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

人β-珠蛋白基因座控制区的结构和功能

• 批准号: 8532881
• 负责人: JORG BUNGERT
• 金额: $ 28.13万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2014-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532881
• 关键词: Address; Adult; Affect; Amino Acids; Anemia; Binding Sites; Biological Assay; C-terminal; Cells; Chromatin; Chromatin Structure; Communication; Complex; DNA; DNA Binding Domain; DNA-Protein Interaction; Deoxyribonuclease I; Development; Disease; Dominant-Negative Mutation; Elements; Erythroid; Erythroid Cells; Event; Gene Expression; Gene Expression Regulation; General Transcription Factors; Genes; Genetic Transcription; Globin; Goals; Hemoglobinopathies; Human; Immunofluorescence Microscopy; In Vitro; Indium; Individual; Knowledge; Lead; Locus Control Region; Maps; Mediating; Methodology; Methyltransferase; Molecular; Monitor; Mus; Mutation; Patients; Pattern; Phosphorylation; Population; Process; Proteins; Protocols documentation; RNA; RNA Polymerase II; Recruitment Activity; Regulatory Element; Resolution; Rest; Role; Sickle Cell; Sickle Cell Anemia; Site; Staging; Structure; Thalassemia; Therapeutic; Time; Tissues; Transgenic Mice; Western Blotting; Zinc Fingers; beta Globin; chromatin immunoprecipitation; design; fetal; gene interaction; gene synthesis; in vivo; mutant; novel; novel strategies; promoter; public health relevance; reconstitution; single molecule; therapy development; tool; transcription factor; transcription factor USF

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.

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一种是导致镰状细胞病（SCD）的成人2-珠蛋白链中的氨基酸变化。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，对于血红蛋白病还没有令人满意的治疗方法可以使所有患者受益。2-珠蛋白基因座由五个基因组成，在发育过程中由一个基因座控制区（LCR）竞争性调控。LCR由5个DNA酶I超敏（HS）位点组成，这些位点分布在超过10 kbp的DNA上，共同作用以介导高水平的珠蛋白基因表达。尽管在过去的三十年里在理解珠蛋白基因调控方面取得了进展，但LCR如何介导高水平珠蛋白基因表达尚不清楚。详细了解导致珠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的血红蛋白病治疗方法。镰状细胞突变纯合子成人中胎儿3-球蛋白的表达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-珠蛋白基因并同时激活3-珠蛋白基因表达的策略的发展将为各种血红蛋白病提供新的疗法。显然，详细了解基因如何被LCR竞争性调节对于开发靶向或利用LCR介导的珠蛋白基因调节机制的疗法至关重要。方法：本申请的目的是破译LCR调节2-珠蛋白基因表达的机制。有人建议，LCR作为主要的附着和组装网站的延伸活性转录复合物中的2-珠蛋白基因座。延伸活性转录复合物以发育阶段特异性方式转移到珠蛋白基因启动子。通过检查LCR是否足以在红系细胞中形成转录灶以及转录因子上游刺激因子（USF）如何与红系特异性转录因子一起调节该过程（特异性目的1），将在转基因小鼠中解决这一假设。此外，为了分析转录复合物募集和组装的机制，募集至LCR和成人2-珠蛋白基因启动子的转录复合物的组分将使用新的体外组装测定以及使用完整细胞中的染色质免疫沉淀（ChIP）进行表征（特异性目的2）。我们将使用一种称为MAPit的新方法，在分化过程中绘制LCR和2-珠蛋白基因位点的其余部分的可访问性，该方法旨在以高分辨率对单分子进行蛋白质DNA相互作用的测定（具体目标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节DNA元件，我们将表达中和转录因子结合位点的锌指DNA结合结构域，并检查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（具体目标4）。 公共卫生关系：这项研究的目的是增加我们对2-珠蛋白基因是如何被基因座控制区调控的知识，并开发新的工具来帮助找到更好的血红蛋白病治疗方法。本申请的主要重点是确定转录因子和顺式调节元件在2-球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性可能导致镰状细胞病患者治疗性3-珠蛋白基因表达增加。