Fetal Globin Silencing

描述（由申请人提供）：血红蛋白转换是一个进化保守的过程，发生在脊椎动物红细胞生成过程中。人类胎儿γ -珠蛋白基因在从胎儿到成人发育阶段的过渡过程中沉默，但参与这一过程的因素仍有待确定。本课题拟在斑马鱼中研究胎儿珠蛋白沉默。我们以前利用突变斑马鱼作为人类疾病的动物模型，通过诱变筛选，我们分离出了参与红细胞生成的新基因。斑马鱼表现出从胚胎到幼虫，以及从幼虫到成虫阶段的珠蛋白转换。斑马鱼β -e1珠蛋白基因具有与人类γ -珠蛋白基因相似的发育表达模式。它在幼虫时期表达，在25日龄时，表达停止。我们已经证明，在羟基脲和丁酸盐处理下，幼虫β -e1珠蛋白表达被诱导。这两种药物已在哺乳动物红细胞中显示出防止γ -珠蛋白基因沉默的作用。我们计划采用经典的遗传和化学遗传方法来了解调节β -e1珠蛋白沉默的因素。首先，我们将利用雌性二倍体斑马鱼对β -e1珠蛋白沉默缺陷突变体进行温度敏感筛选。将创建一个在β -e1珠蛋白调控元件下表达的绿色荧光蛋白转基因系，使β -e1珠蛋白基因在成人体内的表达可视化。我们还将创建一个稳定的转基因斑马鱼系，其BAC包含整个人类珠蛋白位点。在基因筛选中获得的斑马鱼突变体可以用于人类γ -珠蛋白沉默测试。最后，我们计划对药物进行化学基因筛选，以防止斑马鱼成年早期珠蛋白基因沉默。通过这一建议发现的基因和化合物可以在哺乳动物系统中进一步研究。这些研究将提高我们对珠蛋白基因调控的基本分子生物学的理解，并可能对镰状贫血和地中海贫血患者的治疗有用。

DESCRIPTION (provided by applicant): Hemoglobin switching is an evolutionarily conserved process that occurs during vertebrate erythropoiesis. The fetal gamma-globin gene in humans is silenced during the transition from the fetal to adult stage of development, yet the factors involved in this process remain to be defined. In the grant, we propose to study fetal globin silencing in zebrafish. We have previously utilized mutant zebrafish as animal models of human disease and through mutagenesis screens, we have isolated novel genes involved in erythropoiesis. The zebrafish exhibit globin switching from embryonic to larval life, and from larval to adult stages. The zebrafish beta-e1 globin gene has a similar developmental pattern of expression as the human gamma-globin gene. It is expressed during larval life and at 25 days of age, expression is silenced. We have shown that larval beta-e1 globin expression is induced in adults treated with hydroxyurea and butyrate. These two drugs have been shown in mammalian erythroid cells to prevent gamma-globin gene silencing. We plan to take a classical genetic and chemical genetic approach to understanding factors that regulate beta-e1 globin silencing. First, we will undertake a temperature sensitive screen for mutants with defects in beta-e1 globin silencing using gynogenetic diploid zebrafish. A transgenic line will be created with green fluorescent protein expressed under the beta-e1 globin regulatory elements, allowing in vivo visualization of beta-e1 globin gene expression in adults. We will also create a stable transgenic zebrafish line with a BAC containing the entire human globin locus. Zebrafish mutants obtained in the genetic screen can then be tested for human gamma-globin silencing. Finally, we plan to undertake a chemical genetic screen for drugs that will prevent globin gene silencing in the early zebrafish adulthood. The genes and compounds found by this proposal can be further studied in mammalian systems. These studies should improve our understanding of the basic molecular biology of globin gene regulation and could be therapeutically useful for patients with sickle anemia and thalassemia.