Structure and function of the human beta-globin locus control region

描述(申请人提供)：据估计，世界上超过3%的人口携带2-珠蛋白基因座突变，这种突变或多或少会导致严重的贫血病例。最著名和最具特征的突变之一是成人2-珠蛋白链中的氨基酸变化，导致镰状细胞病(SCD)。其他突变会导致地中海贫血，其特征是珠蛋白基因合成减少。目前，还没有一种令人满意的治疗方法可以使所有患者受益。2-珠蛋白基因座由5个基因组成，它们在发育过程中受到一个位点控制区(LCR)的竞争性调控。LCR由5个DNase I超敏(HS)位点组成，它们分布在超过10kBP的DNA上，共同作用于介导珠蛋白基因的高水平表达。尽管在过去的三十年里，对珠蛋白基因调控的理解取得了进展，但LCR是如何调节珠蛋白基因的高水平表达的尚不清楚。对导致珠蛋白基因阶段特异性表达的分子机制的详细了解将有助于开发新的、更广泛适用的治疗血红蛋白疾病的方法。在镰状细胞突变纯合子的成年人中表达胎儿3-珠蛋白可以改善疾病的严重形式，这一事实最好地说明了这一点。沉默突变的2-珠蛋白基因，同时激活3-珠蛋白基因的表达的策略的发展将为各种血红蛋白疾病提供新的治疗方法。显然，对LCR如何竞争性调节基因的详细了解对于开发靶向或利用LCR介导的珠蛋白基因调节机制的治疗方法至关重要。方法：本应用的目的是破译LCR调节2-珠蛋白基因表达的机制(S)。有人认为LCR是2-珠蛋白基因座上伸长活性转录复合体的主要附着和组装部位。延伸活性转录复合体以发育阶段特异性的方式转移到珠蛋白基因启动子上。这一假说将在转基因小鼠中解决，方法是检查LCR是否足以在红系细胞中形成转录焦点，以及转录因子上游刺激因子(USF)如何与红系特异性转录因子一起调节这一过程(特定目标1)。此外，为了分析转录复合体招募和组装的机制，将使用一种新的体外组装试验以及在完整细胞中使用染色质免疫沉淀(ChIP)(特定目标2)来表征招募到LCR和成体2-珠蛋白基因启动子的转录复合体的成分。我们将使用一种名为MapIt的新方法，在分化过程中绘制LCR和其余2-珠蛋白基因座的可及性图，该方法旨在以高分辨率分析单分子上的蛋白质DNA相互作用(特定目标3)。最后，为了确定介导LCR和珠蛋白基因之间相互作用的顺式调控DNA元件，我们将表达中和转录因子结合位点的锌指DNA结合域，并检测这些蛋白质对珠蛋白基因表达和LCR珠蛋白基因相互作用的影响(特定目标4)。 公共卫生相关性：这项研究的目标是增加我们对2-珠蛋白基因如何受基因位点控制区调控的了解，并开发新的工具，帮助找到更好的治疗血红蛋白疾病的方法。这一应用的主要焦点是确定转录因子和顺式调节元件在2-珠蛋白基因高水平表达中的作用。干扰介导成人2-珠蛋白基因高水平表达的活动可能会导致镰状细胞疾病患者治疗性3-珠蛋白基因表达增加。

DESCRIPTION (provided by applicant): It is estimated that more than 3% of the world's population carry mutations in the 2-globin gene locus that cause more or less severe cases of anemia. One of the best known and characterized of these mutations is an amino acid change in the adult 2-globin chain that causes sickle cell disease (SCD). Other mutations lead to thalassemias, which are characterized by reduction in globin gene synthesis. Currently, there is no satisfactory treatment for hemoglobinopathies that will benefit all patients. The 2-globin gene locus consists of five genes that are competitively regulated by a locus control region (LCR) during development. The LCR is composed of five DNase I hypersensitive (HS) sites that are spread over more than 10 kbp of DNA and which act together to mediate high-level globin gene expression. Despite advances in understanding globin gene regulation over the last three decades, how the LCR operates to mediate high level globin gene expression is not understood. A detailed knowledge of the molecular mechanisms leading to stage specific expression of the globin genes will aid the development of novel and more broadly applicable therapies for hemoglobinopathies. This is best illustrated by the fact that expression of the fetal 3-globin in adults homozygous for the sickle cell mutation ameliorates the severe form of the disease. The development of strategies that silence the mutant 2-globin gene and at the same time activate expression of the 3-globin genes would offer novel therapies for a variety of hemoglobinopathies. Clearly, a detailed understanding of how the genes are competitively regulated by the LCR is crucial for the development of therapies that target or take advantage of LCR mediated mechanisms of globin gene regulation. Approach: The goal of this application is to decipher the mechanism(s) by which the LCR regulates 2-globin gene expression. It is proposed that the LCR serves as the primary attachment and assembly site for elongation active transcription complexes in the 2-globin gene locus. The elongation active transcription complexes are transferred to globin gene promoters in a developmental stage specific manner. This hypothesis will be addressed in transgenic mice by examining if the LCR is sufficient to form transcription foci in erythroid cells and how transcription factor upstream stimulatory factor (USF) in conjunction with erythroid specific transcription factors regulates this process (Specific Aim 1). Furthermore, to analyze the mechanism of transcription complex recruitment and assembly, components of transcription complexes recruited to the LCR and to the adult 2-globin gene promoter will be characterized using a novel in vitro assembly assay as well as using chromatin immunoprecipitation (ChIP) in intact cells (Specific Aim 2). We will map accessibility in the LCR and the rest of the 2-globin gene locus during differentiation using a novel approach, called MAPit, designed to assay protein DNA interactions at high resolution on single molecules (Specific Aim 3). Finally, to identify cis-regulatory DNA elements that mediate interactions between the LCR and the globin genes we will express zinc finger DNA binding domains that neutralize transcription factor binding sites and examine the effect of these proteins on globin gene expression and LCR globin gene interactions (Specific Aim 4). PUBLIC HEALTH RELEVANCE: The goal of this study is to increase our knowledge of how the 2-globin genes are regulated by the locus control region and to develop new tools that could aid in finding better cures for hemoglobinopathies. The major focus of this application is to determine the role of transcription factors and cis-regulatory elements in the high-level expression of the 2-globin gene. Interfering with activities that mediate high-level expression of the adult 2-globin gene could lead to increased expression of the therapeutic 3-globin genes in patients with sickle cell disease.