Transcriptional Control of Hemoglobin Synthesis

描述(申请人提供)：由血红蛋白合成异常引起的人类疾病困扰着大量患者，而这些疾病的治疗方法还远远不是最理想的。结合使用β-珠蛋白基因作为模型来理解细胞类型特定的转录机制的优点，这个问题已经受到了相当大的关注。利用创新的方法，我们确定了控制成体类珠蛋白基因的因素和机制。GATA-1及其相互作用分子通过分布在β-珠蛋白基因和编码包括血红素生物合成酶在内的红细胞生物学关键调控基因的基因位点发挥作用。了解因子如何选择染色质部位，招募共同调节因子修饰/重塑染色质，参与转录机制，以及赋予基因亚核定位是至关重要的。鉴于强大的技术、隐含的因素和独特的专业知识，我们将解决关键问题，以开发一个综合模型来解释血红蛋白合成的转录控制。具体目标1-测试模型，以解释控制珠蛋白基因和编码血红素生物合成酶的基因之间的关系。我们假设GATA-1通过一组独特的反式作用因子和共调节因子发挥作用，将珠蛋白基因和编码血红素生物合成限速酶的基因-4-氨基乙酰丙酸合成酶(Alas2)从核外清除。这将建立高水平转录所需的亚核定位，并协调珠蛋白链的产生和血红素的合成。我们将测试模型来解释GATA-1调节因子如何在外围和内部位置启动和/或维持这些基因座的锚定或排斥。特定目的2-阐明GATA-1依赖转录的启动和维持机制。对于差异启动转录和维持转录的机制几乎一无所知，这是一个特别重要的问题，对于控制转录与治疗结果的策略。我们将使用一种强大的RNA干扰来检验这一假设，即调节β-珠蛋白和Alas2转录的成分在支持启动和维持方面的能力不同。具体目标3-确定GATA-1突变如何影响人类血液病对GATA因子依赖的遗传网络的调节。关于疾病突变如何损害GATA-1活性的许多问题仍然没有答案。我们将确定N端锌指Arg216W和Arg216Gln突变是如何影响GATA-1活性的，这些突变是先天性红细胞生成性卟啉症、X连锁灰色血小板综合征和伴有地中海贫血的血小板减少的基础。这些研究将对控制血红蛋白合成、正常和恶性造血以及其他发育过程的机制产生广泛的见解。

DESCRIPTION (provided by applicant): Human diseases resulting from aberrant hemoglobin synthesis afflict large numbers of patients, and therapeutics for these disorders are far from optimal. Taken together with the merits of using the ?-globin locus as a model to understand cell type-specific transcriptional mechanisms, this problem has received considerable attention. Using innovative approaches, we identified factors and mechanisms controlling the adult ?-like globin genes. GATA-1 and its interactors function through dispersed sites at the ?-globin locus and loci encoding pivotal regulators of red cell biology, including heme biosynthetic enzymes. It is crucial to understand how factors select chromatin sites, recruit coregulators that modify/remodel chromatin, engage transcriptional machinery, and confer gene subnuclear localizations. Given powerful technologies, implicated factors, and unique expertise, we will address key issues to develop an integrative model to explain the transcriptional control of hemoglobin synthesis. Specific Aim 1 - To test models to explain the relationship between mechanisms controlling globin genes and genes encoding heme biosynthetic enzymes. We hypothesize that GATA-1 functions with a unique cohort of trans-acting factors and coregulators to expel globin genes and the gene encoding a rate-limiting enzyme in heme biosynthesis, 4- aminolevulinic acid synthase (Alas2), from the nuclear periphery. This would establish a subnuclear positioning required for high-level transcription and coordinate globin chain production and heme synthesis. We will test models to explain how GATA-1-regulating factors initiate and/or maintain anchoring or repulsion of these loci at peripheral and internal sites. Specific Aim 2 - To elucidate mechanisms underlying initiation and maintenance of GATA-1-dependent transcription. Nearly nothing is known about mechanisms that differentially initiate vs. maintain transcription, a problem particularly important for strategies to control transcription with a therapeutic outcome. We will use a powerful RNA interference asay to test the hypothesis that components regulating ?-globin and Alas2 transcription differ in their capacities to support initiation vs. maintenance. Specific Aim 3 - To establish how GATA-1 mutations underlying human hematologic disorders dysregulate GATA factor-dependent genetic networks. Many questions remain unanswered regarding how disease mutations impair GATA-1 activity. We will determine how Arg216W and Arg216Gln mutations within the N-terminal zinc finger that underlie congenital erythropoietic porphyria, X- linked grey platelet syndrome, and thrombocytopenia with ?-thalassemia afect GATA-1 activity. These studies will yield broad insights into mechanisms controling hemoglobin synthesis, normal and malignant hematopoiesis, and additional developmental processes.