H19--A MODEL FOR PARENTAL IMPRINTING IN THE MOUSE

本申请的目的是研究功能和 在哺乳动物的父母印迹机制，作为一个模型系统， 鼠Igf 2和H19基因。igf 2编码胎儿特异性生长因子 胰岛素样生长因子II，并在整个发育过程中表达 主要来自父系遗传的染色体。紧密相连的H19 基因以与Igf 2非常相似的模式表达，但仅从 母亲的染色体这些基因的印记和连锁是 在人类中是保守的， 与Beckwith-Wiedeman综合征、Wilm肿瘤和易感性有关 儿童肿瘤如横纹肌肉瘤和肝细胞癌 癌将在小鼠中产生一系列突变，以测试 这些基因在这些疾病中的作用第一个具体目标是测试 一个模型来解释Igf 2和H19的相互印记。模型 提出基因竞争顺式共同调控元件。的 竞争在两条染色体上有不同的偏向， 父亲特异性DNA甲基化，其作用是沉默H19 启动子和促进Igf 2转录。实验被提议为 测试位于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。的 删除的母系和父系遗传的影响 将检查对Igf 2和H19的增强子。 的基础 H19在明显未标记的母体上的优先转录 将通过检测野生型和突变体来研究染色体 酵母人工染色体携带的基因导入组织培养细胞。 将研究体内Igf 2印迹的弛豫以了解 体细胞中的印记是如何被抹去的第二个具体目标 将集中在H19基因的功能，及其可能的参与 在印记的机制中。一系列的基因突变及其 转录控制区将通过同源 胚胎干细胞中的基因重组小鼠的表型 将确定遗传的母本或父本无效突变。 H19的额外拷贝引起的晚期胎儿致死的基础将 通过引入基因的野生型和突变型拷贝来研究 植入胚胎干细胞，以建立小鼠品系， 传递额外的基因拷贝。蛋白质的性质复杂， H19颗粒将通过纯化颗粒和克隆来确定 第三个具体目标是阐明 真兽目哺乳动物的印记功能， 进化了Igf 2和H19基因将从 有袋类和单孔类，并测试等位基因特异性表达。的 H19基因在哺乳动物物种形成过程中出现的可能性， XIST的复制，XIST是一种定位于X染色体失活的基因， 中心，并专门从非活性X染色体表达，将 得到考验

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.