19基因将被鉴定和克隆 有袋类和单孔动物，并测试等位基因特异性表达。这个 H19基因在哺乳动物物种形成过程中作为一种 X染色体失活基因XIST的复制 中心，并且只在不活跃的X染色体上表达，将 接受测试。