A Comparative Approach To Globin Regulation/Thalassemia

珠蛋白调节/地中海贫血的比较方法

• 批准号: 7806589
• 负责人: LEONARD Ira ZON
• 金额: $ 35.55万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7806589
• 关键词: Adult; Affect; Antisense Oligonucleotides; Biochemistry; Biological Assay; Blood Circulation; Categories; Cell Lineage; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Chromosomes, Human, Pair 3; Code; Color; Complex; Deoxyribonuclease I; Development; Disease; Embryo; Epigenetic Process; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Event; Family; Fishes; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Globin; Goals; Grant; Grx5 protein; Heme Iron; Hemoglobin; Human; Hypersensitivity; Hypochromic anemia; Injection of therapeutic agent; Iron; Lead; Life; Locus Control Region; Maps; Methodology; Methods; Minor; Modeling; Mutate; Mutation; Orthologous Gene; Oxygen; Patients; Process; Production; Regulation; Regulatory Element; Resolution; Sickle Cell Anemia; Study models; Sulfur; System; Technology; Thalassemia; Transgenic Organisms; Work; Zebrafish; chromatin immunoprecipitation; comparative; embryo cell; falls; gene function; human disease; insight; knock-down; metal transporting protein 1; microcytic/hypochromic anemia; mutant; novel; overexpression; positional cloning; transcription factor

DESCRIPTION (provided by applicant): Hemoglobin production is an evolutionarily conserved process that is specific to the red blood cell lineage. We have utilized the zebrafish as a model for studying this process. Our analysis of mutant fish with a hypochromic microcytic anemia has provided key insights into factors that regulate hemoglobin synthesis. These factors fall into four categories: the production of heme, iron utilization, globin expression, and iron sulfur cluster production. We have found several novel genes involved in hemoglobin production, including ferroportin, mitoferrin, and glutaredoxin 5. The first two of these factors were found to be mutated in patients with human disorders, establishing the zebrafish system as a model for human disease. To date, we have uncovered over eight complementation groups of hypochromic anemia. In this proposal, we plan to extend our observations by characterizing the globin locus of the zebrafish, including its associated chromatin structure. Regulatory elements will also be examined. The zebrafish locus control region (LCR) is representative of an ?LCR. In transgenics, bringing the zebrafish LCR into apposition with globin genes drives high level of expression within erythroid cells. Transgenic fish are being created with human globin BACs to evaluate conservation of regulatory elements. The mutant zinfandel lacks embryonic globin production, but recovers and has normal adult hemoglobin formation. This gene was mapped to the globin locus on chromosome 3 and no globin coding regions are mutated. A high-resolution positional cloning project is underway to define this novel embryonic-specific globin regulatory mutation. We also developed a method for genetically analyzing chromatin factors that regulate hemoglobin production. The technology of morpholinos in which antisense oligonucleotides against specific genes are injected into the one cell embryo can knock-down gene function and will reveal changes in erythropoiesis. A compendium of over 300 chromatin remodeling factors that are orthologs of human genes has been created, and each will be studied by gene knock-down in zebrafish embryos. The effect of gene knock-down on erythroid gene expression (including LCR activation and globin gene expression) will be evaluated. These factors will be studied genetically in the zebrafish and interactions with specific transcription factors will be investigated by double knock-down and overexpression studies. Using human erythroid cells, we plan to biochemically purify a complex of chromatin factor orthologs found through this genetic screen. The understanding of chromatin factors that affect hemoglobin production will have a tremendous impact on our understanding of hemoglobin production and for the treatment of patients with thalassemia, sickle cell anemia, and globin mutations.

描述（由申请人提供）：血红蛋白的产生是一个进化保守的过程，是红细胞谱系特有的。我们利用斑马鱼作为研究这一过程的模型。我们的分析突变鱼与低色素小细胞贫血提供了关键的见解，调节血红蛋白合成的因素。这些因素分为四类：血红素的产生、铁的利用、珠蛋白的表达和铁硫簇的产生。我们已经发现了几个与血红蛋白产生有关的新基因，包括铁转运蛋白、丝裂铁蛋白和glutaredoxin 5。前两个因子在人类疾病患者中被发现发生突变，建立了斑马鱼系统作为人类疾病的模型。迄今为止，我们已经发现了超过8个低色素贫血的互补群。在本提案中，我们计划通过表征斑马鱼的珠蛋白位点，包括其相关的染色质结构来扩展我们的观察。监管因素也将被审查。斑马鱼基因座控制区（LCR）是一种具有代表性的基因控制区。电感电容电阻测量。在转基因中，将斑马鱼LCR与珠蛋白基因相结合，可在红细胞内驱动高水平的表达。人们正在用人类珠蛋白BACs创造转基因鱼，以评估调节元件的保护。突变的仙粉黛缺乏胚胎球蛋白的产生，但恢复并具有正常的成年血红蛋白形成。该基因定位于3号染色体上的珠蛋白位点，无珠蛋白编码区突变。一个高分辨率的定位克隆项目正在进行中，以确定这种新的胚胎特异性珠蛋白调控突变。我们还开发了一种基因分析调节血红蛋白产生的染色质因子的方法。morpholinos技术将针对特定基因的反义寡核苷酸注射到单个细胞胚胎中，可以破坏基因功能并揭示红细胞生成的变化。一个超过300个染色质重塑因子的纲要，这些因子是人类基因的同源物，每个都将通过斑马鱼胚胎的基因敲除来研究。基因敲除对红系基因表达（包括LCR激活和珠蛋白基因表达）的影响将被评估。这些因子将在斑马鱼中进行遗传研究，并通过双敲除和过表达研究来研究与特定转录因子的相互作用。使用人类红细胞，我们计划生化纯化通过这种遗传筛选发现的染色质因子同源物的复合体。对影响血红蛋白产生的染色质因子的了解将对我们对血红蛋白产生的理解以及对地中海贫血、镰状细胞性贫血和珠蛋白突变患者的治疗产生巨大影响。