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

描述（由申请人提供）：据估计，世界上超过3%的人口携带2-珠蛋白基因位点突变，导致或多或少严重的贫血病例。这些突变中最著名和最具特征的是导致镰状细胞病（SCD）的成人2-珠蛋白链氨基酸变化。其他突变导致地中海贫血，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，在发育过程中由一个基因座控制区（locus control region， LCR）竞争性调控。LCR由5个DNA酶I超敏（HS）位点组成，分布在超过10kbp的DNA上，它们共同作用介导高水平的珠蛋白基因表达。尽管在过去三十年中对珠蛋白基因调控的理解有所进展，但LCR如何调节高水平珠蛋白基因表达尚不清楚。详细了解导致球蛋白基因阶段特异性表达的分子机制将有助于开发新的和更广泛适用于血红蛋白病的治疗方法。胎儿3-珠蛋白在成人镰状细胞突变纯合子中的表达可以改善严重形式的疾病，这一事实最好地说明了这一点。在抑制突变的2-珠蛋白基因的同时激活3-珠蛋白基因表达的策略的发展将为多种血红蛋白病提供新的治疗方法。显然，详细了解这些基因是如何被LCR竞争性调控的，对于开发靶向或利用LCR介导的珠蛋白基因调控机制的疗法至关重要。方法：本应用程序的目的是破译LCR调节2-珠蛋白基因表达的机制。这表明LCR是2-珠蛋白基因座上延伸活性转录复合物的主要附着和组装位点。延伸活性转录复合物以特定发育阶段的方式转移到珠蛋白基因启动子上。通过检测LCR是否足以在红细胞中形成转录灶，以及转录因子上游刺激因子（USF）如何与红细胞特异性转录因子一起调节这一过程，将在转基因小鼠中解决这一假设（specific Aim 1）。此外，为了分析转录复合物募集和组装的机制，转录复合物募集到LCR和成人2-珠蛋白基因启动子的组分将使用一种新的体外组装试验以及完整细胞中的染色质免疫沉淀（ChIP）来表征（Specific Aim 2）。我们将使用一种名为MAPit的新方法，在分化过程中绘制LCR和2-珠蛋白基因位点的可达性，该方法旨在以高分辨率在单分子上测定蛋白质DNA相互作用（Specific Aim 3）。最后，为了鉴定介导LCR和珠蛋白基因之间相互作用的顺式调控DNA元件，我们将表达锌指DNA结合域来中和转录因子结合位点，并检查这些蛋白对珠蛋白基因表达和LCR珠蛋白基因相互作用的影响（Specific Aim 4）。

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.